Precision metabolic engineering: The design of responsive, selective, and controllable metabolic systems.

Science.gov (United States)

McNerney, Monica P; Watstein, Daniel M; Styczynski, Mark P

2015-09-01

Metabolic engineering is generally focused on static optimization of cells to maximize production of a desired product, though recently dynamic metabolic engineering has explored how metabolic programs can be varied over time to improve titer. However, these are not the only types of applications where metabolic engineering could make a significant impact. Here, we discuss a new conceptual framework, termed "precision metabolic engineering," involving the design and engineering of systems that make different products in response to different signals. Rather than focusing on maximizing titer, these types of applications typically have three hallmarks: sensing signals that determine the desired metabolic target, completely directing metabolic flux in response to those signals, and producing sharp responses at specific signal thresholds. In this review, we will first discuss and provide examples of precision metabolic engineering. We will then discuss each of these hallmarks and identify which existing metabolic engineering methods can be applied to accomplish those tasks, as well as some of their shortcomings. Ultimately, precise control of metabolic systems has the potential to enable a host of new metabolic engineering and synthetic biology applications for any problem where flexibility of response to an external signal could be useful. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Modular co-culture engineering, a new approach for metabolic engineering.

Science.gov (United States)

Zhang, Haoran; Wang, Xiaonan

2016-09-01

With the development of metabolic engineering, employment of a selected microbial host for accommodation of a designed biosynthetic pathway to produce a target compound has achieved tremendous success in the past several decades. Yet, increasing requirements for sophisticated microbial biosynthesis call for establishment and application of more advanced metabolic engineering methodologies. Recently, important progress has been made towards employing more than one engineered microbial strains to constitute synthetic co-cultures and modularizing the biosynthetic labor between the co-culture members in order to improve bioproduction performance. This emerging approach, referred to as modular co-culture engineering in this review, presents a valuable opportunity for expanding the scope of the broad field of metabolic engineering. We highlight representative research accomplishments using this approach, especially those utilizing metabolic engineering tools for microbial co-culture manipulation. Key benefits and major challenges associated with modular co-culture engineering are also presented and discussed. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Synthetic biology and regulatory networks: where metabolic systems biology meets control engineering.

Science.gov (United States)

He, Fei; Murabito, Ettore; Westerhoff, Hans V

2016-04-01

Metabolic pathways can be engineered to maximize the synthesis of various products of interest. With the advent of computational systems biology, this endeavour is usually carried out through in silico theoretical studies with the aim to guide and complement further in vitro and in vivo experimental efforts. Clearly, what counts is the result in vivo, not only in terms of maximal productivity but also robustness against environmental perturbations. Engineering an organism towards an increased production flux, however, often compromises that robustness. In this contribution, we review and investigate how various analytical approaches used in metabolic engineering and synthetic biology are related to concepts developed by systems and control engineering. While trade-offs between production optimality and cellular robustness have already been studied diagnostically and statically, the dynamics also matter. Integration of the dynamic design aspects of control engineering with the more diagnostic aspects of metabolic, hierarchical control and regulation analysis is leading to the new, conceptual and operational framework required for the design of robust and productive dynamic pathways. © 2016 The Author(s).

Synthetic biology and metabolic engineering.

Science.gov (United States)

Stephanopoulos, Gregory

2012-11-16

Metabolic engineering emerged 20 years ago as the discipline occupied with the directed modification of metabolic pathways for the microbial synthesis of various products. As such, it deals with the engineering (design, construction, and optimization) of native as well as non-natural routes of product synthesis, aided in this task by the availability of synthetic DNA, the core enabling technology of synthetic biology. The two fields, however, only partially overlap in their interest in pathway engineering. While fabrication of biobricks, synthetic cells, genetic circuits, and nonlinear cell dynamics, along with pathway engineering, have occupied researchers in the field of synthetic biology, the sum total of these areas does not constitute a coherent definition of synthetic biology with a distinct intellectual foundation and well-defined areas of application. This paper reviews the origins of the two fields and advances two distinct paradigms for each of them: that of unit operations for metabolic engineering and electronic circuits for synthetic biology. In this context, metabolic engineering is about engineering cell factories for the biological manufacturing of chemical and pharmaceutical products, whereas the main focus of synthetic biology is fundamental biological research facilitated by the use of synthetic DNA and genetic circuits.

Protein engineering for metabolic engineering: current and next-generation tools

Science.gov (United States)

Marcheschi, Ryan J.; Gronenberg, Luisa S.; Liao, James C.

2014-01-01

Protein engineering in the context of metabolic engineering is increasingly important to the field of industrial biotechnology. As the demand for biologically-produced food, fuels, chemicals, food additives, and pharmaceuticals continues to grow, the ability to design and modify proteins to accomplish new functions will be required to meet the high productivity demands for the metabolism of engineered organisms. This article reviews advances of selecting, modeling, and engineering proteins to improve or alter their activity. Some of the methods have only recently been developed for general use and are just beginning to find greater application in the metabolic engineering community. We also discuss methods of generating random and targeted diversity in proteins to generate mutant libraries for analysis. Recent uses of these techniques to alter cofactor use, produce non-natural amino acids, alcohols, and carboxylic acids, and alter organism phenotypes are presented and discussed as examples of the successful engineering of proteins for metabolic engineering purposes. PMID:23589443

Metabolic engineering of yeast for lignocellulosic biofuel production.

Science.gov (United States)

Jin, Yong-Su; Cate, Jamie Hd

2017-12-01

Production of biofuels from lignocellulosic biomass remains an unsolved challenge in industrial biotechnology. Efforts to use yeast for conversion face the question of which host organism to use, counterbalancing the ease of genetic manipulation with the promise of robust industrial phenotypes. Saccharomyces cerevisiae remains the premier host for metabolic engineering of biofuel pathways, due to its many genetic, systems and synthetic biology tools. Numerous engineering strategies for expanding substrate ranges and diversifying products of S. cerevisiae have been developed. Other yeasts generally lack these tools, yet harbor superior phenotypes that could be exploited in the harsh processes required for lignocellulosic biofuel production. These include thermotolerance, resistance to toxic compounds generated during plant biomass deconstruction, and wider carbon consumption capabilities. Although promising, these yeasts have yet to be widely exploited. By contrast, oleaginous yeasts such as Yarrowia lipolytica capable of producing high titers of lipids are rapidly advancing in terms of the tools available for their metabolic manipulation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genome-scale modeling for metabolic engineering.

Science.gov (United States)

Simeonidis, Evangelos; Price, Nathan D

2015-03-01

We focus on the application of constraint-based methodologies and, more specifically, flux balance analysis in the field of metabolic engineering, and enumerate recent developments and successes of the field. We also review computational frameworks that have been developed with the express purpose of automatically selecting optimal gene deletions for achieving improved production of a chemical of interest. The application of flux balance analysis methods in rational metabolic engineering requires a metabolic network reconstruction and a corresponding in silico metabolic model for the microorganism in question. For this reason, we additionally present a brief overview of automated reconstruction techniques. Finally, we emphasize the importance of integrating metabolic networks with regulatory information-an area which we expect will become increasingly important for metabolic engineering-and present recent developments in the field of metabolic and regulatory integration.

Plant metabolic modeling: achieving new insight into metabolism and metabolic engineering.

Science.gov (United States)

Baghalian, Kambiz; Hajirezaei, Mohammad-Reza; Schreiber, Falk

2014-10-01

Models are used to represent aspects of the real world for specific purposes, and mathematical models have opened up new approaches in studying the behavior and complexity of biological systems. However, modeling is often time-consuming and requires significant computational resources for data development, data analysis, and simulation. Computational modeling has been successfully applied as an aid for metabolic engineering in microorganisms. But such model-based approaches have only recently been extended to plant metabolic engineering, mainly due to greater pathway complexity in plants and their highly compartmentalized cellular structure. Recent progress in plant systems biology and bioinformatics has begun to disentangle this complexity and facilitate the creation of efficient plant metabolic models. This review highlights several aspects of plant metabolic modeling in the context of understanding, predicting and modifying complex plant metabolism. We discuss opportunities for engineering photosynthetic carbon metabolism, sucrose synthesis, and the tricarboxylic acid cycle in leaves and oil synthesis in seeds and the application of metabolic modeling to the study of plant acclimation to the environment. The aim of the review is to offer a current perspective for plant biologists without requiring specialized knowledge of bioinformatics or systems biology. © 2014 American Society of Plant Biologists. All rights reserved.

Modularization of genetic elements promotes synthetic metabolic engineering.

Science.gov (United States)

Qi, Hao; Li, Bing-Zhi; Zhang, Wen-Qian; Liu, Duo; Yuan, Ying-Jin

2015-11-15

In the context of emerging synthetic biology, metabolic engineering is moving to the next stage powered by new technologies. Systematical modularization of genetic elements makes it more convenient to engineer biological systems for chemical production or other desired purposes. In the past few years, progresses were made in engineering metabolic pathway using synthetic biology tools. Here, we spotlighted the topic of implementation of modularized genetic elements in metabolic engineering. First, we overviewed the principle developed for modularizing genetic elements and then discussed how the genetic modules advanced metabolic engineering studies. Next, we picked up some milestones of engineered metabolic pathway achieved in the past few years. Last, we discussed the rapid raised synthetic biology field of "building a genome" and the potential in metabolic engineering. Copyright © 2015 Elsevier Inc. All rights reserved.

Metabolic engineering in chemolithoautotrophic hosts for the production of fuels and chemicals.

Science.gov (United States)

Nybo, S Eric; Khan, Nymul E; Woolston, Benjamin M; Curtis, Wayne R

2015-07-01

The ability of autotrophic organisms to fix CO2 presents an opportunity to utilize this 'greenhouse gas' as an inexpensive substrate for biochemical production. Unlike conventional heterotrophic microorganisms that consume carbohydrates and amino acids, prokaryotic chemolithoautotrophs have evolved the capacity to utilize reduced chemical compounds to fix CO2 and drive metabolic processes. The use of chemolithoautotrophic hosts as production platforms has been renewed by the prospect of metabolically engineered commodity chemicals and fuels. Efforts such as the ARPA-E electrofuels program highlight both the potential and obstacles that chemolithoautotrophic biosynthetic platforms provide. This review surveys the numerous advances that have been made in chemolithoautotrophic metabolic engineering with a focus on hydrogen oxidizing bacteria such as the model chemolithoautotrophic organism (Ralstonia), the purple photosynthetic bacteria (Rhodobacter), and anaerobic acetogens. Two alternative strategies of microbial chassis development are considered: (1) introducing or enhancing autotrophic capabilities (carbon fixation, hydrogen utilization) in model heterotrophic organisms, or (2) improving tools for pathway engineering (transformation methods, promoters, vectors etc.) in native autotrophic organisms. Unique characteristics of autotrophic growth as they relate to bioreactor design and process development are also discussed in the context of challenges and opportunities for genetic manipulation of organisms as production platforms. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Perspectives in metabolic engineering: understanding cellular regulation towards the control of metabolic routes.

Science.gov (United States)

Zadran, Sohila; Levine, Raphael D

2013-01-01

Metabolic engineering seeks to redirect metabolic pathways through the modification of specific biochemical reactions or the introduction of new ones with the use of recombinant technology. Many of the chemicals synthesized via introduction of product-specific enzymes or the reconstruction of entire metabolic pathways into engineered hosts that can sustain production and can synthesize high yields of the desired product as yields of natural product-derived compounds are frequently low, and chemical processes can be both energy and material expensive; current endeavors have focused on using biologically derived processes as alternatives to chemical synthesis. Such economically favorable manufacturing processes pursue goals related to sustainable development and "green chemistry". Metabolic engineering is a multidisciplinary approach, involving chemical engineering, molecular biology, biochemistry, and analytical chemistry. Recent advances in molecular biology, genome-scale models, theoretical understanding, and kinetic modeling has increased interest in using metabolic engineering to redirect metabolic fluxes for industrial and therapeutic purposes. The use of metabolic engineering has increased the productivity of industrially pertinent small molecules, alcohol-based biofuels, and biodiesel. Here, we highlight developments in the practical and theoretical strategies and technologies available for the metabolic engineering of simple systems and address current limitations.

Modeling of Zymomonas mobilis central metabolism for novel metabolic engineering strategies.

Science.gov (United States)

Kalnenieks, Uldis; Pentjuss, Agris; Rutkis, Reinis; Stalidzans, Egils; Fell, David A

2014-01-01

Mathematical modeling of metabolism is essential for rational metabolic engineering. The present work focuses on several types of modeling approach to quantitative understanding of central metabolic network and energetics in the bioethanol-producing bacterium Zymomonas mobilis. Combined use of Flux Balance, Elementary Flux Mode, and thermodynamic analysis of its central metabolism, together with dynamic modeling of the core catabolic pathways, can help to design novel substrate and product pathways by systematically analyzing the solution space for metabolic engineering, and yields insights into the function of metabolic network, hardly achievable without applying modeling tools.

Computer-aided design for metabolic engineering.

Science.gov (United States)

Fernández-Castané, Alfred; Fehér, Tamás; Carbonell, Pablo; Pauthenier, Cyrille; Faulon, Jean-Loup

2014-12-20

The development and application of biotechnology-based strategies has had a great socio-economical impact and is likely to play a crucial role in the foundation of more sustainable and efficient industrial processes. Within biotechnology, metabolic engineering aims at the directed improvement of cellular properties, often with the goal of synthesizing a target chemical compound. The use of computer-aided design (CAD) tools, along with the continuously emerging advanced genetic engineering techniques have allowed metabolic engineering to broaden and streamline the process of heterologous compound-production. In this work, we review the CAD tools available for metabolic engineering with an emphasis, on retrosynthesis methodologies. Recent advances in genetic engineering strategies for pathway implementation and optimization are also reviewed as well as a range of bionalytical tools to validate in silico predictions. A case study applying retrosynthesis is presented as an experimental verification of the output from Retropath, the first complete automated computational pipeline applicable to metabolic engineering. Applying this CAD pipeline, together with genetic reassembly and optimization of culture conditions led to improved production of the plant flavonoid pinocembrin. Coupling CAD tools with advanced genetic engineering strategies and bioprocess optimization is crucial for enhanced product yields and will be of great value for the development of non-natural products through sustainable biotechnological processes. Copyright © 2014 Elsevier B.V. All rights reserved.

Metabolic engineering in methanotrophic bacteria

Energy Technology Data Exchange (ETDEWEB)

Kalyuzhnaya, MG; Puri, AW; Lidstrom, ME

2015-05-01

Methane, as natural gas or biogas, is the least expensive source of carbon for (bio)chemical synthesis. Scalable biological upgrading of this simple alkane to chemicals and fuels can bring new sustainable solutions to a number of industries with large environmental footprints, such as natural gas/petroleum production, landfills, wastewater treatment, and livestock. Microbial biocatalysis with methane as a feedstock has been pursued off and on for almost a half century, with little enduring success. Today, biological engineering and systems biology provide new opportunities for metabolic system modulation and give new optimism to the concept of a methane-based bio-industry. Here we present an overview of the most recent advances pertaining to metabolic engineering of microbial methane utilization. Some ideas concerning metabolic improvements for production of acetyl-CoA and pyruvate, two main precursors for bioconversion, are presented. We also discuss main gaps in the current knowledge of aerobic methane utilization, which must be solved in order to release the full potential of methane-based biosystems. (C) 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Metabolic engineering for the microbial production of isoprenoids: Carotenoids and isoprenoid-based biofuels

Directory of Open Access Journals (Sweden)

Fu-Xing Niu

2017-09-01

Full Text Available Isoprenoids are the most abundant and highly diverse group of natural products. Many isoprenoids have been used for pharmaceuticals, nutraceuticals, flavors, cosmetics, food additives and biofuels. Carotenoids and isoprenoid-based biofuels are two classes of important isoprenoids. These isoprenoids have been produced microbially through metabolic engineering and synthetic biology efforts. Herein, we briefly review the engineered biosynthetic pathways in well-characterized microbial systems for the production of carotenoids and several isoprenoid-based biofuels.

Biofuel production in Escherichia coli. The role of metabolic engineering and synthetic biology

Energy Technology Data Exchange (ETDEWEB)

Clomburg, James M. [Rice Univ., Houston, TX (United States). Dept. of Chemical and Biomolecular Engineering; Gonzalez, Ramon [Rice Univ., Houston, TX (United States). Dept. of Chemical and Biomolecular Engineering; Rice Univ., Houston, TX (United States). Dept. of Bioengineering

2010-03-15

The microbial production of biofuels is a promising avenue for the development of viable processes for the generation of fuels from sustainable resources. In order to become cost and energy effective, these processes must utilize organisms that can be optimized to efficiently produce candidate fuels from a variety of feedstocks. Escherichia coli has become a promising host organism for the microbial production of biofuels in part due to the ease at which this organism can be manipulated. Advancements in metabolic engineering and synthetic biology have led to the ability to efficiently engineer E. coli as a biocatalyst for the production of a wide variety of potential biofuels from several biomass constituents. This review focuses on recent efforts devoted to engineering E. coli for the production of biofuels, with emphasis on the key aspects of both the utilization of a variety of substrates as well as the synthesis of several promising biofuels. Strategies for the efficient utilization of carbohydrates, carbohydrate mixtures, and noncarbohydrate carbon sources will be discussed along with engineering efforts for the exploitation of both fermentative and nonfermentative pathways for the production of candidate biofuels such as alcohols and higher carbon biofuels derived from fatty acid and isoprenoid pathways. Continued advancements in metabolic engineering and synthetic biology will help improve not only the titers, yields, and productivities of biofuels discussed herein, but also increase the potential range of compounds that can be produced. (orig.)

Metabolic engineering with plants for a sustainable biobased economy.

Science.gov (United States)

Yoon, Jong Moon; Zhao, Le; Shanks, Jacqueline V

2013-01-01

Plants are bona fide sustainable organisms because they accumulate carbon and synthesize beneficial metabolites from photosynthesis. To meet the challenges to food security and health threatened by increasing population growth and depletion of nonrenewable natural resources, recent metabolic engineering efforts have shifted from single pathways to holistic approaches with multiple genes owing to integration of omics technologies. Successful engineering of plants results in the high yield of biomass components for primary food sources and biofuel feedstocks, pharmaceuticals, and platform chemicals through synthetic biology and systems biology strategies. Further discovery of undefined biosynthesis pathways in plants, integrative analysis of discrete omics data, and diversified process developments for production of platform chemicals are essential to overcome the hurdles for sustainable production of value-added biomolecules from plants.

The logics of metabolic regulation in bacteria challenges biosensor-based metabolic engineering

Directory of Open Access Journals (Sweden)

Matthieu Jules

2017-12-01

Full Text Available Synthetic Biology (SB aims at the rational design and engineering of novel biological functions and systems. By facilitating the engineering of living organisms, SB promises to facilitate the development of many new applications for health, biomanufacturing, and the environment. Over the last decade, SB promoted the construction of libraries of components enabling the fine-tuning of genetic circuits expression and the development of novel genome engineering methodologies for many organisms of interest. SB thus opened new perspectives in the field of metabolic engineering, which was until then mainly limited to (overproducing naturally synthesized metabolic compounds. To engineer efficient cell factories, it is key to precisely reroute cellular resources from the central carbon metabolism (CCM to the synthetic circuitry. This task is however difficult as there is still significant lack of knowledge regarding both the function of several metabolic components and the regulation of the CCM fluxes for many industrially important bacteria. Pyruvate is a pivotal metabolite at the heart of the CCM and a key precursor for the synthesis of several commodity compounds and fine chemicals. Numerous bacterial species can also use it as a carbon source when present in the environment but bacterial, pyruvate-specific uptake systems were to be discovered. This is an issue for metabolic engineering as one can imagine to make use of pyruvate transport systems to replenish synthetic metabolic pathways towards the synthesis of chemicals of interest. Here we describe a recent study (MBio 8(5: e00976-17, which identified and characterized a pyruvate transport system in the Gram-positive (G+ve bacterium Bacillus subtilis, a well-established biotechnological workhorse for the production of enzymes, fine chemicals and antibiotics. This study also revealed that the activity of the two-component system (TCS responsible for its induction is retro-inhibited by the level of

Engineering yeast metabolism for production of fuels and chemicals

DEFF Research Database (Denmark)

Nielsen, Jens

2016-01-01

faster development of metabolically engineered strains that can be used for production of fuels and chemicals. The yeast Saccharomyces cerevisiae is widely used for production of fuels, chemicals, pharmaceuticals and materials. Through metabolic engineering of this yeast a number of novel industrial...... as for metabolic design. In this lecture it will be demonstrated how the Design-Build-Test cycle of metabolic engineering has allowed for development of yeast cell factories for production of a range of different fuels and chemicals. Some examples of different technologies will be presented together with examples......Metabolic engineering relies on the Design-Build-Test cycle. This cycle includes technologies like mathematical modeling of metabolism, genome editing and advanced tools for phenotypic characterization. In recent years there have been advances in several of these technologies, which has enabled...

Recent advances in systems metabolic engineering tools and strategies.

Science.gov (United States)

Chae, Tong Un; Choi, So Young; Kim, Je Woong; Ko, Yoo-Sung; Lee, Sang Yup

2017-10-01

Metabolic engineering has been playing increasingly important roles in developing microbial cell factories for the production of various chemicals and materials to achieve sustainable chemical industry. Nowadays, many tools and strategies are available for performing systems metabolic engineering that allows systems-level metabolic engineering in more sophisticated and diverse ways by adopting rapidly advancing methodologies and tools of systems biology, synthetic biology and evolutionary engineering. As an outcome, development of more efficient microbial cell factories has become possible. Here, we review recent advances in systems metabolic engineering tools and strategies together with accompanying application examples. In addition, we describe how these tools and strategies work together in simultaneous and synergistic ways to develop novel microbial cell factories. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genetic and metabolic engineering in diatoms.

Science.gov (United States)

Huang, Weichao; Daboussi, Fayza

2017-09-05

Diatoms have attracted considerable attention due to their success in diverse environmental conditions, which probably is a consequence of their complex origins. Studies of their metabolism will provide insight into their adaptation capacity and are a prerequisite for metabolic engineering. Several years of investigation have led to the development of the genome engineering tools required for such studies, and a profusion of appropriate tools is now available for exploring and exploiting the metabolism of these organisms. Diatoms are highly prized in industrial biotechnology, due to both their richness in natural lipids and carotenoids and their ability to produce recombinant proteins, of considerable value in diverse markets. This review provides an overview of recent advances in genetic engineering methods for diatoms, from the development of gene expression cassettes and gene delivery methods, to cutting-edge genome-editing technologies. It also highlights the contributions of these rapid developments to both basic and applied research: they have improved our understanding of key physiological processes; and they have made it possible to modify the natural metabolism to favour the production of specific compounds or to produce new compounds for green chemistry and pharmaceutical applications.This article is part of the themed issue 'The peculiar carbon metabolism in diatoms'. © 2017 The Author(s).

Engineering central metabolism – a grand challenge for plant biologists

DEFF Research Database (Denmark)

Sweetlove, Lee J.; Nielsen, Jens; Fernie, Alisdair R.

2017-01-01

The goal of increasing crop productivity and nutrient-use efficiency is being addressed by a number of ambitious research projects seeking to re-engineer photosynthetic biochemistry. Many of these projects will require the engineering of substantial changes in fluxes of central metabolism. However......, as has been amply demonstrated in simpler systems such as microbes, central metabolism is extremely difficult to rationally engineer. This is because of multiple layers of regulation that operate to maintain metabolic steady state and because of the highly connected nature of central metabolism....... In this review we discuss new approaches for metabolic engineering that have the potential to address these problems and dramatically improve the success with which we can rationally engineer central metabolism in plants. In particular, we advocate the adoption of an iterative ‘design-build-test-learn’ cycle...

Protein design in systems metabolic engineering for industrial strain development.

Science.gov (United States)

Chen, Zhen; Zeng, An-Ping

2013-05-01

Accelerating the process of industrial bacterial host strain development, aimed at increasing productivity, generating new bio-products or utilizing alternative feedstocks, requires the integration of complementary approaches to manipulate cellular metabolism and regulatory networks. Systems metabolic engineering extends the concept of classical metabolic engineering to the systems level by incorporating the techniques used in systems biology and synthetic biology, and offers a framework for the development of the next generation of industrial strains. As one of the most useful tools of systems metabolic engineering, protein design allows us to design and optimize cellular metabolism at a molecular level. Here, we review the current strategies of protein design for engineering cellular synthetic pathways, metabolic control systems and signaling pathways, and highlight the challenges of this subfield within the context of systems metabolic engineering. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Metabolite damage and repair in metabolic engineering design.

Science.gov (United States)

Sun, Jiayi; Jeffryes, James G; Henry, Christopher S; Bruner, Steven D; Hanson, Andrew D

2017-11-01

The necessarily sharp focus of metabolic engineering and metabolic synthetic biology on pathways and their fluxes has tended to divert attention from the damaging enzymatic and chemical side-reactions that pathway metabolites can undergo. Although historically overlooked and underappreciated, such metabolite damage reactions are now known to occur throughout metabolism and to generate (formerly enigmatic) peaks detected in metabolomics datasets. It is also now known that metabolite damage is often countered by dedicated repair enzymes that undo or prevent it. Metabolite damage and repair are highly relevant to engineered pathway design: metabolite damage reactions can reduce flux rates and product yields, and repair enzymes can provide robust, host-independent solutions. Herein, after introducing the core principles of metabolite damage and repair, we use case histories to document how damage and repair processes affect efficient operation of engineered pathways - particularly those that are heterologous, non-natural, or cell-free. We then review how metabolite damage reactions can be predicted, how repair reactions can be prospected, and how metabolite damage and repair can be built into genome-scale metabolic models. Lastly, we propose a versatile 'plug and play' set of well-characterized metabolite repair enzymes to solve metabolite damage problems known or likely to occur in metabolic engineering and synthetic biology projects. Copyright © 2017 International Metabolic Engineering Society. All rights reserved.

Systems metabolic engineering in an industrial setting.

Science.gov (United States)

Sagt, Cees M J

2013-03-01

Systems metabolic engineering is based on systems biology, synthetic biology, and evolutionary engineering and is now also applied in industry. Industrial use of systems metabolic engineering focuses on strain and process optimization. Since ambitious yields, titers, productivities, and low costs are key in an industrial setting, the use of effective and robust methods in systems metabolic engineering is becoming very important. Major improvements in the field of proteomics and metabolomics have been crucial in the development of genome-wide approaches in strain and process development. This is accompanied by a rapid increase in DNA sequencing and synthesis capacity. These developments enable the use of systems metabolic engineering in an industrial setting. Industrial systems metabolic engineering can be defined as the combined use of genome-wide genomics, transcriptomics, proteomics, and metabolomics to modify strains or processes. This approach has become very common since the technology for generating large data sets of all levels of the cellular processes has developed quite fast into robust, reliable, and affordable methods. The main challenge and scope of this mini review is how to translate these large data sets in relevant biological leads which can be tested for strain or process improvements. Experimental setup, heterogeneity of the culture, and sample pretreatment are important issues which are easily underrated. In addition, the process of structuring, filtering, and visualization of data is important, but also, the availability of a genetic toolbox and equipment for medium/high-throughput fermentation is a key success factor. For an efficient bioprocess, all the different components in this process have to work together. Therefore, mutual tuning of these components is an important strategy.

Advancing metabolic engineering through systems biology of industrial microorganisms

DEFF Research Database (Denmark)

Dai, Zongjie; Nielsen, Jens

2015-01-01

resources. The objective of systems biology is to gain a comprehensive and quantitative understanding of living cells and can hereby enhance our ability to characterize and predict cellular behavior. Systems biology of industrial microorganisms is therefore valuable for metabolic engineering. Here we review......Development of sustainable processes to produce bio-based compounds is necessary due to the severe environmental problems caused by the use of fossil resources. Metabolic engineering can facilitate the development of highly efficient cell factories to produce these compounds from renewable...... the application of systems biology tools for the identification of metabolic engineering targets which may lead to reduced development time for efficient cell factories. Finally, we present some perspectives of systems biology for advancing metabolic engineering further....

Advances and prospects in metabolic engineering of Zymomonas mobilis.

Science.gov (United States)

Wang, Xia; He, Qiaoning; Yang, Yongfu; Wang, Jingwen; Haning, Katie; Hu, Yun; Wu, Bo; He, Mingxiong; Zhang, Yaoping; Bao, Jie; Contreras, Lydia M; Yang, Shihui

2018-04-05

Biorefinery of biomass-based biofuels and biochemicals by microorganisms is a competitive alternative of traditional petroleum refineries. Zymomonas mobilis is a natural ethanologen with many desirable characteristics, which makes it an ideal industrial microbial biocatalyst for commercial production of desirable bioproducts through metabolic engineering. In this review, we summarize the metabolic engineering progress achieved in Z. mobilis to expand its substrate and product ranges as well as to enhance its robustness against stressful conditions such as inhibitory compounds within the lignocellulosic hydrolysates and slurries. We also discuss a few metabolic engineering strategies that can be applied in Z. mobilis to further develop it as a robust workhorse for economic lignocellulosic bioproducts. In addition, we briefly review the progress of metabolic engineering in Z. mobilis related to the classical synthetic biology cycle of "Design-Build-Test-Learn", as well as the progress and potential to develop Z. mobilis as a model chassis for biorefinery practices in the synthetic biology era. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Metabolic engineering of Yarrowia lipolytica for industrial applications.

Science.gov (United States)

Zhu, Quinn; Jackson, Ethel N

2015-12-01

Yarrowia lipolytica is a safe and robust yeast that has a history of industrial applications. Its physiological, metabolic and genomic characteristics have made it a superior host for metabolic engineering. The results of optimizing internal pathways and introducing new pathways have demonstrated that Y. lipolytica can be a platform cell factory for cost-effective production of chemicals and fuels derived from fatty acids, lipids and acetyl-CoA. Two products have been commercialized from metabolically engineered Y. lipolytica strains producing high amounts of omega-3 eicosapentaenoic acid, and more products are on the way to be produced at industrial scale. Here we review recent progress in metabolic engineering of Y. lipolytica for production of biodiesel fuel, functional fatty acids and carotenoids. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Metabolic engineering is key to a sustainable chemical industry.

Science.gov (United States)

Murphy, Annabel C

2011-08-01

The depletion of fossil fuel stocks will prohibit their use as the main feedstock of future industrial processes. Biocatalysis is being increasingly used to reduce fossil fuel reliance and to improve the sustainability, efficiency and cost of chemical production. Even with their current small market share, biocatalyzed processes already generate approximately US$50 billion and it has been estimated that they could be used to produce up to 20% of fine chemicals by 2020. Until the advent of molecular biological technologies, the compounds that were readily accessible from renewable biomass were restricted to naturally-occurring metabolites. However, metabolic engineering has considerably broadened the range of compounds now accessible, providing access to compounds that cannot be otherwise reliably sourced, as well as replacing established chemical processes. This review presents the case for continued efforts to promote the adoption of biocatalyzed processes, highlighting successful examples of industrial chemical production from biomass and/or via biocatalyzed processes. A selection of emerging technologies that may further extend the potential and sustainability of biocatalysis are also presented. As the field matures, metabolic engineering will be increasingly crucial in maintaining our quality of life into a future where our current resources and feedstocks cannot be relied upon.

Advancing metabolic engineering through systems biology of industrial microorganisms.

Science.gov (United States)

Dai, Zongjie; Nielsen, Jens

2015-12-01

Development of sustainable processes to produce bio-based compounds is necessary due to the severe environmental problems caused by the use of fossil resources. Metabolic engineering can facilitate the development of highly efficient cell factories to produce these compounds from renewable resources. The objective of systems biology is to gain a comprehensive and quantitative understanding of living cells and can hereby enhance our ability to characterize and predict cellular behavior. Systems biology of industrial microorganisms is therefore valuable for metabolic engineering. Here we review the application of systems biology tools for the identification of metabolic engineering targets which may lead to reduced development time for efficient cell factories. Finally, we present some perspectives of systems biology for advancing metabolic engineering further. Copyright © 2015 Elsevier Ltd. All rights reserved.

Applications of computational modeling in metabolic engineering of yeast

DEFF Research Database (Denmark)

Kerkhoven, Eduard J.; Lahtvee, Petri-Jaan; Nielsen, Jens

2015-01-01

a preferred flux distribution. These methods point to strategies for altering gene expression; however, fluxes are often controlled by post-transcriptional events. Moreover, GEMs are usually not taking into account metabolic regulation, thermodynamics and enzyme kinetics. To facilitate metabolic engineering......, it is necessary to expand the modeling of metabolism to consider kinetics of individual processes. This review will give an overview about models available for metabolic engineering of yeast and discusses their applications....

[Improving industrial microbial stress resistance by metabolic engineering: a review].

Science.gov (United States)

Fu, Ruiyan; Li, Yin

2010-09-01

Metabolic engineering is a technologic platform for industrial strain improvement and aims not only at modifying microbial metabolic fluxes, but also improving the physiological performance of industrial microbes. Microbes will meet multiple stresses in industrial processes. Consequently, elicited gene responses might result in a decrease in overall cell fitness and the efficiency of biotransformation. Thus, it is crucial to develop robust and productive microbial strains that can be integrated into industrial-scale bioprocesses. In this review, we focus on the progress of these novel methods and strategies for engineering stress-tolerance phenotypes referring to rational metabolic engineering and inverse metabolic engineering in recent years. In addition, we also address problems existing in this area and future research needs of microbial physiological functionality engineering.

13C Metabolic Flux Analysis for systematic metabolic engineering of S. cerevisiae for overproduction of fatty acids.

Directory of Open Access Journals (Sweden)

Amit Ghosh

2016-10-01

Full Text Available Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here we used flux-based modeling approaches to improve yields of fatty acids in S. cerevisiae. We combined 13C labeling data with comprehensive genome-scale models to shed light onto microbial metabolism and improve metabolic engineering efforts. We concentrated on studying the balance of acetyl-CoA, a precursor metabolite for the biosynthesis of fatty acids. A genome-wide acetyl-CoA balance study showed ATP citrate lyase from Y. lipolytica as a robust source of cytoplasmic acetyl-CoA and malate synthase as a desirable target for down-regulation in terms of acetyl-CoA consumption. These genetic modifications were applied to S. cerevisiae WRY2, a strain that is capable of producing 460 mg L of free fatty acids. With the addition of ATP citrate lyase and down-regulation of malate synthase the engineered strain produced 26 per cent more free fatty acids. Further increases in free fatty acid production of 33 per cent were obtained by knocking out the cytoplasmic glycerol-3-phosphate dehydrogenase, which flux analysis had shown was competing for carbon flux upstream with the carbon flux through the acetyl-CoA production pathway in the cytoplasm. In total, the genetic interventions applied in this work increased fatty acid production by 70 per cent.

Engineering strategy of yeast metabolism for higher alcohol production

Directory of Open Access Journals (Sweden)

Shimizu Hiroshi

2011-09-01

Full Text Available Abstract Background While Saccharomyces cerevisiae is a promising host for cost-effective biorefinary processes due to its tolerance to various stresses during fermentation, the metabolically engineered S. cerevisiae strains exhibited rather limited production of higher alcohols than that of Escherichia coli. Since the structure of the central metabolism of S. cerevisiae is distinct from that of E. coli, there might be a problem in the structure of the central metabolism of S. cerevisiae. In this study, the potential production of higher alcohols by S. cerevisiae is compared to that of E. coli by employing metabolic simulation techniques. Based on the simulation results, novel metabolic engineering strategies for improving higher alcohol production by S. cerevisiae were investigated by in silico modifications of the metabolic models of S. cerevisiae. Results The metabolic simulations confirmed that the high production of butanols and propanols by the metabolically engineered E. coli strains is derived from the flexible behavior of their central metabolism. Reducing this flexibility by gene deletion is an effective strategy to restrict the metabolic states for producing target alcohols. In contrast, the lower yield using S. cerevisiae originates from the structurally limited flexibility of its central metabolism in which gene deletions severely reduced cell growth. Conclusions The metabolic simulation demonstrated that the poor productivity of S. cerevisiae was improved by the introduction of E. coli genes to compensate the structural difference. This suggested that gene supplementation is a promising strategy for the metabolic engineering of S. cerevisiae to produce higher alcohols which should be the next challenge for the synthetic bioengineering of S. cerevisiae for the efficient production of higher alcohols.

Next-generation genome-scale models for metabolic engineering

DEFF Research Database (Denmark)

King, Zachary A.; Lloyd, Colton J.; Feist, Adam M.

2015-01-01

Constraint-based reconstruction and analysis (COBRA) methods have become widely used tools for metabolic engineering in both academic and industrial laboratories. By employing a genome-scale in silico representation of the metabolic network of a host organism, COBRA methods can be used to predict...... examples of applying COBRA methods to strain optimization are presented and discussed. Then, an outlook is provided on the next generation of COBRA models and the new types of predictions they will enable for systems metabolic engineering....

Applications of computational modeling in metabolic engineering of yeast.

Science.gov (United States)

Kerkhoven, Eduard J; Lahtvee, Petri-Jaan; Nielsen, Jens

2015-02-01

Generally, a microorganism's phenotype can be described by its pattern of metabolic fluxes. Although fluxes cannot be measured directly, inference of fluxes is well established. In biotechnology the aim is often to increase the capacity of specific fluxes. For this, metabolic engineering methods have been developed and applied extensively. Many of these rely on balancing of intracellular metabolites, redox, and energy fluxes, using genome-scale models (GEMs) that in combination with appropriate objective functions and constraints can be used to predict potential gene targets for obtaining a preferred flux distribution. These methods point to strategies for altering gene expression; however, fluxes are often controlled by post-transcriptional events. Moreover, GEMs are usually not taking into account metabolic regulation, thermodynamics and enzyme kinetics. To facilitate metabolic engineering, tools from synthetic biology have emerged, enabling integration and assembly of naturally nonexistent, but well-characterized components into a living organism. To describe these systems kinetic models are often used and to integrate these systems with the standard metabolic engineering approach, it is necessary to expand the modeling of metabolism to consider kinetics of individual processes. This review will give an overview about models available for metabolic engineering of yeast and discusses their applications. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

Metabolite damage and repair in metabolic engineering design

Energy Technology Data Exchange (ETDEWEB)

Sun, Jiayi; Jeffryes, James G.; Henry, Christopher S.; Bruner, Steven D.; Hanson, Andrew D.

2017-11-01

The necessarily sharp focus of metabolic engineering and metabolic synthetic biology on pathways and their fluxes has tended to divert attention from the damaging enzymatic and chemical side-reactions that pathway metabolites can undergo. Although historically overlooked and underappreciated, such metabolite damage reactions are now known to occur throughout metabolism and to generate (formerly enigmatic) peaks detected in metabolomics datasets. It is also now known that metabolite damage is often countered by dedicated repair enzymes that undo or prevent it. Metabolite damage and repair are highly relevant to engineered pathway design: metabolite damage reactions can reduce flux rates and product yields, and repair enzymes can provide robust, host-independent solutions. Herein, after introducing the core principles of metabolite damage and repair, we use case histories to document how damage and repair processes affect efficient operation of engineered pathways - particularly those that are heterologous, non-natural, or cell-free. We then review how metabolite damage reactions can be predicted, how repair reactions can be prospected, and how metabolite damage and repair can be built into genome-scale metabolic models. Lastly, we propose a versatile 'plug and play' set of well-characterized metabolite repair enzymes to solve metabolite damage problems known or likely to occur in metabolic engineering and synthetic biology projects.

The Future of Metabolic Engineering and Synthetic Biology: Towards a Systematic Practice

Science.gov (United States)

Yadav, Vikramaditya G.; De Mey, Marjan; Lim, Chin Giaw; Ajikumar, Parayil Kumaran; Stephanopoulos, Gregory

2012-01-01

Industrial biotechnology promises to revolutionize conventional chemical manufacturing in the years ahead, largely owing to the excellent progress in our ability to re-engineer cellular metabolism. However, most successes of metabolic engineering have been confined to over-producing natively synthesized metabolites in E. coli and S. cerevisiae. A major reason for this development has been the descent of metabolic engineering, particularly secondary metabolic engineering, to a collection of demonstrations rather than a systematic practice with generalizable tools. Synthetic biology, a more recent development, faces similar criticisms. Herein, we attempt to lay down a framework around which bioreaction engineering can systematize itself just like chemical reaction engineering. Central to this undertaking is a new approach to engineering secondary metabolism known as ‘multivariate modular metabolic engineering’ (MMME), whose novelty lies in its assessment and elimination of regulatory and pathway bottlenecks by re-defining the metabolic network as a collection of distinct modules. After introducing the core principles of MMME, we shall then present a number of recent developments in secondary metabolic engineering that could potentially serve as its facilitators. It is hoped that the ever-declining costs of de novo gene synthesis; the improved use of bioinformatic tools to mine, sort and analyze biological data; and the increasing sensitivity and sophistication of investigational tools will make the maturation of microbial metabolic engineering an autocatalytic process. Encouraged by these advances, research groups across the world would take up the challenge of secondary metabolite production in simple hosts with renewed vigor, thereby adding to the range of products synthesized using metabolic engineering. PMID:22629571

The future of metabolic engineering and synthetic biology: towards a systematic practice.

Science.gov (United States)

Yadav, Vikramaditya G; De Mey, Marjan; Lim, Chin Giaw; Ajikumar, Parayil Kumaran; Stephanopoulos, Gregory

2012-05-01

Industrial biotechnology promises to revolutionize conventional chemical manufacturing in the years ahead, largely owing to the excellent progress in our ability to re-engineer cellular metabolism. However, most successes of metabolic engineering have been confined to over-producing natively synthesized metabolites in E. coli and S. cerevisiae. A major reason for this development has been the descent of metabolic engineering, particularly secondary metabolic engineering, to a collection of demonstrations rather than a systematic practice with generalizable tools. Synthetic biology, a more recent development, faces similar criticisms. Herein, we attempt to lay down a framework around which bioreaction engineering can systematize itself just like chemical reaction engineering. Central to this undertaking is a new approach to engineering secondary metabolism known as 'multivariate modular metabolic engineering' (MMME), whose novelty lies in its assessment and elimination of regulatory and pathway bottlenecks by re-defining the metabolic network as a collection of distinct modules. After introducing the core principles of MMME, we shall then present a number of recent developments in secondary metabolic engineering that could potentially serve as its facilitators. It is hoped that the ever-declining costs of de novo gene synthesis; the improved use of bioinformatic tools to mine, sort and analyze biological data; and the increasing sensitivity and sophistication of investigational tools will make the maturation of microbial metabolic engineering an autocatalytic process. Encouraged by these advances, research groups across the world would take up the challenge of secondary metabolite production in simple hosts with renewed vigor, thereby adding to the range of products synthesized using metabolic engineering. Copyright © 2011 Elsevier Inc. All rights reserved.

Production of amino acids - Genetic and metabolic engineering approaches.

Science.gov (United States)

Lee, Jin-Ho; Wendisch, Volker F

2017-12-01

The biotechnological production of amino acids occurs at the million-ton scale and annually about 6milliontons of l-glutamate and l-lysine are produced by Escherichia coli and Corynebacterium glutamicum strains. l-glutamate and l-lysine production from starch hydrolysates and molasses is very efficient and access to alternative carbon sources and new products has been enabled by metabolic engineering. This review focusses on genetic and metabolic engineering of amino acid producing strains. In particular, rational approaches involving modulation of transcriptional regulators, regulons, and attenuators will be discussed. To address current limitations of metabolic engineering, this article gives insights on recent systems metabolic engineering approaches based on functional tools and method such as genome reduction, amino acid sensors based on transcriptional regulators and riboswitches, CRISPR interference, small regulatory RNAs, DNA scaffolding, and optogenetic control, and discusses future prospects. Copyright © 2017 Elsevier Ltd. All rights reserved.

Balancing cellular redox metabolism in microbial electrosynthesis and electro fermentation - A chance for metabolic engineering.

Science.gov (United States)

Kracke, Frauke; Lai, Bin; Yu, Shiqin; Krömer, Jens O

2018-01-01

More and more microbes are discovered that are capable of extracellular electron transfer, a process in which they use external electrodes as electron donors or acceptors for metabolic reactions. This feature can be used to overcome cellular redox limitations and thus optimizing microbial production. The technologies, termed microbial electrosynthesis and electro-fermentation, have the potential to open novel bio-electro production platforms from sustainable energy and carbon sources. However, the performance of reported systems is currently limited by low electron transport rates between microbes and electrodes and our limited ability for targeted engineering of these systems due to remaining knowledge gaps about the underlying fundamental processes. Metabolic engineering offers many opportunities to optimize these processes, for instance by genetic engineering of pathways for electron transfer on the one hand and target product synthesis on the other hand. With this review, we summarize the status quo of knowledge and engineering attempts around chemical production in bio-electrochemical systems from a microbe perspective. Challenges associated with the introduction or enhancement of extracellular electron transfer capabilities into production hosts versus the engineering of target compound synthesis pathways in natural exoelectrogens are discussed. Recent advances of the research community in both directions are examined critically. Further, systems biology approaches, for instance using metabolic modelling, are examined for their potential to provide insight into fundamental processes and to identify targets for metabolic engineering. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Metabolic Engineering: Techniques for analysis of targets for genetic manipulations

DEFF Research Database (Denmark)

Nielsen, Jens Bredal

1998-01-01

Metabolic engineering has been defined as the purposeful modification of intermediary metabolism using recombinant DNA techniques. With this definition metabolic engineering includes: (1) inserting new pathways in microorganisms with the aim of producing novel metabolites, e.g., production...... of polyketides by Streptomyces; (2) production of heterologous peptides, e.g., production of human insulin, erythropoitin, and tPA; and (3) improvement of both new and existing processes, e.g., production of antibiotics and industrial enzymes. Metabolic engineering is a multidisciplinary approach, which involves...... input from chemical engineers, molecular biologists, biochemists, physiologists, and analytical chemists. Obviously, molecular biology is central in the production of novel products, as well as in the improvement of existing processes. However, in the latter case, input from other disciplines is pivotal...

Cytochrome P450-mediated metabolic engineering

DEFF Research Database (Denmark)

Renault, Hugues; Bassard, Jean-Étienne André; Hamberger, Björn Robert

2014-01-01

for the engineered bioproduction of such compounds. Two ground-breaking developments of commercial products driven by the engineering of P450s are the antimalarial drug precursor artemisinic acid and blue roses or carnations. Tedious optimizations were required to generate marketable products. Hurdles encountered...... in P450 engineering and their potential solutions are summarized here. Together with recent technical developments and novel approaches to metabolic engineering, the lessons from this pioneering work should considerably boost exploitation of the amazing P450 toolkit emerging from accelerated sequencing...

Integrating the protein and metabolic engineering toolkits for next-generation chemical biosynthesis.

Science.gov (United States)

Pirie, Christopher M; De Mey, Marjan; Jones Prather, Kristala L; Ajikumar, Parayil Kumaran

2013-04-19

Through microbial engineering, biosynthesis has the potential to produce thousands of chemicals used in everyday life. Metabolic engineering and synthetic biology are fields driven by the manipulation of genes, genetic regulatory systems, and enzymatic pathways for developing highly productive microbial strains. Fundamentally, it is the biochemical characteristics of the enzymes themselves that dictate flux through a biosynthetic pathway toward the product of interest. As metabolic engineers target sophisticated secondary metabolites, there has been little recognition of the reduced catalytic activity and increased substrate/product promiscuity of the corresponding enzymes compared to those of central metabolism. Thus, fine-tuning these enzymatic characteristics through protein engineering is paramount for developing high-productivity microbial strains for secondary metabolites. Here, we describe the importance of protein engineering for advancing metabolic engineering of secondary metabolism pathways. This pathway integrated enzyme optimization can enhance the collective toolkit of microbial engineering to shape the future of chemical manufacturing.

Metabolic Engineering of Probiotic Saccharomyces boulardii.

Science.gov (United States)

Liu, Jing-Jing; Kong, In Iok; Zhang, Guo-Chang; Jayakody, Lahiru N; Kim, Heejin; Xia, Peng-Fei; Kwak, Suryang; Sung, Bong Hyun; Sohn, Jung-Hoon; Walukiewicz, Hanna E; Rao, Christopher V; Jin, Yong-Su

2016-04-01

Saccharomyces boulardiiis a probiotic yeast that has been used for promoting gut health as well as preventing diarrheal diseases. This yeast not only exhibits beneficial phenotypes for gut health but also can stay longer in the gut than Saccharomyces cerevisiae Therefore, S. boulardiiis an attractive host for metabolic engineering to produce biomolecules of interest in the gut. However, the lack of auxotrophic strains with defined genetic backgrounds has hampered the use of this strain for metabolic engineering. Here, we report the development of well-defined auxotrophic mutants (leu2,ura3,his3, and trp1) through clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-based genome editing. The resulting auxotrophic mutants can be used as a host for introducing various genetic perturbations, such as overexpression or deletion of a target gene, using existing genetic tools forS. cerevisiae We demonstrated the overexpression of a heterologous gene (lacZ), the correct localization of a target protein (red fluorescent protein) into mitochondria by using a protein localization signal, and the introduction of a heterologous metabolic pathway (xylose-assimilating pathway) in the genome ofS. boulardii We further demonstrated that human lysozyme, which is beneficial for human gut health, could be secreted by S. boulardii Our results suggest that more sophisticated genetic perturbations to improveS. boulardii can be performed without using a drug resistance marker, which is a prerequisite for in vivo applications using engineeredS. boulardii. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Engineering of sugar metabolism in Lactococcus lactis

NARCIS (Netherlands)

Pool, Weia Arianne

2008-01-01

Short English Summary Lactococcus lactis is a lactic acid bacterium used in the dairy industry. This thesis decribes the genetic engineering performed on the sugar metabolism of L. lactis. Besides our fundamental interest for sugar metabolism and its regulation in L. lactis, this project had the

Metabolic engineering of cyanobacteria for the synthesis of commodity products

NARCIS (Netherlands)

Angermayr, S.A.; Gorchs Rovira, A.; Hellingwerf, K.J.

2015-01-01

Through metabolic engineering cyanobacteria can be employed in biotechnology. Combining the capacity for oxygenic photosynthesis and carbon fixation with an engineered metabolic pathway allows carbon-based product formation from CO2, light, and water directly. Such cyanobacterial 'cell factories'

Metabolic Engineering for Probiotics and their Genome-Wide Expression Profiling.

Science.gov (United States)

Yadav, Ruby; Singh, Puneet K; Shukla, Pratyoosh

2018-01-01

Probiotic supplements in food industry have attracted a lot of attention and shown a remarkable growth in this field. Metabolic engineering (ME) approaches enable understanding their mechanism of action and increases possibility of designing probiotic strains with desired functions. Probiotic microorganisms generally referred as industrially important lactic acid bacteria (LAB) which are involved in fermenting dairy products, food, beverages and produces lactic acid as final product. A number of illustrations of metabolic engineering approaches in industrial probiotic bacteria have been described in this review including transcriptomic studies of Lactobacillus reuteri and improvement in exopolysaccharide (EPS) biosynthesis yield in Lactobacillus casei LC2W. This review summaries various metabolic engineering approaches for exploring metabolic pathways. These approaches enable evaluation of cellular metabolic state and effective editing of microbial genome or introduction of novel enzymes to redirect the carbon fluxes. In addition, various system biology tools such as in silico design commonly used for improving strain performance is also discussed. Finally, we discuss the integration of metabolic engineering and genome profiling which offers a new way to explore metabolic interactions, fluxomics and probiogenomics using probiotic bacteria like Bifidobacterium spp and Lactobacillus spp. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Corynebacterium glutamicum for Sustainable Bioproduction: From Metabolic Physiology to Systems Metabolic Engineering.

Science.gov (United States)

Becker, Judith; Gießelmann, Gideon; Hoffmann, Sarah Lisa; Wittmann, Christoph

Since its discovery 60 years ago, Corynebacterium glutamicum has evolved into a workhorse for industrial biotechnology. Traditionally well known for its remarkable capacity to produce amino acids, this Gram-positive soil bacterium, has become a flexible, efficient production platform for various bulk and fine chemicals, materials, and biofuels. The central turnstile of all these achievements is our excellent understanding of its metabolism and physiology. This knowledge base, together with innovative systems metabolic engineering concepts, which integrate systems and synthetic biology into strain engineering, has upgraded C. glutamicum into one of the most successful industrial microorganisms in the world.

Relationships between Locus of Control, Self-Efficacy, Efforts and Academic Achievement among Engineering Students

Directory of Open Access Journals (Sweden)

Alias Maizam

2016-01-01

Full Text Available The aim of this study is to investigate the relationships between the affective learning needs namely, self-efficacy and locus of control, learning efforts and academic achievement among engineering students. For this purpose, a survey was conducted on first year engineering students from two technical universities in Malaysia. Self-efficacy and locus of control were assessed using existing instruments while learning efforts were assessed using a specifically designed instrument based on Carbonaro’s model of learning effort. Academic achievement data were based on cumulative grade point average (CGPA obtained from self-report by participants. The findings indicate that females engineering students tend to have higher self-efficacy compared to males while both groups have similar locus of control and invest in similar learning efforts. Only locus of control is found to be related to academic achievement while self-efficacy is found to be related to efforts. In conclusion, locus of control seems to be an important factor in predicting academic achievement among engineering students.

SBOLme: a Repository of SBOL Parts for Metabolic Engineering

KAUST Repository

Kuwahara, Hiroyuki

2017-01-12

The Synthetic Biology Open Language (SBOL) is a community-driven open language to promote standardization in synthetic biology. To support the use of SBOL in metabolic engineering, we developed SBOLme, the first open-access repository of SBOL 2-compliant biochemical parts for a wide range of metabolic engineering applications. The URL of our repository is http://www.cbrc.kaust.edu.sa/sbolme.

Cell-free protein synthesis enabled rapid prototyping for metabolic engineering and synthetic biology

Directory of Open Access Journals (Sweden)

Lihong Jiang

2018-06-01

Full Text Available Advances in metabolic engineering and synthetic biology have facilitated the manufacturing of many valuable-added compounds and commodity chemicals using microbial cell factories in the past decade. However, due to complexity of cellular metabolism, the optimization of metabolic pathways for maximal production represents a grand challenge and an unavoidable barrier for metabolic engineering. Recently, cell-free protein synthesis system (CFPS has been emerging as an enabling alternative to address challenges in biomanufacturing. This review summarizes the recent progresses of CFPS in rapid prototyping of biosynthetic pathways and genetic circuits (biosensors to speed up design-build-test (DBT cycles of metabolic engineering and synthetic biology. Keywords: Cell-free protein synthesis, Metabolic pathway optimization, Genetic circuits, Metabolic engineering, Synthetic biology

Metabolic engineering of Bacillus subtilis fueled by systems biology: Recent advances and future directions.

Science.gov (United States)

Liu, Yanfeng; Li, Jianghua; Du, Guocheng; Chen, Jian; Liu, Long

By combining advanced omics technology and computational modeling, systems biologists have identified and inferred thousands of regulatory events and system-wide interactions of the bacterium Bacillus subtilis, which is commonly used both in the laboratory and in industry. This dissection of the multiple layers of regulatory networks and their interactions has provided invaluable information for unraveling regulatory mechanisms and guiding metabolic engineering. In this review, we discuss recent advances in the systems biology and metabolic engineering of B. subtilis and highlight current gaps in our understanding of global metabolism and global pathway engineering in this organism. We also propose future perspectives in the systems biology of B. subtilis and suggest ways that this approach can be used to guide metabolic engineering. Specifically, although hundreds of regulatory events have been identified or inferred via systems biology approaches, systematic investigation of the functionality of these events in vivo has lagged, thereby preventing the elucidation of regulatory mechanisms and further rational pathway engineering. In metabolic engineering, ignoring the engineering of multilayer regulation hinders metabolic flux redistribution. Post-translational engineering, allosteric engineering, and dynamic pathway analyses and control will also contribute to the modulation and control of the metabolism of engineered B. subtilis, ultimately producing the desired cellular traits. We hope this review will aid metabolic engineers in making full use of available systems biology datasets and approaches for the design and perfection of microbial cell factories through global metabolism optimization. Copyright © 2016 Elsevier Inc. All rights reserved.

Metabolic Engineering of Oleaginous Yeasts for Production of Fuels and Chemicals

Directory of Open Access Journals (Sweden)

Shuobo Shi

2017-11-01

Full Text Available Oleaginous yeasts have been increasingly explored for production of chemicals and fuels via metabolic engineering. Particularly, there is a growing interest in using oleaginous yeasts for the synthesis of lipid-related products due to their high lipogenesis capability, robustness, and ability to utilize a variety of substrates. Most of the metabolic engineering studies in oleaginous yeasts focused on Yarrowia that already has plenty of genetic engineering tools. However, recent advances in systems biology and synthetic biology have provided new strategies and tools to engineer those oleaginous yeasts that have naturally high lipid accumulation but lack genetic tools, such as Rhodosporidium, Trichosporon, and Lipomyces. This review highlights recent accomplishments in metabolic engineering of oleaginous yeasts and recent advances in the development of genetic engineering tools in oleaginous yeasts within the last 3 years.

Essences in Metabolic Engineering of Lignan Biosynthesis

Directory of Open Access Journals (Sweden)

Honoo Satake

2015-05-01

Full Text Available Lignans are structurally and functionally diverse phytochemicals biosynthesized in diverse plant species and have received wide attentions as leading compounds of novel drugs for tumor treatment and healthy diets to reduce of the risks of lifestyle-related non-communicable diseases. However, the lineage-specific distribution and the low-amount of production in natural plants, some of which are endangered species, hinder the efficient and stable production of beneficial lignans. Accordingly, the development of new procedures for lignan production is of keen interest. Recent marked advances in the molecular and functional characterization of lignan biosynthetic enzymes and endogenous and exogenous factors for lignan biosynthesis have suggested new methods for the metabolic engineering of lignan biosynthesis cascades leading to the efficient, sustainable, and stable lignan production in plants, including plant cell/organ cultures. Optimization of light conditions, utilization of a wide range of elicitor treatments, and construction of transiently gene-transfected or transgenic lignan-biosynthesizing plants are mainly being attempted. This review will present the basic and latest knowledge regarding metabolic engineering of lignans based on their biosynthetic pathways and biological activities, and the perspectives in lignan production via metabolic engineering.

2005 Plant Metabolic Engineering Gordon Conference - July 10-15, 2005

Energy Technology Data Exchange (ETDEWEB)

Eleanore T. Wurtzel

2006-06-30

The post-genomic era presents new opportunities for manipulating plant chemistry for improvement of plant traits such as disease and stress resistance and nutritional qualities. This conference will provide a setting for developing multidisciplinary collaborations needed to unravel the dynamic complexity of plant metabolic networks and advance basic and applied research in plant metabolic engineering. The conference will integrate recent advances in genomics, with metabolite and gene expression analyses. Research discussions will explore how biosynthetic pathways interact with regard to substrate competition and channeling, plasticity of biosynthetic enzymes, and investigate the localization, structure, and assembly of biosynthetic metabolons in native and nonnative environments. The meeting will develop new perspectives for plant transgenic research with regard to how transgene expression may influence cellular metabolism. Incorporation of spectroscopic approaches for metabolic profiling and flux analysis combined with mathematical modeling will contribute to the development of rational metabolic engineering strategies and lead to the development of new tools to assess temporal and subcellular changes in metabolite pools. The conference will also highlight new technologies for pathway engineering, including use of heterologous systems, directed enzyme evolution, engineering of transcription factors and application of molecular/genetic techniques for controlling biosynthetic pathways.

2007 Plant Metabolic Engineering Gordon Conference and Graduate Research Seminar

Energy Technology Data Exchange (ETDEWEB)

Erich Grotewold

2008-09-15

Plant Metabolic Engineering is an emerging field that integrates a diverse range of disciplines including plant genetics, genomics, biochemistry, chemistry and cell biology. The Gordon-Kenan Graduate Research Seminar (GRS) in Plant Metabolic Engineering was initiated to provide a unique opportunity for future researcher leaders to present their work in this field. It also creates an environment allowing for peer-review and critical assessment of work without the intimidation usually associated with the presence of senior investigators. The GRS immediately precedes the Plant Metabolic Engineering Gordon Research Conference and will be for and by graduate students and post-docs, with the assistance of the organizers listed.

SYSTEMS BIOLOGY AND METABOLIC ENGINEERING OF ARTHROSPIRA CELL FACTORIES

Directory of Open Access Journals (Sweden)

Amornpan Klanchui

2012-10-01

Full Text Available Arthrospira are attractive candidates to serve as cell factories for production of many valuable compounds useful for food, feed, fuel and pharmaceutical industries. In connection with the development of sustainable bioprocessing, it is a challenge to design and develop efficient Arthrospira cell factories which can certify effective conversion from the raw materials (i.e. CO2 and sun light into desired products. With the current availability of the genome sequences and metabolic models of Arthrospira, the development of Arthrospira factories can now be accelerated by means of systems biology and the metabolic engineering approach. Here, we review recent research involving the use of Arthrospira cell factories for industrial applications, as well as the exploitation of systems biology and the metabolic engineering approach for studying Arthrospira. The current status of genomics and proteomics through the development of the genome-scale metabolic model of Arthrospira, as well as the use of mathematical modeling to simulate the phenotypes resulting from the different metabolic engineering strategies are discussed. At the end, the perspective and future direction on Arthrospira cell factories for industrial biotechnology are presented.

Modeling to Mars: a NASA Model Based Systems Engineering Pathfinder Effort

Science.gov (United States)

Phojanamongkolkij, Nipa; Lee, Kristopher A.; Miller, Scott T.; Vorndran, Kenneth A.; Vaden, Karl R.; Ross, Eric P.; Powell, Bobby C.; Moses, Robert W.

2017-01-01

The NASA Engineering Safety Center (NESC) Systems Engineering (SE) Technical Discipline Team (TDT) initiated the Model Based Systems Engineering (MBSE) Pathfinder effort in FY16. The goals and objectives of the MBSE Pathfinder include developing and advancing MBSE capability across NASA, applying MBSE to real NASA issues, and capturing issues and opportunities surrounding MBSE. The Pathfinder effort consisted of four teams, with each team addressing a particular focus area. This paper focuses on Pathfinder team 1 with the focus area of architectures and mission campaigns. These efforts covered the timeframe of February 2016 through September 2016. The team was comprised of eight team members from seven NASA Centers (Glenn Research Center, Langley Research Center, Ames Research Center, Goddard Space Flight Center IV&V Facility, Johnson Space Center, Marshall Space Flight Center, and Stennis Space Center). Collectively, the team had varying levels of knowledge, skills and expertise in systems engineering and MBSE. The team applied their existing and newly acquired system modeling knowledge and expertise to develop modeling products for a campaign (Program) of crew and cargo missions (Projects) to establish a human presence on Mars utilizing In-Situ Resource Utilization (ISRU). Pathfinder team 1 developed a subset of modeling products that are required for a Program System Requirement Review (SRR)/System Design Review (SDR) and Project Mission Concept Review (MCR)/SRR as defined in NASA Procedural Requirements. Additionally, Team 1 was able to perform and demonstrate some trades and constraint analyses. At the end of these efforts, over twenty lessons learned and recommended next steps have been identified.

Evolutionary programming as a platform for in silico metabolic engineering

Directory of Open Access Journals (Sweden)

Förster Jochen

2005-12-01

Full Text Available Abstract Background Through genetic engineering it is possible to introduce targeted genetic changes and hereby engineer the metabolism of microbial cells with the objective to obtain desirable phenotypes. However, owing to the complexity of metabolic networks, both in terms of structure and regulation, it is often difficult to predict the effects of genetic modifications on the resulting phenotype. Recently genome-scale metabolic models have been compiled for several different microorganisms where structural and stoichiometric complexity is inherently accounted for. New algorithms are being developed by using genome-scale metabolic models that enable identification of gene knockout strategies for obtaining improved phenotypes. However, the problem of finding optimal gene deletion strategy is combinatorial and consequently the computational time increases exponentially with the size of the problem, and it is therefore interesting to develop new faster algorithms. Results In this study we report an evolutionary programming based method to rapidly identify gene deletion strategies for optimization of a desired phenotypic objective function. We illustrate the proposed method for two important design parameters in industrial fermentations, one linear and other non-linear, by using a genome-scale model of the yeast Saccharomyces cerevisiae. Potential metabolic engineering targets for improved production of succinic acid, glycerol and vanillin are identified and underlying flux changes for the predicted mutants are discussed. Conclusion We show that evolutionary programming enables solving large gene knockout problems in relatively short computational time. The proposed algorithm also allows the optimization of non-linear objective functions or incorporation of non-linear constraints and additionally provides a family of close to optimal solutions. The identified metabolic engineering strategies suggest that non-intuitive genetic modifications span

Mini-review: In vitro Metabolic Engineering for Biomanufacturing of High-value Products

Directory of Open Access Journals (Sweden)

Weihua Guo

Full Text Available With the breakthroughs in biomolecular engineering and synthetic biology, many valuable biologically active compound and commodity chemicals have been successfully manufactured using cell-based approaches in the past decade. However, because of the high complexity of cell metabolism, the identification and optimization of rate-limiting metabolic pathways for improving the product yield is often difficult, which represents a significant and unavoidable barrier of traditional in vivo metabolic engineering. Recently, some in vitro engineering approaches were proposed as alternative strategies to solve this problem. In brief, by reconstituting a biosynthetic pathway in a cell-free environment with the supplement of cofactors and substrates, the performance of each biosynthetic pathway could be evaluated and optimized systematically. Several value-added products, including chemicals, nutraceuticals, and drug precursors, have been biosynthesized as proof-of-concept demonstrations of in vitro metabolic engineering. This mini-review summarizes the recent progresses on the emerging topic of in vitro metabolic engineering and comments on the potential application of cell-free technology to speed up the “design-build-test” cycles of biomanufacturing. Keywords: Cell-free, Biosynthesis, Metabolic pathways, Design-build-test cycle

Microbial Development and Metabolic Engineering | Bioenergy | NREL

Science.gov (United States)

Diversity Our genetically engineered microbes utilize a variety of feedstock including cellulose, xylan , syngas, simple sugars, organic acids, and carbon dioxide (CO2). We have modified the metabolic pathways

Deriving metabolic engineering strategies from genome-scale modeling with flux ratio constraints.

Science.gov (United States)

Yen, Jiun Y; Nazem-Bokaee, Hadi; Freedman, Benjamin G; Athamneh, Ahmad I M; Senger, Ryan S

2013-05-01

Optimized production of bio-based fuels and chemicals from microbial cell factories is a central goal of systems metabolic engineering. To achieve this goal, a new computational method of using flux balance analysis with flux ratios (FBrAtio) was further developed in this research and applied to five case studies to evaluate and design metabolic engineering strategies. The approach was implemented using publicly available genome-scale metabolic flux models. Synthetic pathways were added to these models along with flux ratio constraints by FBrAtio to achieve increased (i) cellulose production from Arabidopsis thaliana; (ii) isobutanol production from Saccharomyces cerevisiae; (iii) acetone production from Synechocystis sp. PCC6803; (iv) H2 production from Escherichia coli MG1655; and (v) isopropanol, butanol, and ethanol (IBE) production from engineered Clostridium acetobutylicum. The FBrAtio approach was applied to each case to simulate a metabolic engineering strategy already implemented experimentally, and flux ratios were continually adjusted to find (i) the end-limit of increased production using the existing strategy, (ii) new potential strategies to increase production, and (iii) the impact of these metabolic engineering strategies on product yield and culture growth. The FBrAtio approach has the potential to design "fine-tuned" metabolic engineering strategies in silico that can be implemented directly with available genomic tools. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biosynthetic Pathway and Metabolic Engineering of Plant Dihydrochalcones.

Science.gov (United States)

Ibdah, Mwafaq; Martens, Stefan; Gang, David R

2018-03-14

Dihydrochalcones are plant natural products containing the phenylpropanoid backbone and derived from the plant-specific phenylpropanoid pathway. Dihydrochalcone compounds are important in plant growth and response to stresses and, thus, can have large impacts on agricultural activity. In recent years, these compounds have also received increased attention from the biomedical community for their potential as anticancer treatments and other benefits for human health. However, they are typically produced at relatively low levels in plants. Therefore, an attractive alternative is to express the plant biosynthetic pathway genes in microbial hosts and to engineer the metabolic pathway/host to improve the production of these metabolites. In the present review, we discuss in detail the functions of genes and enzymes involved in the biosynthetic pathway of the dihydrochalcones and the recent strategies and achievements used in the reconstruction of multi-enzyme pathways in microorganisms in efforts to be able to attain higher amounts of desired dihydrochalcones.

Use of genome-scale microbial models for metabolic engineering

DEFF Research Database (Denmark)

Patil, Kiran Raosaheb; Åkesson, M.; Nielsen, Jens

2004-01-01

Metabolic engineering serves as an integrated approach to design new cell factories by providing rational design procedures and valuable mathematical and experimental tools. Mathematical models have an important role for phenotypic analysis, but can also be used for the design of optimal metaboli...... network structures. The major challenge for metabolic engineering in the post-genomic era is to broaden its design methodologies to incorporate genome-scale biological data. Genome-scale stoichiometric models of microorganisms represent a first step in this direction....

Cyanobacterial metabolic engineering for biofuel and chemical production.

Science.gov (United States)

Oliver, Neal J; Rabinovitch-Deere, Christine A; Carroll, Austin L; Nozzi, Nicole E; Case, Anna E; Atsumi, Shota

2016-12-01

Rising levels of atmospheric CO 2 are contributing to the global greenhouse effect. Large scale use of atmospheric CO 2 may be a sustainable and renewable means of chemical and liquid fuel production to mitigate global climate change. Photosynthetic organisms are an ideal platform for efficient, natural CO 2 conversion to a broad range of chemicals. Cyanobacteria are especially attractive for these purposes, due to their genetic malleability and relatively fast growth rate. Recent years have yielded a range of work in the metabolic engineering of cyanobacteria and have led to greater knowledge of the host metabolism. Understanding of endogenous and heterologous carbon regulation mechanisms leads to the expansion of productive capacity and chemical variety. This review discusses the recent progress in metabolic engineering of cyanobacteria for biofuel and bulk chemical production since 2014. Copyright © 2016 Elsevier Ltd. All rights reserved.

Metabolic Engineering for Substrate Co-utilization

Science.gov (United States)

Gawand, Pratish

Production of biofuels and bio-based chemicals is being increasingly pursued by chemical industry to reduce its dependence on petroleum. Lignocellulosic biomass (LCB) is an abundant source of sugars that can be used for producing biofuels and bio-based chemicals using fermentation. Hydrolysis of LCB results in a mixture of sugars mainly composed of glucose and xylose. Fermentation of such a sugar mixture presents multiple technical challenges at industrial scale. Most industrial microorganisms utilize sugars in a sequential manner due to the regulatory phenomenon of carbon catabolite repression (CCR). Due to sequential utilization of sugars, the LCB-based fermentation processes suffer low productivities and complicated operation. Performance of fermentation processes can be improved by metabolic engineering of microorganisms to obtain superior characteristics such as high product yield. With increased computational power and availability of complete genomes of microorganisms, use of model-based metabolic engineering is now a common practice. The problem of sequential sugar utilization, however, is a regulatory problem, and metabolic models have never been used to solve such regulatory problems. The focus of this thesis is to use model-guided metabolic engineering to construct industrial strains capable of co-utilizing sugars. First, we develop a novel bilevel optimization algorithm SimUp, that uses metabolic models to identify reaction deletion strategies to force co-utilization of two sugars. We then use SimUp to identify reaction deletion strategies to force glucose-xylose co-utilization in Escherichia coli. To validate SimUp predictions, we construct three mutants with multiple gene knockouts and test them for glucose-xylose utilization characteristics. Two mutants, designated as LMSE2 and LMSE5, are shown to co-utilize glucose and xylose in agreement with SimUp predictions. To understand the molecular mechanism involved in glucose-xylose co-utilization of the

Role of glycolytic intermediate in regulation: Improving lycopene production in Escherichia coli by engineering metabolic control

Energy Technology Data Exchange (ETDEWEB)

Farmer, W.R.; Liao, J.C.

2001-06-01

Metabolic engineering in the postgenomic era is expected to benefit from a full understanding of the biosynthetic capability of microorganisms as a result of the progress being made in bioinformatics and functional genomics. The immediate advantage of such information is to allow the rational design of novel pathways and the elimination of native reactions that are detrimental or unnecessary for the desired purpose. However, with the ability to manipulate metabolic pathways becoming more effective, metabolic engineering will need to face a new challenge: the reengineering of the regulatory hierarchy that controls gene expression in those pathways. In addition to constructing the genetic composition of a metabolic pathway, they propose that it will become just as important to consider the dynamics of pathways gene expression. It has been widely observed that high-level induction of a recombinant protein or pathway leads to growth retardation and reduced metabolic activity. These phenotypic characteristics result from the fact that the constant demands of production placed upon the cell interfere with its changing requirements for growth. They believe that this common situation in metabolic engineering can be alleviated by designing a dynamic controller that is able to sense the metabolic state of the cell and regulate the expression of the recombinant pathway accordingly. This approach, which is termed metabolic control engineering, involves redesigning the native regulatory circuits and applying them to the recombinant pathway. The general goal of such an effort will be to control the flux to the recombinant pathway adaptively according to the cell's metabolic state. The dynamically controlled recombinant pathway can potentially lead to enhanced production, minimized growth retardation, and reduced toxic by-product formation. The regulation of gene expression in response to the physiological state is also essential to the success of gene therapy. Here they

Design, Optimization and Application of Small Molecule Biosensor in Metabolic Engineering.

Science.gov (United States)

Liu, Yang; Liu, Ye; Wang, Meng

2017-01-01

The development of synthetic biology and metabolic engineering has painted a great future for the bio-based economy, including fuels, chemicals, and drugs produced from renewable feedstocks. With the rapid advance of genome-scale modeling, pathway assembling and genome engineering/editing, our ability to design and generate microbial cell factories with various phenotype becomes almost limitless. However, our lack of ability to measure and exert precise control over metabolite concentration related phenotypes becomes a bottleneck in metabolic engineering. Genetically encoded small molecule biosensors, which provide the means to couple metabolite concentration to measurable or actionable outputs, are highly promising solutions to the bottleneck. Here we review recent advances in the design, optimization and application of small molecule biosensor in metabolic engineering, with particular focus on optimization strategies for transcription factor (TF) based biosensors.

Dynamic gene expression for metabolic engineering of mammalian cells in culture.

Science.gov (United States)

Le, Huong; Vishwanathan, Nandita; Kantardjieff, Anne; Doo, Inseok; Srienc, Michael; Zheng, Xiaolu; Somia, Nikunj; Hu, Wei-Shou

2013-11-01

Recombinant mammalian cells are the major hosts for the production of protein therapeutics. In addition to high expression of the product gene, a hyper-producer must also harbor superior phenotypic traits related to metabolism, protein secretion, and growth control. Introduction of genes endowing the relevant hyper-productivity traits is a strategy frequently used to enhance the productivity. Most of such cell engineering efforts have been performed using constitutive expression systems. However, cells respond to various environmental cues and cellular events dynamically according to cellular needs. The use of inducible systems allows for time dependent expression, but requires external manipulation. Ideally, a transgene's expression should be synchronous to the host cell's own rhythm, and at levels appropriate for the objective. To that end, we identified genes with different expression dynamics and intensity ranges using pooled transcriptome data. Their promoters may be used to drive the expression of the transgenes following the desired dynamics. We isolated the promoter of the Thioredoxin-interacting protein (Txnip) gene and demonstrated its capability to drive transgene expression in concert with cell growth. We further employed this Chinese hamster promoter to engineer dynamic expression of the mouse GLUT5 fructose transporter in Chinese hamster ovary (CHO) cells, enabling them to utilize sugar according to cellular needs rather than in excess as typically seen in culture. Thus, less lactate was produced, resulting in a better growth rate, prolonged culture duration, and higher product titer. This approach illustrates a novel concept in metabolic engineering which can potentially be used to achieve dynamic control of cellular behaviors for enhanced process characteristics. © 2013 Published by Elsevier Inc.

Pathway elucidation and metabolic engineering of specialized plant metabolites

DEFF Research Database (Denmark)

Salomonsen, Bo

A worldwide need to liberate ourselves from unsustainable petrochemicals has led to numerous metabolic engineering projects, mostly carried out in microbial hosts. Using systems biology for predicting and altering the metabolism of microorganisms towards production of a desired metabolite......, these projects have increased revenues on fermentative production of several biochemicals. The use of systems biology is, however, not limited to microorganisms. Recent advances in biotechnology methods have provided a wealth of data within functional genomics, metabolomics, transcriptomics, proteomics...... and fluxomics for a considerable number of organisms. Unfortunately, transferring the wealth of data to valuable information for metabolic engineering purposes is a non-obvious task. This PhD thesis describes a palate of tools used in generation of cell factories for production of specialized plant metabolites...

Key applications of plant metabolic engineering.

Directory of Open Access Journals (Sweden)

Warren Lau

2014-06-01

Full Text Available Great strides have been made in plant metabolic engineering over the last two decades, with notable success stories including Golden rice. Here, we discuss the field's progress in addressing four long-standing challenges: creating plants that satisfy their own nitrogen requirement, so reducing or eliminating the need for nitrogen fertilizer; enhancing the nutrient content of crop plants; engineering biofuel feed stocks that harbor easy-to-access fermentable saccharides by incorporating self-destructing lignin; and increasing photosynthetic efficiency. We also look to the future at emerging areas of research in this field.

Genetic Optimization Algorithm for Metabolic Engineering Revisited

Directory of Open Access Journals (Sweden)

Tobias B. Alter

2018-05-01

Full Text Available To date, several independent methods and algorithms exist for exploiting constraint-based stoichiometric models to find metabolic engineering strategies that optimize microbial production performance. Optimization procedures based on metaheuristics facilitate a straightforward adaption and expansion of engineering objectives, as well as fitness functions, while being particularly suited for solving problems of high complexity. With the increasing interest in multi-scale models and a need for solving advanced engineering problems, we strive to advance genetic algorithms, which stand out due to their intuitive optimization principles and the proven usefulness in this field of research. A drawback of genetic algorithms is that premature convergence to sub-optimal solutions easily occurs if the optimization parameters are not adapted to the specific problem. Here, we conducted comprehensive parameter sensitivity analyses to study their impact on finding optimal strain designs. We further demonstrate the capability of genetic algorithms to simultaneously handle (i multiple, non-linear engineering objectives; (ii the identification of gene target-sets according to logical gene-protein-reaction associations; (iii minimization of the number of network perturbations; and (iv the insertion of non-native reactions, while employing genome-scale metabolic models. This framework adds a level of sophistication in terms of strain design robustness, which is exemplarily tested on succinate overproduction in Escherichia coli.

Production of anthocyanins in metabolically engineered microorganisms: Current status and perspectives

Directory of Open Access Journals (Sweden)

Jian Zha

2017-12-01

Full Text Available Microbial production of plant-derived natural products by engineered microorganisms has achieved great success thanks to large extend to metabolic engineering and synthetic biology. Anthocyanins, the water-soluble colored pigments found in terrestrial plants that are responsible for the red, blue and purple coloration of many flowers and fruits, are extensively used in food and cosmetics industry; however, their current supply heavily relies on complex extraction from plant-based materials. A promising alternative is their sustainable production in metabolically engineered microbes. Here, we review the recent progress on anthocyanin biosynthesis in engineered bacteria, with a special focus on the systematic engineering modifications such as selection and engineering of biosynthetic enzymes, engineering of transportation, regulation of UDP-glucose supply, as well as process optimization. These promising engineering strategies will facilitate successful microbial production of anthocyanins in industry in the near future.

Evolutionary programming as a platform for in silico metabolic engineering

DEFF Research Database (Denmark)

Patil, Kiran Raosaheb; Rocha, Isabel; Förster, Jochen

2005-01-01

, and it is therefore interesting to develop new faster algorithms. Results In this study we report an evolutionary programming based method to rapidly identify gene deletion strategies for optimization of a desired phenotypic objective function. We illustrate the proposed method for two important design parameters...... of close to optimal solutions. The identified metabolic engineering strategies suggest that non-intuitive genetic modifications span several different pathways and may be necessary for solving challenging metabolic engineering problems....

Impact of systems biology on metabolic engineering of Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Nielsen, Jens; Jewett, Michael Christopher

2008-01-01

in the industrial application of this yeast. Developments in genomics and high-throughput systems biology tools are enhancing one's ability to rapidly characterize cellular behaviour, which is valuable in the field of metabolic engineering where strain characterization is often the bottleneck in strain development...... programmes. Here, the impact of systems biology on metabolic engineering is reviewed and perspectives on the role of systems biology in the design of cell factories are given....

The application of microfluidic-based technologies in the cycle of metabolic engineering

Directory of Open Access Journals (Sweden)

Xiaoyan Ma

2016-09-01

Full Text Available The process of metabolic engineering consists of multiple cycles of design, build, test and learn, which is typically laborious and time-consuming. To increase the efficiency and the rate of success of strain engineering, novel instrumentation must be applied. Microfluidics, the control of liquid flow in microstructures, has enabled flexible, accurate, automatic, and high-throughput manipulation of cells in liquid at picoliter to nanoliter scale. These attributes hold great promise in advancing metabolic engineering in terms of the phases of design, build, test and learn. To promote the application of microfluidic-based technologies in strain improvement, this review addressed the potentials of microfluidics and the related approaches in DNA assembly, transformation, strain screening, genotyping and phenotyping, and highlighted their adaptations for single-cell analysis. As a result, this facilitates in-depth understanding of the metabolic network, which in turn promote efficient optimization in the following cycles of strain engineering. Taken together, microfluidic-based technologies enable on-chip workflow, and could greatly accelerate the turnaround of metabolic engineering.

The role of metabolic engineering in the production of secondary metabolites

DEFF Research Database (Denmark)

Nielsen, Jens Bredal

1998-01-01

In the production of secondary metabolites yield and productivity are the most important design parameters. The focus is therefore to direct the carbon fluxes towards the product of interest, and this can be obtained through metabolic engineering whereby directed genetic changes are introduced...... into the production strain. In this process it is, however, important to analyze the metabolic network through measurement of the intracellular metabolites and the flux distributions. Besides playing an important role in the optimization of existing processes, metabolic engineering also offers the possibility...

Hijacking CRISPR-Cas for high-throughput bacterial metabolic engineering: advances and prospects

DEFF Research Database (Denmark)

Mougiakos, Ioannis; Bosma, Elleke F.; Ganguly, Joyshree

2018-01-01

High engineering efficiencies are required for industrial strain development. Due to its user-friendliness and its stringency, CRISPR-Cas-based technologies have strongly increased genome engineering efficiencies in bacteria. This has enabled more rapid metabolic engineering of both the model host...... the range of organisms in which it can be used to create novel production hosts. This review analyses the current status of prokaryotic metabolic engineering towards the production of biotechnologically relevant products, based on the exploitation of different CRISPR-related DNA/RNA endonuclease variants....

The necessity of a theory of biology for tissue engineering: metabolism-repair systems.

Science.gov (United States)

Ganguli, Suman; Hunt, C Anthony

2004-01-01

Since there is no widely accepted global theory of biology, tissue engineering and bioengineering lack a theoretical understanding of the systems being engineered. By default, tissue engineering operates with a "reductionist" theoretical approach, inherited from traditional engineering of non-living materials. Long term, that approach is inadequate, since it ignores essential aspects of biology. Metabolism-repair systems are a theoretical framework which explicitly represents two "functional" aspects of living organisms: self-repair and self-replication. Since repair and replication are central to tissue engineering, we advance metabolism-repair systems as a potential theoretical framework for tissue engineering. We present an overview of the framework, and indicate directions to pursue for extending it to the context of tissue engineering. We focus on biological networks, both metabolic and cellular, as one such direction. The construction of these networks, in turn, depends on biological protocols. Together these concepts may help point the way to a global theory of biology appropriate for tissue engineering.

Genome-scale modeling using flux ratio constraints to enable metabolic engineering of clostridial metabolism in silico.

Science.gov (United States)

McAnulty, Michael J; Yen, Jiun Y; Freedman, Benjamin G; Senger, Ryan S

2012-05-14

Genome-scale metabolic networks and flux models are an effective platform for linking an organism genotype to its phenotype. However, few modeling approaches offer predictive capabilities to evaluate potential metabolic engineering strategies in silico. A new method called "flux balance analysis with flux ratios (FBrAtio)" was developed in this research and applied to a new genome-scale model of Clostridium acetobutylicum ATCC 824 (iCAC490) that contains 707 metabolites and 794 reactions. FBrAtio was used to model wild-type metabolism and metabolically engineered strains of C. acetobutylicum where only flux ratio constraints and thermodynamic reversibility of reactions were required. The FBrAtio approach allowed solutions to be found through standard linear programming. Five flux ratio constraints were required to achieve a qualitative picture of wild-type metabolism for C. acetobutylicum for the production of: (i) acetate, (ii) lactate, (iii) butyrate, (iv) acetone, (v) butanol, (vi) ethanol, (vii) CO2 and (viii) H2. Results of this simulation study coincide with published experimental results and show the knockdown of the acetoacetyl-CoA transferase increases butanol to acetone selectivity, while the simultaneous over-expression of the aldehyde/alcohol dehydrogenase greatly increases ethanol production. FBrAtio is a promising new method for constraining genome-scale models using internal flux ratios. The method was effective for modeling wild-type and engineered strains of C. acetobutylicum.

Alternative Substrate Metabolism in Yarrowia lipolytica

Directory of Open Access Journals (Sweden)

Michael Spagnuolo

2018-05-01

Full Text Available Recent advances in genetic engineering capabilities have enabled the development of oleochemical producing strains of Yarrowia lipolytica. Much of the metabolic engineering effort has focused on pathway engineering of the product using glucose as the feedstock; however, alternative substrates, including various other hexose and pentose sugars, glycerol, lipids, acetate, and less-refined carbon feedstocks, have not received the same attention. In this review, we discuss recent work leading to better utilization of alternative substrates. This review aims to provide a comprehensive understanding of the current state of knowledge for alternative substrate utilization, suggest potential pathways identified through homology in the absence of prior characterization, discuss recent work that either identifies, endogenous or cryptic metabolism, and describe metabolic engineering to improve alternative substrate utilization. Finally, we describe the critical questions and challenges that remain for engineering Y. lipolytica for better alternative substrate utilization.

Metabolic engineering of lactic acid bacteria for the production of nutraceuticals

NARCIS (Netherlands)

Hugenholtz, J.; Sybesma, W.; Groot, M.N.; Wisselink, W.; Ladero, V.; Burgess, K.; Sinderen, van D.; Piard, J.C.; Eggink, G.; Smid, E.J.; Savoy, G.; Sesma, F.; Jansen, T.; Hols, P.; Kleerebezem, M.

2002-01-01

Lactic acid bacteria display a relatively simple and well-described metabolism where the sugar source is converted mainly to lactic acid. Here we will shortly describe metabolic engineering strategies on the level of sugar metabolism, that lead to either the efficient re-routing of the lactococcal

Microalgal bioengineering for sustainable energy development: Recent transgenesis and metabolic engineering strategies.

Science.gov (United States)

Banerjee, Chiranjib; Singh, Puneet Kumar; Shukla, Pratyoosh

2016-03-01

Exploring the efficiency of algae to produce remarkable products can be directly benefitted by studying its mechanism at systems level. Recent advents in biotechnology like flux balance analysis (FBA), genomics and in silico proteomics minimize the wet lab exertion. It is understood that FBA predicts the metabolic products, metabolic pathways and alternative pathway to maximize the desired product, and these are key components for microalgae bio-engineering. This review encompasses recent transgenesis techniques and metabolic engineering strategies applied to different microalgae for improving different traits. Further it also throws light on RNAi and riboswitch engineering based methods which may be advantageous for high throughput microalgal research. A valid and optimally designed microalga can be developed where every engineering strategies meet each other successfully and will definitely fulfill the market needs. It is also to be noted that Omics (viz. genetic and metabolic manipulation with bioinformatics) should be integrated to develop a strain which could prove to be a futuristic solution for sustainable development for energy. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Improved Triacylglycerol Production in Acinetobacter baylyi ADP1 by Metabolic Engineering

Directory of Open Access Journals (Sweden)

Karp Matti

2011-05-01

Full Text Available Abstract Background Triacylglycerols are used in various purposes including food applications, cosmetics, oleochemicals and biofuels. Currently the main sources for triacylglycerol are vegetable oils, and microbial triacylglycerol has been suggested as an alternative for these. Due to the low production rates and yields of microbial processes, the role of metabolic engineering has become more significant. As a robust model organism for genetic and metabolic studies, and for the natural capability to produce triacylglycerol, Acinetobacter baylyi ADP1 serves as an excellent organism for modelling the effects of metabolic engineering for energy molecule biosynthesis. Results Beneficial gene deletions regarding triacylglycerol production were screened by computational means exploiting the metabolic model of ADP1. Four deletions, acr1, poxB, dgkA, and a triacylglycerol lipase were chosen to be studied experimentally both separately and concurrently by constructing a knock-out strain (MT with three of the deletions. Improvements in triacylglycerol production were observed: the strain MT produced 5.6 fold more triacylglycerol (mg/g cell dry weight compared to the wild type strain, and the proportion of triacylglycerol in total lipids was increased by 8-fold. Conclusions In silico predictions of beneficial gene deletions were verified experimentally. The chosen single and multiple gene deletions affected beneficially the natural triacylglycerol metabolism of A. baylyi ADP1. This study demonstrates the importance of single gene deletions in triacylglycerol metabolism, and proposes Acinetobacter sp. ADP1 as a model system for bioenergetic studies regarding metabolic engineering.

Systems metabolic engineering design: fatty acid production as an emerging case study.

Science.gov (United States)

Tee, Ting Wei; Chowdhury, Anupam; Maranas, Costas D; Shanks, Jacqueline V

2014-05-01

Increasing demand for petroleum has stimulated industry to develop sustainable production of chemicals and biofuels using microbial cell factories. Fatty acids of chain lengths from C6 to C16 are propitious intermediates for the catalytic synthesis of industrial chemicals and diesel-like biofuels. The abundance of genetic information available for Escherichia coli and specifically, fatty acid metabolism in E. coli, supports this bacterium as a promising host for engineering a biocatalyst for the microbial production of fatty acids. Recent successes rooted in different features of systems metabolic engineering in the strain design of high-yielding medium chain fatty acid producing E. coli strains provide an emerging case study of design methods for effective strain design. Classical metabolic engineering and synthetic biology approaches enabled different and distinct design paths towards a high-yielding strain. Here we highlight a rational strain design process in systems biology, an integrated computational and experimental approach for carboxylic acid production, as an alternative method. Additional challenges inherent in achieving an optimal strain for commercialization of medium chain-length fatty acids will likely require a collection of strategies from systems metabolic engineering. Not only will the continued advancement in systems metabolic engineering result in these highly productive strains more quickly, this knowledge will extend more rapidly the carboxylic acid platform to the microbial production of carboxylic acids with alternate chain-lengths and functionalities. © 2014 Wiley Periodicals, Inc.

Metabolic engineering of Escherichia coli: a sustainable industrial platform for bio-based chemical production.

Science.gov (United States)

Chen, Xianzhong; Zhou, Li; Tian, Kangming; Kumar, Ashwani; Singh, Suren; Prior, Bernard A; Wang, Zhengxiang

2013-12-01

In order to decrease carbon emissions and negative environmental impacts of various pollutants, more bulk and/or fine chemicals are produced by bioprocesses, replacing the traditional energy and fossil based intensive route. The Gram-negative rod-shaped bacterium, Escherichia coli has been studied extensively on a fundamental and applied level and has become a predominant host microorganism for industrial applications. Furthermore, metabolic engineering of E. coli for the enhanced biochemical production has been significantly promoted by the integrated use of recent developments in systems biology, synthetic biology and evolutionary engineering. In this review, we focus on recent efforts devoted to the use of genetically engineered E. coli as a sustainable platform for the production of industrially important biochemicals such as biofuels, organic acids, amino acids, sugar alcohols and biopolymers. In addition, representative secondary metabolites produced by E. coli will be systematically discussed and the successful strategies for strain improvements will be highlighted. Moreover, this review presents guidelines for future developments in the bio-based chemical production using E. coli as an industrial platform. Copyright © 2013 Elsevier Inc. All rights reserved.

Recent Efforts and Experiments in the Construction of Aviation Engines

Science.gov (United States)

SCHWAGER

1920-01-01

It became evident during World War I that ever-increasing demands were being placed on the mean power of aircraft engines as a result of the increased on board equipment and the demands of aerial combat. The need was for increased climbing efficiency and climbing speed. The response to these demands has been in terms of lightweight construction and the adaptation of the aircraft engine to the requirements of its use. Discussed here are specific efforts to increase flying efficiency, such as reduction of the number of revolutions of the propeller from 1400 to about 900 r.p.m. through the use of a reduction gear, increasing piston velocity, locating two crankshafts in one gear box, and using the two-cycle stroke. Also discussed are improvements in the transformation of fuel energy into engine power, the raising of compression ratios, the use of super-compression with carburetors constructed for high altitudes, the use of turbo-compressors, rotary engines, and the use of variable pitch propellers.

Modulation of sulfur metabolism enables efficient glucosinolate engineering

Directory of Open Access Journals (Sweden)

Geu-Flores Fernando

2011-01-01

Full Text Available Abstract Background Metabolic engineering in heterologous organisms is an attractive approach to achieve efficient production of valuable natural products. Glucosinolates represent a good example of such compounds as they are thought to be the cancer-preventive agents in cruciferous plants. We have recently demonstrated that it is feasible to engineer benzylglucosinolate (BGLS in the non-cruciferous plant Nicotiana benthamiana by transient expression of five genes from Arabidopsis thaliana. In the same study, we showed that co-expression of a sixth Arabidopsis gene, γ-glutamyl peptidase 1 (GGP1, resolved a metabolic bottleneck, thereby increasing BGLS accumulation. However, the accumulation did not reach the expected levels, leaving room for further optimization. Results To optimize heterologous glucosinolate production, we have in this study performed a comparative metabolite analysis of BGLS-producing N. benthamiana leaves in the presence or absence of GGP1. The analysis revealed that the increased BGLS levels in the presence of GGP1 were accompanied by a high accumulation of the last intermediate, desulfoBGLS, and a derivative thereof. This evidenced a bottleneck in the last step of the pathway, the transfer of sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS to desulfoBGLS by the sulfotransferase AtSOT16. While substitution of AtSOT16 with alternative sulfotransferases did not alleviate the bottleneck, experiments with the three genes involved in the formation and recycling of PAPS showed that co-expression of adenosine 5'-phosphosulfate kinase 2 (APK2 alone reduced the accumulation of desulfoBGLS and its derivative by more than 98% and increased BGLS accumulation 16-fold. Conclusion Adjusting sulfur metabolism by directing sulfur from primary to secondary metabolism leads to a remarkable improvement in BGLS accumulation and thereby represents an important step towards a clean and efficient production of glucosinolates in

The Genome-Based Metabolic Systems Engineering to Boost Levan Production in a Halophilic Bacterial Model.

Science.gov (United States)

Aydin, Busra; Ozer, Tugba; Oner, Ebru Toksoy; Arga, Kazim Yalcin

2018-03-01

Metabolic systems engineering is being used to redirect microbial metabolism for the overproduction of chemicals of interest with the aim of transforming microbial hosts into cellular factories. In this study, a genome-based metabolic systems engineering approach was designed and performed to improve biopolymer biosynthesis capability of a moderately halophilic bacterium Halomonas smyrnensis AAD6 T producing levan, which is a fructose homopolymer with many potential uses in various industries and medicine. For this purpose, the genome-scale metabolic model for AAD6 T was used to characterize the metabolic resource allocation, specifically to design metabolic engineering strategies for engineered bacteria with enhanced levan production capability. Simulations were performed in silico to determine optimal gene knockout strategies to develop new strains with enhanced levan production capability. The majority of the gene knockout strategies emphasized the vital role of the fructose uptake mechanism, and pointed out the fructose-specific phosphotransferase system (PTS fru ) as the most promising target for further metabolic engineering studies. Therefore, the PTS fru of AAD6 T was restructured with insertional mutagenesis and triparental mating techniques to construct a novel, engineered H. smyrnensis strain, BMA14. Fermentation experiments were carried out to demonstrate the high efficiency of the mutant strain BMA14 in terms of final levan concentration, sucrose consumption rate, and sucrose conversion efficiency, when compared to the AAD6 T . The genome-based metabolic systems engineering approach presented in this study might be considered an efficient framework to redirect microbial metabolism for the overproduction of chemicals of interest, and the novel strain BMA14 might be considered a potential microbial cell factory for further studies aimed to design levan production processes with lower production costs.

CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum.

Science.gov (United States)

Cho, Jae Sung; Choi, Kyeong Rok; Prabowo, Cindy Pricilia Surya; Shin, Jae Ho; Yang, Dongsoo; Jang, Jaedong; Lee, Sang Yup

2017-07-01

Genome engineering of Corynebacterium glutamicum, an important industrial microorganism for amino acids production, currently relies on random mutagenesis and inefficient double crossover events. Here we report a rapid genome engineering strategy to scarlessly knock out one or more genes in C. glutamicum in sequential and iterative manner. Recombinase RecT is used to incorporate synthetic single-stranded oligodeoxyribonucleotides into the genome and CRISPR/Cas9 to counter-select negative mutants. We completed the system by engineering the respective plasmids harboring CRISPR/Cas9 and RecT for efficient curing such that multiple gene targets can be done iteratively and final strains will be free of plasmids. To demonstrate the system, seven different mutants were constructed within two weeks to study the combinatorial deletion effects of three different genes on the production of γ-aminobutyric acid, an industrially relevant chemical of much interest. This genome engineering strategy will expedite metabolic engineering of C. glutamicum. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Metabolic engineering of strains: from industrial-scale to lab-scale chemical production.

Science.gov (United States)

Sun, Jie; Alper, Hal S

2015-03-01

A plethora of successful metabolic engineering case studies have been published over the past several decades. Here, we highlight a collection of microbially produced chemicals using a historical framework, starting with titers ranging from industrial scale (more than 50 g/L), to medium-scale (5-50 g/L), and lab-scale (0-5 g/L). Although engineered Escherichia coli and Saccharomyces cerevisiae emerge as prominent hosts in the literature as a result of well-developed genetic engineering tools, several novel native-producing strains are gaining attention. This review catalogs the current progress of metabolic engineering towards production of compounds such as acids, alcohols, amino acids, natural organic compounds, and others.

Fumaric acid production in Saccharomyces cerevisiae by in silico aided metabolic engineering.

Directory of Open Access Journals (Sweden)

Guoqiang Xu

Full Text Available Fumaric acid (FA is a promising biomass-derived building-block chemical. Bio-based FA production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here we report on FA production by direct fermentation using metabolically engineered Saccharomyces cerevisiae with the aid of in silico analysis of a genome-scale metabolic model. First, FUM1 was selected as the target gene on the basis of extensive literature mining. Flux balance analysis (FBA revealed that FUM1 deletion can lead to FA production and slightly lower growth of S. cerevisiae. The engineered S. cerevisiae strain obtained by deleting FUM1 can produce FA up to a concentration of 610±31 mg L(-1 without any apparent change in growth in fed-batch culture. FT-IR and (1H and (13C NMR spectra confirmed that FA was synthesized by the engineered S. cerevisiae strain. FBA identified pyruvate carboxylase as one of the factors limiting higher FA production. When the RoPYC gene was introduced, S. cerevisiae produced 1134±48 mg L(-1 FA. Furthermore, the final engineered S. cerevisiae strain was able to produce 1675±52 mg L(-1 FA in batch culture when the SFC1 gene encoding a succinate-fumarate transporter was introduced. These results demonstrate that the model shows great predictive capability for metabolic engineering. Moreover, FA production in S. cerevisiae can be efficiently developed with the aid of in silico metabolic engineering.

Non-photosynthetic plastids as hosts for metabolic engineering.

Science.gov (United States)

Mellor, Silas Busck; Behrendorff, James B Y H; Nielsen, Agnieszka Zygadlo; Jensen, Poul Erik; Pribil, Mathias

2018-04-13

Using plants as hosts for production of complex, high-value compounds and therapeutic proteins has gained increasing momentum over the past decade. Recent advances in metabolic engineering techniques using synthetic biology have set the stage for production yields to become economically attractive, but more refined design strategies are required to increase product yields without compromising development and growth of the host system. The ability of plant cells to differentiate into various tissues in combination with a high level of cellular compartmentalization represents so far the most unexploited plant-specific resource. Plant cells contain organelles called plastids that retain their own genome, harbour unique biosynthetic pathways and differentiate into distinct plastid types upon environmental and developmental cues. Chloroplasts, the plastid type hosting the photosynthetic processes in green tissues, have proven to be suitable for high yield protein and bio-compound production. Unfortunately, chloroplast manipulation often affects photosynthetic efficiency and therefore plant fitness. In this respect, plastids of non-photosynthetic tissues, which have focused metabolisms for synthesis and storage of particular classes of compounds, might prove more suitable for engineering the production and storage of non-native metabolites without affecting plant fitness. This review provides the current state of knowledge on the molecular mechanisms involved in plastid differentiation and focuses on non-photosynthetic plastids as alternative biotechnological platforms for metabolic engineering. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Stable Breathing in Patients With Obstructive Sleep Apnea Is Associated With Increased Effort but Not Lowered Metabolic Rate.

Science.gov (United States)

de Melo, Camila M; Taranto-Montemurro, Luigi; Butler, James P; White, David P; Loring, Stephen H; Azarbarzin, Ali; Marques, Melania; Berger, Philip J; Wellman, Andrew; Sands, Scott A

2017-10-01

In principle, if metabolic rate were to fall during sleep in a patient with obstructive sleep apnea (OSA), ventilatory requirements could be met without increased respiratory effort thereby favoring stable breathing. Indeed, most patients achieve periods of stable flow-limited breathing without respiratory events for periods during the night for reasons that are unclear. Thus, we tested the hypothesis that in patients with OSA, periods of stable breathing occur when metabolic rate (VO2) declines. Twelve OSA patients (apnea-hypopnea index >15 events/h) completed overnight polysomnography including measurements of VO2 (using ventilation and intranasal PO2) and respiratory effort (esophageal pressure). Contrary to our hypothesis, VO2 did not differ between stable and unstable breathing periods in non-REM stage 2 (208 ± 20 vs. 213 ± 18 mL/min), despite elevated respiratory effort during stable breathing (26 ± 2 versus 23 ± 2 cmH2O, p = .03). However, VO2 was lowered during deeper sleep (244 to 179 mL/min from non-REM stages 1 to 3, p = .04) in conjunction with more stable breathing. Further analysis revealed that airflow obstruction curtailed metabolism in both stable and unstable periods, since CPAP increased VO2 by 14% in both cases (p = .02, .03, respectively). Patients whose VO2 fell most during sleep avoided an increase in PCO2 and respiratory effort. OSA patients typically convert from unstable to stable breathing without lowering metabolic rate. During sleep, OSA patients labor with increased respiratory effort but fail to satisfy metabolic demand even in the absence of overt respiratory events. © Sleep Research Society 2017. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.

Engineering the fatty acid metabolic pathway in Saccharomyces cerevisiae for advanced biofuel production

Directory of Open Access Journals (Sweden)

Xiaoling Tang

2015-12-01

Full Text Available Fatty acid-derived fuels and chemicals have attracted a great deal of attention in recent decades, due to their following properties of high compatibility to gasoline-based fuels and existing infrastructure for their direct utilization, storage and distribution. The yeast Saccharomyces cerevisiae is the ideal biofuel producing candidate, based on the wealth of available genetic information and versatile tools designed to manipulate its metabolic pathways. Engineering the fatty acid metabolic pathways in S. cerevisiae is an effective strategy to increase its fatty acid biosynthesis and provide more pathway precursors for production of targeted products. This review summarizes the recent progress in metabolic engineering of yeast cells for fatty acids and fatty acid derivatives production, including the regulation of acetyl-CoA biosynthesis, NADPH production, fatty acid elongation, and the accumulation of activated precursors of fatty acids for converting enzymes. By introducing specific enzymes in the engineered strains, a powerful platform with a scalable, controllable and economic route for advanced biofuel production has been established. Keywords: Metabolic engineering, Fatty acid biosynthesis, Fatty acid derivatives, Saccharomyces cerevisiae

De Novo Metabolic Engineering and the Promise of Synthetic DNA

Science.gov (United States)

Klein-Marcuschamer, Daniel; Yadav, Vikramaditya G.; Ghaderi, Adel; Stephanopoulos, Gregory N.

The uncertain price and tight supply of crude oil and the ever-increasing demand for clean energy have prompted heightened attention to the development of sustainable fuel technologies that ensure continued economic development while maintaining stewardship of the environment. In the face of these enormous challenges, biomass has emerged as a viable alternative to petroleum for the production of energy, chemicals, and materials owing to its abundance, inexpensiveness, and carbon-neutrality. Moreover, the immense ease and efficiency of biological systems at converting biomass-derived feedstocks into fuels, chemicals, and materials has generated renewed interest in biotechnology as a replacement for traditional chemical processes. Aided by the ever-expanding repertoire of microbial genetics and plant biotechnology, improved understanding of gene regulation and cellular metabolism, and incessantly accumulating gene and protein data, scientists are now contemplating engineering microbial cell factories to produce fuels, chemical feedstocks, polymers and pharmaceuticals in an economically and environmentally sustainable way. This goal resonates with that of metabolic engineering - the improvement of cellular properties through the intelligent design, rational modification, or directed evolution of biochemical pathways, and arguably, metabolic engineering seems best positioned to achieve the concomittant goals of environmental stewardship and economic prolificity.

Recent applications of synthetic biology tools for yeast metabolic engineering

DEFF Research Database (Denmark)

Jensen, Michael Krogh; Keasling, Jay

2015-01-01

to engineer microbial chemical factories has steadily decreased, improvement is still needed. Through the development of synthetic biology tools for key microbial hosts, it should be possible to further decrease the development times and improve the reliability of the resulting microorganism. Together...... with continuous decreases in price and improvements in DNA synthesis, assembly and sequencing, synthetic biology tools will rationalize time-consuming strain engineering, improve control of metabolic fluxes, and diversify screening assays for cellular metabolism. This review outlines some recently developed...... synthetic biology tools and their application to improve production of chemicals and fuels in yeast. Finally, we provide a perspective for the challenges that lie ahead....

Volatile science? Metabolic engineering of terpenoids in plants

NARCIS (Netherlands)

Aharoni, A.; Jongsma, M.A.; Bouwmeester, H.J.

2005-01-01

Terpenoids are important for plant survival and also possess biological properties that are beneficial to humans. Here, we describe the state of the art in terpenoid metabolic engineering, showing that significant progress has been made over the past few years. Subcellular targeting of enzymes has

Metabolic engineering of Corynebacterium glutamicum aimed at alternative carbon sources and new products

Directory of Open Access Journals (Sweden)

Volker Fritz Wendisch

2012-10-01

Full Text Available Corynebacterium glutamicum is well known as the amino acid-producing workhorse of fermentation industry, being used for multi-million-ton scale production of glutamate and lysine for more than 60 years. However, it is only recently that extensive research has focused on engineering it beyond the scope of amino acids. Meanwhile, a variety of corynebacterial strains allows access to alternative carbon sources and/or allows production of a wide range of industrially relevant compounds. Some of these efforts set new standards in terms of titers and productivities achieved whereas others represent a proof-of-principle. These achievements manifest the position of C. glutamicum as an important industrial microorganism with capabilities far beyond the traditional amino acid production. In this review we focus on the state of the art of metabolic engineering of C. glutamicum for utilization of alternative carbon sources, (e.g. coming from wastes and unprocessed sources, and construction of C. glutamicum strains for production of new products such as diamines, organic acids and alcohols.

Systems Biology as an Integrated Platform for Bioinformatics, Systems Synthetic Biology, and Systems Metabolic Engineering

Science.gov (United States)

Chen, Bor-Sen; Wu, Chia-Chou

2013-01-01

Systems biology aims at achieving a system-level understanding of living organisms and applying this knowledge to various fields such as synthetic biology, metabolic engineering, and medicine. System-level understanding of living organisms can be derived from insight into: (i) system structure and the mechanism of biological networks such as gene regulation, protein interactions, signaling, and metabolic pathways; (ii) system dynamics of biological networks, which provides an understanding of stability, robustness, and transduction ability through system identification, and through system analysis methods; (iii) system control methods at different levels of biological networks, which provide an understanding of systematic mechanisms to robustly control system states, minimize malfunctions, and provide potential therapeutic targets in disease treatment; (iv) systematic design methods for the modification and construction of biological networks with desired behaviors, which provide system design principles and system simulations for synthetic biology designs and systems metabolic engineering. This review describes current developments in systems biology, systems synthetic biology, and systems metabolic engineering for engineering and biology researchers. We also discuss challenges and future prospects for systems biology and the concept of systems biology as an integrated platform for bioinformatics, systems synthetic biology, and systems metabolic engineering. PMID:24709875

Systems Biology as an Integrated Platform for Bioinformatics, Systems Synthetic Biology, and Systems Metabolic Engineering

Directory of Open Access Journals (Sweden)

Bor-Sen Chen

2013-10-01

Full Text Available Systems biology aims at achieving a system-level understanding of living organisms and applying this knowledge to various fields such as synthetic biology, metabolic engineering, and medicine. System-level understanding of living organisms can be derived from insight into: (i system structure and the mechanism of biological networks such as gene regulation, protein interactions, signaling, and metabolic pathways; (ii system dynamics of biological networks, which provides an understanding of stability, robustness, and transduction ability through system identification, and through system analysis methods; (iii system control methods at different levels of biological networks, which provide an understanding of systematic mechanisms to robustly control system states, minimize malfunctions, and provide potential therapeutic targets in disease treatment; (iv systematic design methods for the modification and construction of biological networks with desired behaviors, which provide system design principles and system simulations for synthetic biology designs and systems metabolic engineering. This review describes current developments in systems biology, systems synthetic biology, and systems metabolic engineering for engineering and biology researchers. We also discuss challenges and future prospects for systems biology and the concept of systems biology as an integrated platform for bioinformatics, systems synthetic biology, and systems metabolic engineering.

Metabolic Engineering of Probiotic Saccharomyces boulardii

OpenAIRE

Liu, Jing-Jing; Kong, In Iok; Zhang, Guo-Chang; Jayakody, Lahiru N.; Kim, Heejin; Xia, Peng-Fei; Kwak, Suryang; Sung, Bong Hyun; Sohn, Jung-Hoon; Walukiewicz, Hanna E.; Rao, Christopher V.; Jin, Yong-Su

2016-01-01

Saccharomyces boulardii is a probiotic yeast that has been used for promoting gut health as well as preventing diarrheal diseases. This yeast not only exhibits beneficial phenotypes for gut health but also can stay longer in the gut than Saccharomyces cerevisiae. Therefore, S. boulardii is an attractive host for metabolic engineering to produce biomolecules of interest in the gut. However, the lack of auxotrophic strains with defined genetic backgrounds has hampered the use of this strain for...

Metabolic engineering of volatile isoprenoids in plants and microbes.

Science.gov (United States)

Vickers, Claudia E; Bongers, Mareike; Liu, Qing; Delatte, Thierry; Bouwmeester, Harro

2014-08-01

The chemical properties and diversity of volatile isoprenoids lends them to a broad variety of biological roles. It also lends them to a host of biotechnological applications, both by taking advantage of their natural functions and by using them as industrial chemicals/chemical feedstocks. Natural functions include roles as insect attractants and repellents, abiotic stress protectants in pathogen defense, etc. Industrial applications include use as pharmaceuticals, flavours, fragrances, fuels, fuel additives, etc. Here we will examine the ways in which researchers have so far found to exploit volatile isoprenoids using biotechnology. Production and/or modification of volatiles using metabolic engineering in both plants and microorganisms are reviewed, including engineering through both mevalonate and methylerythritol diphosphate pathways. Recent advances are illustrated using several case studies (herbivores and bodyguards, isoprene, and monoterpene production in microbes). Systems and synthetic biology tools with particular utility for metabolic engineering are also reviewed. Finally, we discuss the practical realities of various applications in modern biotechnology, explore possible future applications, and examine the challenges of moving these technologies forward so that they can deliver tangible benefits. While this review focuses on volatile isoprenoids, many of the engineering approaches described here are also applicable to non-isoprenoid volatiles and to non-volatile isoprenoids. © 2014 John Wiley & Sons Ltd.

Recent advances in microbial production of fuels and chemicals using tools and strategies of systems metabolic engineering

DEFF Research Database (Denmark)

Cho, Changhee; Choi, So Young; Luo, Zi Wei

2015-01-01

The advent of various systems metabolic engineering tools and strategies has enabled more sophisticated engineering of microorganisms for the production of industrially useful fuels and chemicals. Advances in systems metabolic engineering have been made in overproducing natural chemicals...... and producing novel non-natural chemicals. In this paper, we review the tools and strategies of systems metabolic engineering employed for the development of microorganisms for the production of various industrially useful chemicals belonging to fuels, building block chemicals, and specialty chemicals......, in particular focusing on those reported in the last three years. It was aimed at providing the current landscape of systems metabolic engineering and suggesting directions to address future challenges towards successfully establishing processes for the bio-based production of fuels and chemicals from renewable...

Metabolic engineering of microalgal based biofuel production: prospects and challenges

Directory of Open Access Journals (Sweden)

Chiranjib eBanerjee

2016-03-01

Full Text Available The current scenario in renewable energy is focused on development of alternate and sustainable energy sources, amongst which microalgae stands as one of the promising feedstock for biofuel production. It is well known that microalgae generate much larger amounts of biofuels in a shorter time than other sources based on plant seeds. However, the greatest challenge in a transition to algae-based biofuel production is the various other complications involved in microalgal cultivation, its harvesting, concentration, drying and lipid extraction. Several green microalgae accumulate lipids, especially triacylglycerols (TAGs, which are main precursors in the production of lipid. The various aspects on metabolic pathway analysis of an oleaginous microalgae i.e. Chlamydomonas reinhardtii have elucidated some novel metabolically important genes and this enhances the lipid production in this microalgae. Adding to it, various other aspects in metabolic engineering using OptFlux and effectual bioprocess design also gives an interactive snapshot of enhancing lipid production which ultimately improvises the oil yield. This article reviews the current status of microalgal based technologies for biofuel production, bioreactor process design, flux analysis and it also provides various strategies to increase lipids accumulation via metabolic engineering.

Biobased production of alkanes and alkenes through metabolic engineering of microorganisms

DEFF Research Database (Denmark)

Kang, Min Kyoung; Nielsen, Jens

2017-01-01

Advancement in metabolic engineering of microorganisms has enabled bio-based production of a range of chemicals, and such engineered microorganism can be used for sustainable production leading to reduced carbon dioxide emission there. One area that has attained much interest is microbial...... hydrocarbon biosynthesis, and in particular, alkanes and alkenes are important high-value chemicals as they can be utilized for a broad range of industrial purposes as well as ‘drop-in’ biofuels. Some microorganisms have the ability to biosynthesize alkanes and alkenes naturally, but their production level...... is extremely low. Therefore, there have been various attempts to recruit other microbial cell factories for production of alkanes and alkenes by applying metabolic engineering strategies. Here we review different pathways and involved enzymes for alkane and alkene production and discuss bottlenecks...

SBOLme: a Repository of SBOL Parts for Metabolic Engineering

KAUST Repository

Kuwahara, Hiroyuki; Cui, Xuefeng; Umarov, Ramzan; Grunberg, Raik; Myers, Chris J.; Gao, Xin

2017-01-01

The Synthetic Biology Open Language (SBOL) is a community-driven open language to promote standardization in synthetic biology. To support the use of SBOL in metabolic engineering, we developed SBOLme, the first open-access repository of SBOL 2

Recent advances in microbial production of fuels and chemicals using tools and strategies of systems metabolic engineering.

Science.gov (United States)

Cho, Changhee; Choi, So Young; Luo, Zi Wei; Lee, Sang Yup

2015-11-15

The advent of various systems metabolic engineering tools and strategies has enabled more sophisticated engineering of microorganisms for the production of industrially useful fuels and chemicals. Advances in systems metabolic engineering have been made in overproducing natural chemicals and producing novel non-natural chemicals. In this paper, we review the tools and strategies of systems metabolic engineering employed for the development of microorganisms for the production of various industrially useful chemicals belonging to fuels, building block chemicals, and specialty chemicals, in particular focusing on those reported in the last three years. It was aimed at providing the current landscape of systems metabolic engineering and suggesting directions to address future challenges towards successfully establishing processes for the bio-based production of fuels and chemicals from renewable resources. Copyright © 2014 Elsevier Inc. All rights reserved.

Metabolic engineering with systems biology tools to optimize production of prokaryotic secondary metabolites

DEFF Research Database (Denmark)

Kim, Hyun Uk; Charusanti, Pep; Lee, Sang Yup

2016-01-01

Metabolic engineering using systems biology tools is increasingly applied to overproduce secondary metabolites for their potential industrial production. In this Highlight, recent relevant metabolic engineering studies are analyzed with emphasis on host selection and engineering approaches...... for the optimal production of various prokaryotic secondary metabolites: native versus heterologous hosts (e.g., Escherichia coli) and rational versus random approaches. This comparative analysis is followed by discussions on systems biology tools deployed in optimizing the production of secondary metabolites....... The potential contributions of additional systems biology tools are also discussed in the context of current challenges encountered during optimization of secondary metabolite production....

Quantifying the metabolic capabilities of engineered Zymomonas mobilis using linear programming analysis

Directory of Open Access Journals (Sweden)

Tsantili Ivi C

2007-03-01

Full Text Available Abstract Background The need for discovery of alternative, renewable, environmentally friendly energy sources and the development of cost-efficient, "clean" methods for their conversion into higher fuels becomes imperative. Ethanol, whose significance as fuel has dramatically increased in the last decade, can be produced from hexoses and pentoses through microbial fermentation. Importantly, plant biomass, if appropriately and effectively decomposed, is a potential inexpensive and highly renewable source of the hexose and pentose mixture. Recently, the engineered (to also catabolize pentoses anaerobic bacterium Zymomonas mobilis has been widely discussed among the most promising microorganisms for the microbial production of ethanol fuel. However, Z. mobilis genome having been fully sequenced in 2005, there is still a small number of published studies of its in vivo physiology and limited use of the metabolic engineering experimental and computational toolboxes to understand its metabolic pathway interconnectivity and regulation towards the optimization of its hexose and pentose fermentation into ethanol. Results In this paper, we reconstructed the metabolic network of the engineered Z. mobilis to a level that it could be modelled using the metabolic engineering methodologies. We then used linear programming (LP analysis and identified the Z. mobilis metabolic boundaries with respect to various biological objectives, these boundaries being determined only by Z. mobilis network's stoichiometric connectivity. This study revealed the essential for bacterial growth reactions and elucidated the association between the metabolic pathways, especially regarding main product and byproduct formation. More specifically, the study indicated that ethanol and biomass production depend directly on anaerobic respiration stoichiometry and activity. Thus, enhanced understanding and improved means for analyzing anaerobic respiration and redox potential in vivo are

Genomics:GTL Contractor-Grantee Workshop IV and Metabolic Engineering Working Group Inter-Agency Conference on Metabolic Engineering 2006

Energy Technology Data Exchange (ETDEWEB)

Mansfield, Betty Kay [ORNL; Martin, Sheryl A [ORNL

2006-02-01

Welcome to the 2006 joint meeting of the fourth Genomics:GTL Contractor-Grantee Workshop and the six Metabolic Engineering Working Group Inter-Agency Conference. The vision and scope of the Genomics:GTL program continue to expand and encompass research and technology issues from diverse scientific disciplines, attracting broad interest and support from researchers at universities, DOE national laboratories, and industry. Metabolic engineering's vision is the targeted and purposeful alteration of metabolic pathways to improve the understanding and use of cellular pathways for chemical transformation, energy transduction, and supramolecular assembly. These two programs have much complementarity in both vision and technological approaches, as reflected in this joint workshop. GLT's challenge to the scientific community remains the further development and use of a broad array of innovative technologies and computational tools to systematically leverage the knowledge and capabilities brought to us by DNA sequencing projects. The goal is to seek a broad and predictive understanding of the functioning and control of complex systems--individual microbes, microbial communities, and plants. GTL's prominent position at the interface of the physical, computational, and biological sciences is both a strength and challenge. Microbes remain GTL's principal biological focus. In the complex 'simplicity' of microbes, they find capabilities needed by DOE and the nation for clean and secure energy, cleanup of environmental contamination, and sequestration of atmospheric carbon dioxide that contributes to global warming. An ongoing challenge for the entire GTL community is to demonstrate that the fundamental science conducted in each of your research projects brings us a step closer to biology-based solutions for these important national energy and environmental needs.

The Need for Integrated Approaches in Metabolic Engineering

Energy Technology Data Exchange (ETDEWEB)

Lechner, Anna; Brunk, Elizabeth; Keasling, Jay D.

2016-08-15

This review highlights state-of-the-art procedures for heterologous small-molecule biosynthesis, the associated bottlenecks, and new strategies that have the potential to accelerate future accomplishments in metabolic engineering. We emphasize that a combination of different approaches over multiple time and size scales must b e considered for successful pathway engineering in a heterologous host. We have classified these optimization procedures based on the "system" that is being manipulated: transcriptome, translatome, proteome, or reactome. By bridging multiple disciplines, including molecular biology, biochemistry, biophysics, and computational sciences, we can create an integral framework for the discovery and implementation of novel biosynthetic production routes.

Engineering of metabolic pathways by artificial enzyme channels

Directory of Open Access Journals (Sweden)

Marlene ePröschel

2015-10-01

Full Text Available Application of industrial enzymes for production of valuable chemical compounds has greatly benefited from recent developments in Systems and Synthetic Biology. Both, in vivo and in vitro systems have been established, allowing conversion of simple into complex compounds. Metabolic engineering in living cells needs to be balanced which is achieved by controlling gene expression levels, translation, scaffolding, compartmentation and flux control. In vitro applications are often hampered by limited protein stability/half-life and insufficient rates of substrate conversion. To improve stability and catalytic activity, proteins are post-translationally modified and arranged in artificial metabolic channels. Within the review article we will first discuss the supramolecular organization of enzymes in living systems and secondly summarize current and future approaches to design artificial metabolic channels by additive manufacturing for the efficient production of desired products.

Review of Microfluidic Photobioreactor Technology for Metabolic Engineering and Synthetic Biology of Cyanobacteria and Microalgae

Directory of Open Access Journals (Sweden)

Ya-Tang Yang

2016-10-01

Full Text Available One goal of metabolic engineering and synthetic biology for cyanobacteria and microalgae is to engineer strains that can optimally produce biofuels and commodity chemicals. However, the current workflow is slow and labor intensive with respect to assembly of genetic parts and characterization of production yields because of the slow growth rates of these organisms. Here, we review recent progress in the microfluidic photobioreactors and identify opportunities and unmet needs in metabolic engineering and synthetic biology. Because of the unprecedented experimental resolution down to the single cell level, long-term real-time monitoring capability, and high throughput with low cost, microfluidic photobioreactor technology will be an indispensible tool to speed up the development process, advance fundamental knowledge, and realize the full potential of metabolic engineering and synthetic biology for cyanobacteria and microalgae.

Transcriptome data modeling for targeted plant metabolic engineering.

Science.gov (United States)

Yonekura-Sakakibara, Keiko; Fukushima, Atsushi; Saito, Kazuki

2013-04-01

The massive data generated by omics technologies require the power of bioinformatics, especially network analysis, for data mining and doing data-driven biology. Gene coexpression analysis, a network approach based on comprehensive gene expression data using microarrays, is becoming a standard tool for predicting gene function and elucidating the relationship between metabolic pathways. Differential and comparative gene coexpression analyses suggest a change in coexpression relationships and regulators controlling common and/or specific biological processes. In conjunction with the newly emerging genome editing technology, network analysis integrated with other omics data should pave the way for robust and practical plant metabolic engineering. Copyright © 2012 Elsevier Ltd. All rights reserved.

Current progress of targetron technology: development, improvement and application in metabolic engineering.

Science.gov (United States)

Liu, Ya-Jun; Zhang, Jie; Cui, Gu-Zhen; Cui, Qiu

2015-06-01

Targetrons are mobile group II introns that can recognize their DNA target sites by base-pairing RNA-DNA interactions with the aid of site-specific binding reverse transcriptases. Targetron technology stands out from recently developed gene targeting methods because of the flexibility, feasibility, and efficiency, and is particularly suitable for the genetic engineering of difficult microorganisms, including cellulolytic bacteria that are considered promising candidates for biomass conversion via consolidated bioprocessing. Along with the development of the thermotargetron method for thermophiles, targetron technology becomes increasingly important for the metabolic engineering of industrial microorganisms aiming at biofuel/chemical production. To summarize the current progress of targetron technology and provide new insights on the use of the technology, this paper reviews the retrohoming mechanisms of both mesophilic and thermophilic targetron methods based on various group II introns, investigates the improvement of targetron tools for high target efficiency and specificity, and discusses the current applications in the metabolic engineering for bacterial producers. Although there are still intellectual property and technical restrictions in targetron applications, we propose that targetron technology will contribute to both biochemistry research and the metabolic engineering for industrial productions. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Advances in Metabolic Engineering of Cyanobacteria for Photosynthetic Biochemical Production

OpenAIRE

Lai, Martin C.; Lan, Ethan I.

2015-01-01

Engineering cyanobacteria into photosynthetic microbial cell factories for the production of biochemicals and biofuels is a promising approach toward sustainability. Cyanobacteria naturally grow on light and carbon dioxide, bypassing the need of fermentable plant biomass and arable land. By tapping into the central metabolism and rerouting carbon flux towards desirable compound production, cyanobacteria are engineered to directly convert CO2 into various chemicals. This review discusses the d...

Mitigating health risks associated with alcoholic beverages through metabolic engineering.

Science.gov (United States)

Jayakody, Lahiru N; Lane, Stephan; Kim, Heejin; Jin, Yong-Su

2016-02-01

Epidemiological studies have established a positive relationship between the occurrence of cancer and consumption of alcoholic beverages. Metabolic engineering of brewing yeast to reduce potential carcinogenic compounds in alcoholic beverage is technically feasible as well as economically promising. This review presents the mechanisms of formation of potentially carcinogenic components in alcoholic beverages, such as formaldehyde, acetaldehyde, ethyl carbamate, acrylamide, and heavy metals, and introduces effective genetic perturbations to minimize the concentrations of these harmful components. As precise and effective genome editing tools for polyploid yeast are now available, we envision that yeast metabolic engineering might open up new research directions for improving brewing yeast in order to ensure product safety as well as to increase overall quality of alcoholic beverages. Copyright © 2015 Elsevier Ltd. All rights reserved.

Engineering of aromatic amino acid metabolism in Saccharomyces cerevisiae

NARCIS (Netherlands)

Vuralhan, Z.

2006-01-01

Saccharomyces cerevisiae is a popular industrial microorganism. It has since long been used in bread, beer and wine making. More recently it is also being applied for heterologous protein production and as a target organism for metabolic engineering. The work presented in this thesis describes how

Requirements for guidelines systems: implementation challenges and lessons from existing software-engineering efforts.

Science.gov (United States)

Shah, Hemant; Allard, Raymond D; Enberg, Robert; Krishnan, Ganesh; Williams, Patricia; Nadkarni, Prakash M

2012-03-09

A large body of work in the clinical guidelines field has identified requirements for guideline systems, but there are formidable challenges in translating such requirements into production-quality systems that can be used in routine patient care. Detailed analysis of requirements from an implementation perspective can be useful in helping define sub-requirements to the point where they are implementable. Further, additional requirements emerge as a result of such analysis. During such an analysis, study of examples of existing, software-engineering efforts in non-biomedical fields can provide useful signposts to the implementer of a clinical guideline system. In addition to requirements described by guideline-system authors, comparative reviews of such systems, and publications discussing information needs for guideline systems and clinical decision support systems in general, we have incorporated additional requirements related to production-system robustness and functionality from publications in the business workflow domain, in addition to drawing on our own experience in the development of the Proteus guideline system (http://proteme.org). The sub-requirements are discussed by conveniently grouping them into the categories used by the review of Isern and Moreno 2008. We cite previous work under each category and then provide sub-requirements under each category, and provide example of similar work in software-engineering efforts that have addressed a similar problem in a non-biomedical context. When analyzing requirements from the implementation viewpoint, knowledge of successes and failures in related software-engineering efforts can guide implementers in the choice of effective design and development strategies.

Enhancing gold recovery from electronic waste via lixiviant metabolic engineering in Chromobacterium violaceum

Science.gov (United States)

Tay, Song Buck; Natarajan, Gayathri; Rahim, Muhammad Nadjad bin Abdul; Tan, Hwee Tong; Chung, Maxey Ching Ming; Ting, Yen Peng; Yew, Wen Shan

2013-01-01

Conventional leaching (extraction) methods for gold recovery from electronic waste involve the use of strong acids and pose considerable threat to the environment. The alternative use of bioleaching microbes for gold recovery is non-pollutive and relies on the secretion of a lixiviant or (bio)chemical such as cyanide for extraction of gold from electronic waste. However, widespread industrial use of bioleaching microbes has been constrained by the limited cyanogenic capabilities of lixiviant-producing microorganisms such as Chromobacterium violaceum. Here we show the construction of a metabolically-engineered strain of Chromobacterium violaceum that produces more (70%) cyanide lixiviant and recovers more than twice as much gold from electronic waste compared to wild-type bacteria. Comparative proteome analyses suggested the possibility of further enhancement in cyanogenesis through subsequent metabolic engineering. Our results demonstrated the utility of lixiviant metabolic engineering in the construction of enhanced bioleaching microbes for the bioleaching of precious metals from electronic waste. PMID:23868689

Engineering the spatial organization of metabolic pathways

DEFF Research Database (Denmark)

Albertsen, Line; Maury, Jerome; Bach, Lars Stougaard

One of the goals of metabolic engineering is to optimize the production of valuable metabolites in cell factories. In this context, modulating the gene expression and activity of enzymes are tools that have been extensively used. Another approach that is gaining interest is the engineering...... of the spatial organization of biosynthetic pathways. Several natural systems for ensuring optimal spatial arrangement of biosynthetic enzymes exist. Sequentially acting enzymes can for example be positioned in close proximity by attachment to cellular structures, up-concentration in membrane enclosed organelles...... or assembly into large complexes. The vision is that by positioning sequentially acting enzymes in close proximity, the cell can accelerate reaction rates and thereby prevent loss of intermediates through diffusion, degradation or competing pathways. The production of valuable metabolites in cell factories...

Systems biology and metabolic engineering of lactic acid bacteria for improved fermented foods

NARCIS (Netherlands)

Flahaut, N.A.L.; Vos, de W.M.

2014-01-01

Lactic acid bacteria have long been used in industrial dairy and other food fermentations that make use of their metabolic activities leading to products with specific organoleptic properties. Metabolic engineering is a rational approach to steer fermentations toward the production of desired

Metabolic engineering of Candida glabrata for diacetyl production.

Directory of Open Access Journals (Sweden)

Xiang Gao

Full Text Available In this study, Candida glabrata, an efficient pyruvate-producing strain, was metabolically engineered for the production of the food ingredient diacetyl. A diacetyl biosynthetic pathway was reconstructed based on genetic modifications and medium optimization. The former included (i channeling carbon flux into the diacetyl biosynthetic pathway by amplification of acetolactate synthase, (ii elimination of the branched pathway of α-acetolactate by deleting the ILV5 gene, and (iii restriction of diacetyl degradation by deleting the BDH gene. The resultant strain showed an almost 1∶1 co-production of α-acetolactate and diacetyl (0.95 g L(-1. Furthermore, addition of Fe3+ to the medium enhanced the conversion of α-acetolactate to diacetyl and resulted in a two-fold increase in diacetyl production (2.1 g L(-1. In addition, increased carbon flux was further channeled into diacetyl biosynthetic pathway and a titer of 4.7 g L(-1 of diacetyl was achieved by altering the vitamin level in the flask culture. Thus, this study illustrates that C. glabrata could be tailored as an attractive platform for enhanced biosynthesis of beneficial products from pyruvate by metabolic engineering strategies.

Hydrogen production and metabolic flux analysis of metabolically engineered Escherichia coli strains

Energy Technology Data Exchange (ETDEWEB)

Kim, Seohyoung; Seol, Eunhee; Park, Sunghoon [Department of Chemical and Biochemical Engineering, Pusan National University, Busan 609-735 (Korea); Oh, You-Kwan [Bioenergy Research Center, Korea Institute of Energy Research, Daejeon 305-543 (Korea); Wang, G.Y. [Department of Oceanography, University of Hawaii at Manoa Honolulu, HI 96822 (United States)

2009-09-15

Escherichia coli can produce H{sub 2} from glucose via formate hydrogen lyase (FHL). In order to improve the H{sub 2} production rate and yield, metabolically engineered E. coli strains, which included pathway alterations in their H{sub 2} production and central carbon metabolism, were developed and characterized by batch experiments and metabolic flux analysis. Deletion of hycA, a negative regulator for FHL, resulted in twofold increase of FHL activity. Deletion of two uptake hydrogenases (1 (hya) and hydrogenase 2 (hyb)) increased H{sub 2} production yield from 1.20 mol/mol glucose to 1.48 mol/mol glucose. Deletion of lactate dehydrogenase (ldhA) and fumarate reductase (frdAB) further improved the H{sub 2} yield; 1.80 mol/mol glucose under high H{sub 2} pressure or 2.11 mol/mol glucose under reduced H{sub 2} pressure. Several batch experiments at varying concentrations of glucose (2.5-10 g/L) and yeast extract (0.3 or 3.0 g/L) were conducted for the strain containing all these genetic alternations, and their carbon and energy balances were analyzed. The metabolic flux analysis revealed that deletion of ldhA and frdAB directed most of the carbons from glucose to the glycolytic pathway leading to H{sub 2} production by FHL, not to the pentose phosphate pathway. (author)

Genome and metabolic engineering in non-conventional yeasts: Current advances and applications.

Science.gov (United States)

Löbs, Ann-Kathrin; Schwartz, Cory; Wheeldon, Ian

2017-09-01

Microbial production of chemicals and proteins from biomass-derived and waste sugar streams is a rapidly growing area of research and development. While the model yeast Saccharomyces cerevisia e is an excellent host for the conversion of glucose to ethanol, production of other chemicals from alternative substrates often requires extensive strain engineering. To avoid complex and intensive engineering of S. cerevisiae, other yeasts are often selected as hosts for bioprocessing based on their natural capacity to produce a desired product: for example, the efficient production and secretion of proteins, lipids, and primary metabolites that have value as commodity chemicals. Even when using yeasts with beneficial native phenotypes, metabolic engineering to increase yield, titer, and production rate is essential. The non-conventional yeasts Kluyveromyces lactis, K. marxianus, Scheffersomyces stipitis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris have been developed as eukaryotic hosts because of their desirable phenotypes, including thermotolerance, assimilation of diverse carbon sources, and high protein secretion. However, advanced metabolic engineering in these yeasts has been limited. This review outlines the challenges of using non-conventional yeasts for strain and pathway engineering, and discusses the developed solutions to these problems and the resulting applications in industrial biotechnology.

Metabolic Engineering for Production of Biorenewable Fuels and Chemicals: Contributions of Synthetic Biology

Directory of Open Access Journals (Sweden)

Laura R. Jarboe

2010-01-01

Full Text Available Production of fuels and chemicals through microbial fermentation of plant material is a desirable alternative to petrochemical-based production. Fermentative production of biorenewable fuels and chemicals requires the engineering of biocatalysts that can quickly and efficiently convert sugars to target products at a cost that is competitive with existing petrochemical-based processes. It is also important that biocatalysts be robust to extreme fermentation conditions, biomass-derived inhibitors, and their target products. Traditional metabolic engineering has made great advances in this area, but synthetic biology has contributed and will continue to contribute to this field, particularly with next-generation biofuels. This work reviews the use of metabolic engineering and synthetic biology in biocatalyst engineering for biorenewable fuels and chemicals production, such as ethanol, butanol, acetate, lactate, succinate, alanine, and xylitol. We also examine the existing challenges in this area and discuss strategies for improving biocatalyst tolerance to chemical inhibitors.

Metabolic Engineering of Microorganisms for the Production of Higher Alcohols

Science.gov (United States)

Choi, Yong Jun; Lee, Joungmin; Jang, Yu-Sin

2014-01-01

ABSTRACT Due to the increasing concerns about limited fossil resources and environmental problems, there has been much interest in developing biofuels from renewable biomass. Ethanol is currently used as a major biofuel, as it can be easily produced by existing fermentation technology, but it is not the best biofuel due to its low energy density, high vapor pressure, hygroscopy, and incompatibility with current infrastructure. Higher alcohols, including 1-propanol, 1-butanol, isobutanol, 2-methyl-1-butanol, and 3-methyl-1-butanol, which possess fuel properties more similar to those of petroleum-based fuel, have attracted particular interest as alternatives to ethanol. Since microorganisms isolated from nature do not allow production of these alcohols at high enough efficiencies, metabolic engineering has been employed to enhance their production. Here, we review recent advances in metabolic engineering of microorganisms for the production of higher alcohols. PMID:25182323

Non-photosynthetic plastids as hosts for metabolic engineering

DEFF Research Database (Denmark)

Mellor, Silas Busck; Behrendorff, James Bruce Yarnton H; Nielsen, Agnieszka Janina Zygadlo

2018-01-01

Using plants as hosts for production of complex, high-value compounds and therapeutic proteins has gained increasing momentum over the past decade. Recent advances in metabolic engineering techniques using synthetic biology have set the stage for production yields to become economically attractive......, but more refined design strategies are required to increase product yields without compromising development and growth of the host system. The ability of plant cells to differentiate into various tissues in combination with a high level of cellular compartmentalization represents so far the most...... in green tissues, have proven to be suitable for high yield protein and bio-compound production. Unfortunately, chloroplast manipulation often affects photosynthetic efficiency and therefore plant fitness. In this respect, plastids of non-photosynthetic tissues, which have focused metabolisms for synthesis...

Metabollic Engineering of Saccharomyces Cereviae a,omi acid metabolism for production of products of industrial interest

DEFF Research Database (Denmark)

Chen, Xiao

-based processes. This study has focused on metabolic engineering of the amino acid metabolism in S. cerevisiae for production of two types of chemicals of industrial interest. The first chemical is δ-(L-α-aminoadipyl)–L-cysteinyl–D-valine (LLD-ACV). ACV belongs to non-ribosomal peptides (NRPs), which......Saccharomyces cerevisiae is widely used in microbial production of chemicals, metabolites and proteins, mainly because genetic manipulation of S. cerevisiae is relatively easy and experiences from its wide application in the existing industrial fermentations directly benefit new S. cerevisiae...

Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels

Energy Technology Data Exchange (ETDEWEB)

Kuk Lee, Sung; Chou, Howard; Ham, Timothy S.; Soon Lee, Taek; Keasling, Jay D.

2009-12-02

The ability to generate microorganisms that can produce biofuels similar to petroleum-based transportation fuels would allow the use of existing engines and infrastructure and would save an enormous amount of capital required for replacing the current infrastructure to accommodate biofuels that have properties significantly different from petroleum-based fuels. Several groups have demonstrated the feasibility of manipulating microbes to produce molecules similar to petroleum-derived products, albeit at relatively low productivity (e.g. maximum butanol production is around 20 g/L). For cost-effective production of biofuels, the fuel-producing hosts and pathways must be engineered and optimized. Advances in metabolic engineering and synthetic biology will provide new tools for metabolic engineers to better understand how to rewire the cell in order to create the desired phenotypes for the production of economically viable biofuels.

From pathways to genomes and beyond. The metabolic engineering toolbox and its place in biofuels production

Energy Technology Data Exchange (ETDEWEB)

Liu, Leqian; Reed, Ben; Alper, Hal [Texas Univ., Austin, TX (United States). Dept. of Chemical Engineering

2011-07-01

Concerns about the availability of petroleum-derived fuels and chemicals have led to the exploration of metabolically engineered organisms as novel hosts for biofuels and chemicals production. However, the complexity inherent in metabolic and regulatory networks makes this undertaking a complex task. To address these limitations, metabolic engineering has adapted a wide-variety of tools for altering phenotypes. In this review, we will highlight traditional and recent metabolic engineering tools for optimizing cells including pathway-based, global, and genomic-enabled approaches. Specifically, we describe these tools as well as provide demonstrations of their effectiveness in optimizing biofuels production. However, each of these tools provides stepping stones towards the grand goal of biofuels production. Thus, developing methods for large-scale cellular optimization and integrative approaches are invaluable for further cell optimization. This review highlights the challenges that still must be met to accomplish this goal. (orig.)

Metabolically engineered cells for the production of polyunsaturated fatty acids

DEFF Research Database (Denmark)

2005-01-01

The present invention relates to the construction and engineering of cells, more particularly microorganisms for producing PUFAs with four or more double bonds from non-fatty acid substrates through heterologous expression of an oxygen requiring pathway. The invention especially involves...... improvement of the PUFA content in the host organism through fermentation optimization, e.g. decreasing the temperature and/or designing an optimal medium, or through improving the flux towards fatty acids by metabolic engineering, e.g. through over-expression of fatty acid synthases, over-expression of other...

Value-added biotransformation of cellulosic sugars by engineered Saccharomyces cerevisiae.

Science.gov (United States)

Lane, Stephan; Dong, Jia; Jin, Yong-Su

2018-07-01

The substantial research efforts into lignocellulosic biofuels have generated an abundance of valuable knowledge and technologies for metabolic engineering. In particular, these investments have led to a vast growth in proficiency of engineering the yeast Saccharomyces cerevisiae for consuming lignocellulosic sugars, enabling the simultaneous assimilation of multiple carbon sources, and producing a large variety of value-added products by introduction of heterologous metabolic pathways. While microbial conversion of cellulosic sugars into large-volume low-value biofuels is not currently economically feasible, there may still be opportunities to produce other value-added chemicals as regulation of cellulosic sugar metabolism is quite different from glucose metabolism. This review summarizes these recent advances with an emphasis on employing engineered yeast for the bioconversion of lignocellulosic sugars into a variety of non-ethanol value-added products. Copyright © 2018 Elsevier Ltd. All rights reserved.

Toward systems metabolic engineering of Aspergillus and Pichia species for the production of chemicals and biofuels

DEFF Research Database (Denmark)

Caspeta, Luis; Nielsen, Jens

2013-01-01

trends in systems biology of Aspergillus and Pichia species, highlighting the relevance of these developments for systems metabolic engineering of these organisms for the production of hydrolytic enzymes, biofuels and chemicals from biomass. Metabolic engineering is moving from traditional methods...... for the production of hydrolytic enzymes, biofuels and chemicals from biomass. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim....

Resveratrol biosynthesis: plant metabolic engineering for nutritional improvement of food.

Science.gov (United States)

Giovinazzo, Giovanna; Ingrosso, Ilaria; Paradiso, Annalisa; De Gara, Laura; Santino, Angelo

2012-09-01

The plant polyphenol trans-resveratrol (3, 5, 4'-trihydroxystilbene) mainly found in grape, peanut and other few plants, displays a wide range of biological effects. Numerous in vitro studies have described various biological effects of resveratrol. In order to provide more information regarding absorption, metabolism, and bioavailability of resveratrol, various research approaches have been performed, including in vitro, ex vivo, and in vivo models. In recent years, the induction of resveratrol synthesis in plants which normally do not accumulate such polyphenol, has been successfully achieved by molecular engineering. In this context, the ectopic production of resveratrol has been reported to have positive effects both on plant resistance to biotic stress and the enhancement of the nutritional value of several widely consumed fruits and vegetables. The metabolic engineering of plants offers the opportunity to change the content of specific phytonutrients in plant - derived foods. This review focuses on the latest findings regarding on resveratrol bioproduction and its effects on the prevention of the major pathological conditions in man.

Production of biopharmaceutical proteins by yeast: Advances through metabolic engineering

DEFF Research Database (Denmark)

Nielsen, Jens

2013-01-01

Production of recombinant proteins for use as pharmaceuticals, so-called biopharmaceuticals, is a multi-billion dollar industry. Many different cell factories are used for the production of biopharmaceuticals, but the yeast Saccharomyces cerevisiae is an important cell factory as it is used for p...... production. The involvement of directed metabolic engineering through the integration of tools from genetic engineering, systems biology and mathematical modeling, is also discussed....... by yeast are human serum albumin, hepatitis vaccines and virus like particles used for vaccination against human papillomavirus. Here is given a brief overview of biopharmaceutical production by yeast and it is discussed how the secretory pathway can be engineered to ensure more efficient protein...

Genome and metabolic engineering in non-conventional yeasts: Current advances and applications

Directory of Open Access Journals (Sweden)

Ann-Kathrin Löbs

2017-09-01

Full Text Available Microbial production of chemicals and proteins from biomass-derived and waste sugar streams is a rapidly growing area of research and development. While the model yeast Saccharomyces cerevisiae is an excellent host for the conversion of glucose to ethanol, production of other chemicals from alternative substrates often requires extensive strain engineering. To avoid complex and intensive engineering of S. cerevisiae, other yeasts are often selected as hosts for bioprocessing based on their natural capacity to produce a desired product: for example, the efficient production and secretion of proteins, lipids, and primary metabolites that have value as commodity chemicals. Even when using yeasts with beneficial native phenotypes, metabolic engineering to increase yield, titer, and production rate is essential. The non-conventional yeasts Kluyveromyces lactis, K. marxianus, Scheffersomyces stipitis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris have been developed as eukaryotic hosts because of their desirable phenotypes, including thermotolerance, assimilation of diverse carbon sources, and high protein secretion. However, advanced metabolic engineering in these yeasts has been limited. This review outlines the challenges of using non-conventional yeasts for strain and pathway engineering, and discusses the developed solutions to these problems and the resulting applications in industrial biotechnology.

Biobased production of alkanes and alkenes through metabolic engineering of microorganisms

OpenAIRE

Kang, Min Kyoung; Nielsen, Jens

2017-01-01

Advancement in metabolic engineering of microorganisms has enabled bio-based production of a range of chemicals, and such engineered microorganism can be used for sustainable production leading to reduced carbon dioxide emission there. One area that has attained much interest is microbial hydrocarbon biosynthesis, and in particular, alkanes and alkenes are important high-value chemicals as they can be utilized for a broad range of industrial purposes as well as ?drop-in? biofuels. Some microo...

A Consensus Genome-scale Reconstruction of Chinese Hamster Ovary Cell Metabolism

KAUST Repository

Hefzi, Hooman

2016-11-23

Chinese hamster ovary (CHO) cells dominate biotherapeutic protein production and are widely used in mammalian cell line engineering research. To elucidate metabolic bottlenecks in protein production and to guide cell engineering and bioprocess optimization, we reconstructed the metabolic pathways in CHO and associated them with >1,700 genes in the Cricetulus griseus genome. The genome-scale metabolic model based on this reconstruction, iCHO1766, and cell-line-specific models for CHO-K1, CHO-S, and CHO-DG44 cells provide the biochemical basis of growth and recombinant protein production. The models accurately predict growth phenotypes and known auxotrophies in CHO cells. With the models, we quantify the protein synthesis capacity of CHO cells and demonstrate that common bioprocess treatments, such as histone deacetylase inhibitors, inefficiently increase product yield. However, our simulations show that the metabolic resources in CHO are more than three times more efficiently utilized for growth or recombinant protein synthesis following targeted efforts to engineer the CHO secretory pathway. This model will further accelerate CHO cell engineering and help optimize bioprocesses.

Redox cofactor engineering in industrial microorganisms: strategies, recent applications and future directions.

Science.gov (United States)

Liu, Jiaheng; Li, Huiling; Zhao, Guangrong; Caiyin, Qinggele; Qiao, Jianjun

2018-05-01

NAD and NADP, a pivotal class of cofactors, which function as essential electron donors or acceptors in all biological organisms, drive considerable catabolic and anabolic reactions. Furthermore, they play critical roles in maintaining intracellular redox homeostasis. However, many metabolic engineering efforts in industrial microorganisms towards modification or introduction of metabolic pathways, especially those involving consumption, generation or transformation of NAD/NADP, often induce fluctuations in redox state, which dramatically impede cellular metabolism, resulting in decreased growth performance and biosynthetic capacity. Here, we comprehensively review the cofactor engineering strategies for solving the problematic redox imbalance in metabolism modification, as well as their features, suitabilities and recent applications. Some representative examples of in vitro biocatalysis are also described. In addition, we briefly discuss how tools and methods from the field of synthetic biology can be applied for cofactor engineering. Finally, future directions and challenges for development of cofactor redox engineering are presented.

Metabolic engineering of Ustilago trichophora TZ1 for improved malic acid production

Directory of Open Access Journals (Sweden)

Thiemo Zambanini

2017-06-01

These results open up a wide range of possibilities for further optimization, especially combinatorial metabolic engineering to increase the flux from pyruvate to malic acid and to reduce by-product formation.

In-silico-driven metabolic engineering of Pseudomonas putida for enhanced production of poly-hydroxyalkanoates

NARCIS (Netherlands)

Poblete-Castro, I.; Binger, D.; Rodrigues, A.; Becker, J.; Martins Dos Santos, V.A.P.; Wittmann, C.

2013-01-01

Here, we present systems metabolic engineering driven by in-silico modeling to tailor Pseudomonas putida for synthesis of medium chain length PHAs on glucose. Using physiological properties of the parent wild type as constraints, elementary flux mode analysis of a large-scale model of the metabolism

Metabolic engineering of free-energy (ATP) conserving reactions in Saccharomyces cerevisiae

NARCIS (Netherlands)

De Kok, S.

2012-01-01

Metabolic engineering – the improvement of cellular activities by manipulation of enzymatic, transport and regulatory functions of the cell – has enabled the industrial production of a wide variety of biological molecules from renewable resources. Microbial production of fuels and chemicals thereby

Yeast metabolic engineering--targeting sterol metabolism and terpenoid formation.

Science.gov (United States)

Wriessnegger, Tamara; Pichler, Harald

2013-07-01

Terpenoids comprise various structures conferring versatile functions to eukaryotes, for example in the form of prenyl-anchors they attach proteins to membranes. The physiology of eukaryotic membranes is fine-tuned by another terpenoid class, namely sterols. Evidence is accumulating that numerous membrane proteins require specific sterol structural features for function. Moreover, sterols are intermediates in the synthesis of steroids serving as hormones in higher eukaryotes. Like steroids many compounds of the terpenoid family do not contribute to membrane architecture, but serve as signalling, protective or attractant/repellent molecules. Particularly plants have developed a plenitude of terpenoid biosynthetic routes branching off early in the sterol biosynthesis pathway and, thereby, forming one of the largest groups of naturally occurring organic compounds. Many of these aromatic and volatile molecules are interesting for industrial application ranging from foods to pharmaceuticals. Combining the fortunate situation that sterol biosynthesis is highly conserved in eukaryotes with the amenability of yeasts to genetic and metabolic engineering, basically all naturally occurring terpenoids might be produced involving yeasts. Such engineered yeasts are useful for the study of biological functions and molecular interactions of terpenoids as well as for the large-scale production of high-value compounds, which are unavailable in sufficient amounts from natural sources due to their low abundance. Copyright © 2013 Elsevier Ltd. All rights reserved.

Metabolic engineering of Corynebacterium glutamicum for fermentative production of chemicals in biorefinery.

Science.gov (United States)

Baritugo, Kei-Anne; Kim, Hee Taek; David, Yokimiko; Choi, Jong-Il; Hong, Soon Ho; Jeong, Ki Jun; Choi, Jong Hyun; Joo, Jeong Chan; Park, Si Jae

2018-05-01

Bio-based production of industrially important chemicals provides an eco-friendly alternative to current petrochemical-based processes. Because of the limited supply of fossil fuel reserves, various technologies utilizing microbial host strains for the sustainable production of platform chemicals from renewable biomass have been developed. Corynebacterium glutamicum is a non-pathogenic industrial microbial species traditionally used for L-glutamate and L-lysine production. It is a promising species for industrial production of bio-based chemicals because of its flexible metabolism that allows the utilization of a broad spectrum of carbon sources and the production of various amino acids. Classical breeding, systems, synthetic biology, and metabolic engineering approaches have been used to improve its applications, ranging from traditional amino-acid production to modern biorefinery systems for production of value-added platform chemicals. This review describes recent advances in the development of genetic engineering tools and techniques for the establishment and optimization of metabolic pathways for bio-based production of major C2-C6 platform chemicals using recombinant C. glutamicum.

Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries.

Science.gov (United States)

Hong, Kuk-Ki; Nielsen, Jens

2012-08-01

Metabolic engineering is the enabling science of development of efficient cell factories for the production of fuels, chemicals, pharmaceuticals, and food ingredients through microbial fermentations. The yeast Saccharomyces cerevisiae is a key cell factory already used for the production of a wide range of industrial products, and here we review ongoing work, particularly in industry, on using this organism for the production of butanol, which can be used as biofuel, and isoprenoids, which can find a wide range of applications including as pharmaceuticals and as biodiesel. We also look into how engineering of yeast can lead to improved uptake of sugars that are present in biomass hydrolyzates, and hereby allow for utilization of biomass as feedstock in the production of fuels and chemicals employing S. cerevisiae. Finally, we discuss the perspectives of how technologies from systems biology and synthetic biology can be used to advance metabolic engineering of yeast.

Effort-reward imbalance is associated with the metabolic syndrome — Findings from the Mannheim Industrial Cohort Study (MICS)

NARCIS (Netherlands)

Schmidt, B.; Bosch, J.A.; Jarczok, M.N.; Herr, R.M.; Loerbroks, A.; van Vianen, A.E.M.; Fischer, J.E.

2015-01-01

Background/objectives: Job stress is a predictor of cardiovascular disease incidence and mortality and the metabolic syndrome (MetS) represents one of the key pathways potentially underlying those associations. Effort-reward imbalance (ERI) represents one of the most influential theoretical work

Impact of synthetic biology and metabolic engineering on industrial production of fine chemicals

DEFF Research Database (Denmark)

Jullesson, David; David, Florian; Pfleger, Brian

2015-01-01

Industrial bio-processes for fine chemical production are increasingly relying on cell factories developed through metabolic engineering and synthetic biology. The use of high throughput techniques and automation for the design of cell factories, and especially platform strains, has played...... chemicals that have reached the market, key metabolic engineering tools that have allowed this to happen and some of the companies that are currently utilizing these technologies for developing industrial production processes....... an important role in the transition from laboratory research to industrial production. Model organisms such as Saccharomyces cerevisiae and Escherichia coli remain widely used host strains for industrial production due to their robust and desirable traits. This review describes some of the bio-based fine...

Impact of synthetic biology and metabolic engineering on industrial production of fine chemicals.

Science.gov (United States)

Jullesson, David; David, Florian; Pfleger, Brian; Nielsen, Jens

2015-11-15

Industrial bio-processes for fine chemical production are increasingly relying on cell factories developed through metabolic engineering and synthetic biology. The use of high throughput techniques and automation for the design of cell factories, and especially platform strains, has played an important role in the transition from laboratory research to industrial production. Model organisms such as Saccharomyces cerevisiae and Escherichia coli remain widely used host strains for industrial production due to their robust and desirable traits. This review describes some of the bio-based fine chemicals that have reached the market, key metabolic engineering tools that have allowed this to happen and some of the companies that are currently utilizing these technologies for developing industrial production processes. Copyright © 2015 Elsevier Inc. All rights reserved.

Modeling with a view to target identification in metabolic engineering: a critical evaluation of the available tools.

Science.gov (United States)

Maertens, Jo; Vanrolleghem, Peter A

2010-01-01

The state of the art tools for modeling metabolism, typically used in the domain of metabolic engineering, were reviewed. The tools considered are stoichiometric network analysis (elementary modes and extreme pathways), stoichiometric modeling (metabolic flux analysis, flux balance analysis, and carbon modeling), mechanistic and approximative modeling, cybernetic modeling, and multivariate statistics. In the context of metabolic engineering, one should be aware that the usefulness of these tools to optimize microbial metabolism for overproducing a target compound depends predominantly on the characteristic properties of that compound. Because of their shortcomings not all tools are suitable for every kind of optimization; issues like the dependence of the target compound's synthesis on severe (redox) constraints, the characteristics of its formation pathway, and the achievable/desired flux towards the target compound should play a role when choosing the optimization strategy.

Cytochrome P450-mediated metabolic engineering: current progress and future challenges.

Science.gov (United States)

Renault, Hugues; Bassard, Jean-Etienne; Hamberger, Björn; Werck-Reichhart, Danièle

2014-06-01

Cytochromes P450 catalyze a broad range of regiospecific, stereospecific and irreversible steps in the biosynthetic routes of plant natural metabolites with important applications in pharmaceutical, cosmetic, fragrance and flavour, or polymer industries. They are consequently essential drivers for the engineered bioproduction of such compounds. Two ground-breaking developments of commercial products driven by the engineering of P450s are the antimalarial drug precursor artemisinic acid and blue roses or carnations. Tedious optimizations were required to generate marketable products. Hurdles encountered in P450 engineering and their potential solutions are summarized here. Together with recent technical developments and novel approaches to metabolic engineering, the lessons from this pioneering work should considerably boost exploitation of the amazing P450 toolkit emerging from accelerated sequencing of plant genomes. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Synthetic biology and regulatory networks: where metabolic systems biology meets control engineering

NARCIS (Netherlands)

He, F.; Murabito, E.; Westerhoff, H.V.

2016-01-01

Metabolic pathways can be engineered to maximize the synthesis of various products of interest. With the advent of computational systems biology, this endeavour is usually carried out throughin silicotheoretical studies with the aim to guide and complement furtherin vitroandin vivoexperimental

DEVELOPMENT OF MICROORGANISMS FOR CELLULOSE-BIOFUEL CONSOLIDATED BIOPROCESSINGS: METABOLIC ENGINEERS' TRICKS

Directory of Open Access Journals (Sweden)

Roberto Mazzoli

2012-10-01

By starting from the description of natural enzyme systems for plant biomass degradation and natural metabolic pathways for some of the most valuable product (i.e. butanol, ethanol, and hydrogen biosynthesis, this review describes state-of-the-art bottlenecks and solutions for the development of recombinant microbial strains for cellulosic biofuel CBP by metabolic engineering. Complexed cellulases (i.e. cellulosomes benefit from stronger proximity effects and show enhanced synergy on insoluble substrates (i.e. crystalline cellulose with respect to free enzymes. For this reason, special attention was held on strategies involving cellulosome/designer cellulosome-bearing recombinant microorganisms.

Metabolic network modeling of microbial interactions in natural and engineered environmental systems

Directory of Open Access Journals (Sweden)

Octavio ePerez-Garcia

2016-05-01

Full Text Available We review approaches to characterize metabolic interactions within microbial communities using Stoichiometric Metabolic Network (SMN models for applications in environmental and industrial biotechnology. SMN models are computational tools used to evaluate the metabolic engineering potential of various organisms. They have successfully been applied to design and optimize the microbial production of antibiotics, alcohols and amino acids by single strains. To date however, such models have been rarely applied to analyze and control the metabolism of more complex microbial communities. This is largely attributed to the diversity of microbial community functions, metabolisms and interactions. Here, we firstly review different types of microbial interaction and describe their relevance for natural and engineered environmental processes. Next, we provide a general description of the essential methods of the SMN modeling workflow including the steps of network reconstruction, simulation through Flux Balance Analysis (FBA, experimental data gathering, and model calibration. Then we broadly describe and compare four approaches to model microbial interactions using metabolic networks, i.e. i lumped networks, ii compartment per guild networks, iii bi-level optimization simulations and iv dynamic-SMN methods. These approaches can be used to integrate and analyze diverse microbial physiology, ecology and molecular community data. All of them (except the lumped approach are suitable for incorporating species abundance data but so far they have been used only to model simple communities of two to eight different species. Interactions based on substrate exchange and competition can be directly modeled using the above approaches. However, interactions based on metabolic feedbacks, such as product inhibition and synthropy require extensions to current models, incorporating gene regulation and compounding accumulation mechanisms. SMN models of microbial

EFFECTS OF COMPACTIVE EFFORTS ON GEOTECHNICAL PROPERTIES OF SPENT ENGINE OIL CONTAMINATED LATERITE SOIL

Directory of Open Access Journals (Sweden)

OLUREMI, J. ROTIMI

2017-03-01

Full Text Available Investigation of the effects of compactive efforts and spent engine oil (SEO contamination on the geotechnical properties of lateritic soils was made. Contaminated specimens were prepared by mixing lateritic soil with up to 10 % SEO by dry weight of the soil in step concentration of 2 % and subjected to geotechnical tests. Results indicated a decrease in the fine content, decrease in liquid limit, maximum dry density (MDD and unconfined compressive strength (UCS with up to 10 % SEO content. No general trend was observed in the optimum moisture content (OMC with increasing SEO content. The MDD, OMC and UCS values increased with increase in the compactive effort. Regression analysis of the results showed that optimum moisture content, fine content and compactive effort significantly influence the soils UCS values. Analysis of variance showed that SEO and compactive effort has significant effect on the parameters with the exception in one case. The results of laboratory tests showed that geotechnical properties of the SEO contaminated soil were immensely impaired.

Engineered nanomaterial-mediated changes in the metabolism of terrestrial plants

Energy Technology Data Exchange (ETDEWEB)

Hatami, Mehrnaz, E-mail: m-hatami@araku.ac.ir [Department of Medicinal Plants, Faculty of Agriculture and Natural Resources, Arak University, 38156-8-8349 Arak (Iran, Islamic Republic of); Kariman, Khalil [School of Earth and Environment M004, The University of Western Australia, Crawley, WA 6009 (Australia); Ghorbanpour, Mansour, E-mail: m-ghorbanpour@araku.ac.ir [Department of Medicinal Plants, Faculty of Agriculture and Natural Resources, Arak University, 38156-8-8349 Arak (Iran, Islamic Republic of)

2016-11-15

Engineered nanomaterials (ENMs) possess remarkable physicochemical characteristics suitable for different applications in medicine, pharmaceuticals, biotechnology, energy, cosmetics and electronics. Because of their ultrafine size and high surface reactivity, ENMs can enter plant cells and interact with intracellular structures and metabolic pathways which may produce toxicity or promote plant growth and development by diverse mechanisms. Depending on their type and concentration, ENMs can have positive or negative effects on photosynthesis, photochemical fluorescence and quantum yield as well as photosynthetic pigments status of the plants. Some studies have shown that ENMs can improve photosynthetic efficiency via increasing chlorophyll content and light absorption and also broadening the spectrum of captured light, suggesting that photosynthesis can be nano-engineered for harnessing more solar energy. Both up- and down-regulation of primary metabolites such as proteins and carbohydrates have been observed following exposure of plants to various ENMs. The potential capacity of ENMs for changing the rate of primary metabolites lies in their close relationship with activation and biosynthesis of the key enzymes. Several classes of secondary metabolites such as phenolics, flavonoids, and alkaloids have been shown to be induced (mostly accompanied by stress-related factors) in plants exposed to different ENMs, highlighting their great potential as elicitors to enhance both quantity and quality of biologically active secondary metabolites. Considering reports on both positive and negative effects of ENMs on plant metabolism, in-depth studies are warranted to figure out the most appropriate ENMs (type, size and optimal concentration) in order to achieve the desirable effect on specific metabolites in a given plant species. In this review, we summarize the studies performed on the impacts of ENMs on biosynthesis of plant primary and secondary metabolites and mention the

Engineered nanomaterial-mediated changes in the metabolism of terrestrial plants

International Nuclear Information System (INIS)

Hatami, Mehrnaz; Kariman, Khalil; Ghorbanpour, Mansour

2016-01-01

Engineered nanomaterials (ENMs) possess remarkable physicochemical characteristics suitable for different applications in medicine, pharmaceuticals, biotechnology, energy, cosmetics and electronics. Because of their ultrafine size and high surface reactivity, ENMs can enter plant cells and interact with intracellular structures and metabolic pathways which may produce toxicity or promote plant growth and development by diverse mechanisms. Depending on their type and concentration, ENMs can have positive or negative effects on photosynthesis, photochemical fluorescence and quantum yield as well as photosynthetic pigments status of the plants. Some studies have shown that ENMs can improve photosynthetic efficiency via increasing chlorophyll content and light absorption and also broadening the spectrum of captured light, suggesting that photosynthesis can be nano-engineered for harnessing more solar energy. Both up- and down-regulation of primary metabolites such as proteins and carbohydrates have been observed following exposure of plants to various ENMs. The potential capacity of ENMs for changing the rate of primary metabolites lies in their close relationship with activation and biosynthesis of the key enzymes. Several classes of secondary metabolites such as phenolics, flavonoids, and alkaloids have been shown to be induced (mostly accompanied by stress-related factors) in plants exposed to different ENMs, highlighting their great potential as elicitors to enhance both quantity and quality of biologically active secondary metabolites. Considering reports on both positive and negative effects of ENMs on plant metabolism, in-depth studies are warranted to figure out the most appropriate ENMs (type, size and optimal concentration) in order to achieve the desirable effect on specific metabolites in a given plant species. In this review, we summarize the studies performed on the impacts of ENMs on biosynthesis of plant primary and secondary metabolites and mention the

A review of metabolic and enzymatic engineering strategies for designing and optimizing performance of microbial cell factories

Directory of Open Access Journals (Sweden)

Amanda K. Fisher

2014-08-01

Full Text Available Microbial cell factories (MCFs are of considerable interest to convert low value renewable substrates to biofuels and high value chemicals. This review highlights the progress of computational models for the rational design of an MCF to produce a target bio-commodity. In particular, the rational design of an MCF involves: (i product selection, (ii de novo biosynthetic pathway identification (i.e., rational, heterologous, or artificial, (iii MCF chassis selection, (iv enzyme engineering of promiscuity to enable the formation of new products, and (v metabolic engineering to ensure optimal use of the pathway by the MCF host. Computational tools such as (i de novo biosynthetic pathway builders, (ii docking, (iii molecular dynamics (MD and steered MD (SMD, and (iv genome-scale metabolic flux modeling all play critical roles in the rational design of an MCF. Genome-scale metabolic flux models are of considerable use to the design process since they can reveal metabolic capabilities of MCF hosts. These can be used for host selection as well as optimizing precursors and cofactors of artificial de novo biosynthetic pathways. In addition, recent advances in genome-scale modeling have enabled the derivation of metabolic engineering strategies, which can be implemented using the genomic tools reviewed here as well.

Towards a sustainable bio-based economy: Redirecting primary metabolism to new products with plant synthetic biology.

Science.gov (United States)

Shih, Patrick M

2018-08-01

Humans have domesticated many plant species as indispensable sources of food, materials, and medicines. The dawning era of synthetic biology represents a means to further refine, redesign, and engineer crops to meet various societal and industrial needs. Current and future endeavors will utilize plants as the foundation of a bio-based economy through the photosynthetic production of carbohydrate feedstocks for the microbial fermentation of biofuels and bioproducts, with the end goal of decreasing our dependence on petrochemicals. As our technological capabilities improve, metabolic engineering efforts may expand the utility of plants beyond sugar feedstocks through the direct production of target compounds, including pharmaceuticals, renewable fuels, and commodity chemicals. However, relatively little work has been done to fully realize the potential in redirecting central carbon metabolism in plants for the engineering of novel bioproducts. Although our ability to rationally engineer and manipulate plant metabolism is in its infancy, I highlight some of the opportunities and challenges in applying synthetic biology towards engineering plant primary metabolism. Copyright © 2018 Elsevier B.V. All rights reserved.

Mass spectrometry characterisation of fatty acids from metabolically engineered soybean seeds.

Science.gov (United States)

Murad, André M; Vianna, Giovanni R; Machado, Alex M; da Cunha, Nicolau B; Coelho, Cíntia M; Lacerda, Valquiria A M; Coelho, Marly C; Rech, Elibio L

2014-05-01

Improving the quality and performance of soybean oil as biodiesel depends on the chemical composition of its fatty acids and requires an increase in monounsaturated acids and a reduction in polyunsaturated acids. Despite its current use as a source of biofuel, soybean oil contains an average of 25 % oleic acid and 13 % palmitic acid, which negatively impacts its oxidative stability and freezing point, causing a high rate of nitrogen oxide emission. Gas chromatography and ion mobility mass spectrometry were conducted on soybean fatty acids from metabolically engineered seed extracts to determine the nature of the structural oleic and palmitic acids. The soybean genes FAD2-1 and FatB were placed under the control of the 35SCaMV constitutive promoter, introduced to soybean embryonic axes by particle bombardment and down-regulated using RNA interference technology. Results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 94.58 %) and a reduction in palmitic acid (to seed oil content. No structural differences were observed between the fatty acids of the transgenic and non-transgenic oil extracts.

Production of vanillin by metabolically engineered Escherichia coli.

Science.gov (United States)

Yoon, Sang-Hwal; Li, Cui; Kim, Ju-Eun; Lee, Sook-Hee; Yoon, Ji-Young; Choi, Myung-Suk; Seo, Weon-Taek; Yang, Jae-Kyung; Kim, Jae-Yeon; Kim, Seon-Won

2005-11-01

E. coli was metabolically engineered to produce vanillin by expression of the fcs and ech genes from Amycolatopsis sp. encoding feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase, respectively. Vanillin production was optimized by leaky expression of the genes, under the IPTG-inducible trc promoter, in complex 2YT medium. Supplementation with glucose, fructose, galactose, arabinose or glycerol severely decreased vanillin production. The highest vanillin production of 1.1 g l(-1) was obtained with cultivation for 48 h in 2YT medium with 0.2% (w/v) ferulate, without IPTG and no supplementation of carbon sources.

Metabolic engineering: the ultimate paradigm for continuous pharmaceutical manufacturing.

Science.gov (United States)

Yadav, Vikramaditya G; Stephanopoulos, Gregory

2014-07-01

Research and development (R&D) expenditures by pharmaceutical companies doubled over the past decade, yet candidate attrition rates and development times rose markedly during this period. Understandably, companies have begun downsizing their pipelines and diverting investments away from R&D in favor of manufacturing. It is estimated that transitioning to continuous manufacturing could enable companies to compete for a share in emerging markets. Accordingly, the model for continuous manufacturing that has emerged commences with the conversion of late-stage intermediates into the active pharmaceutical ingredient (API) in a series of continuous flow reactors, followed by continuous solid processing to form finished tablets. The use of flow reactions for API synthesis will certainly generate purer products at higher yields in shorter times compared to equivalent batch reactions. However, transitioning from batch to flow configuration simply alleviates transport limitations within the reaction milieu. As the catalogue of reactions used in flow syntheses is a subset of batch-based chemistries, molecules such as natural products will continue to evade drug prospectors. Also, it is uncertain whether flow synthesis can deliver improvements in the atom and energy economies of API production at the scales that would achieve the levels of revenue growth targeted by companies. Instead, it is argued that implementing metabolic engineering for the production of oxidized scaffolds as gateway molecules for flow-based addition of electrophiles is a more effective and scalable strategy for accessing natural product chemical space. This new paradigm for manufacturing, with metabolic engineering as its engine, would also permit rapid optimization of production variables and allow facile scale-up from gram to ton scale to meet material requirements for clinical trials, thus recasting manufacturing as a tool for discovery. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Transcriptomic Changes in Response to Putrescine Production in Metabolically Engineered Corynebacterium glutamicum

Directory of Open Access Journals (Sweden)

Zhen Li

2017-10-01

Full Text Available Putrescine is widely used in industrial production of bioplastics, pharmaceuticals, agrochemicals, and surfactants. Although engineered Corynebacterium glutamicum has been successfully used to produce high levels of putrescine, the overall cellular physiological and metabolic changes caused by overproduction of putrescine remains unclear. To reveal the transcriptional changes that occur in response to putrescine production in an engineered C. glutamicum strain, a comparative transcriptomic analysis was carried out. Overproduction of putrescine resulted in transcriptional downregulation of genes involved in glycolysis; the TCA cycle, pyruvate degradation, biosynthesis of some amino acids, oxidative phosphorylation; vitamin biosynthesis (thiamine and vitamin 6, metabolism of purine, pyrimidine and sulfur, and ATP-, NAD-, and NADPH-consuming enzymes. The transcriptional levels of genes involved in ornithine biosynthesis and NADPH-forming related enzymes were significantly upregulated in the putrescine producing C. glutamicum strain PUT-ALE. Comparative transcriptomic analysis provided some genetic modification strategies to further improve putrescine production. Repressing ATP- and NADPH-consuming enzyme coding gene expression via CRISPRi enhanced putrescine production.

Transcriptomic Changes in Response to Putrescine Production in Metabolically Engineered Corynebacterium glutamicum

Science.gov (United States)

Li, Zhen; Liu, Jian-Zhong

2017-01-01

Putrescine is widely used in industrial production of bioplastics, pharmaceuticals, agrochemicals, and surfactants. Although engineered Corynebacterium glutamicum has been successfully used to produce high levels of putrescine, the overall cellular physiological and metabolic changes caused by overproduction of putrescine remains unclear. To reveal the transcriptional changes that occur in response to putrescine production in an engineered C. glutamicum strain, a comparative transcriptomic analysis was carried out. Overproduction of putrescine resulted in transcriptional downregulation of genes involved in glycolysis; the TCA cycle, pyruvate degradation, biosynthesis of some amino acids, oxidative phosphorylation; vitamin biosynthesis (thiamine and vitamin 6), metabolism of purine, pyrimidine and sulfur, and ATP-, NAD-, and NADPH-consuming enzymes. The transcriptional levels of genes involved in ornithine biosynthesis and NADPH-forming related enzymes were significantly upregulated in the putrescine producing C. glutamicum strain PUT-ALE. Comparative transcriptomic analysis provided some genetic modification strategies to further improve putrescine production. Repressing ATP- and NADPH-consuming enzyme coding gene expression via CRISPRi enhanced putrescine production. PMID:29089930

Genome Sequencing of Streptomyces atratus SCSIOZH16 and Activation Production of Nocardamine via Metabolic Engineering

Directory of Open Access Journals (Sweden)

Yan Li

2018-06-01

Full Text Available The Actinomycetes are metabolically flexible microorganisms capable of producing a wide range of interesting compounds, including but by no means limited to, siderophores which have high affinity for ferric iron. In this study, we report the complete genome sequence of marine-derived Streptomyces atratus ZH16 and the activation of an embedded siderophore gene cluster via the application of metabolic engineering methods. The S. atratus ZH16 genome reveals that this strain has the potential to produce 26 categories of natural products (NPs barring the ilamycins. Our activation studies revealed S. atratus SCSIO ZH16 to be a promising source of the production of nocardamine-type (desferrioxamine compounds which are important in treating acute iron intoxication and performing ecological remediation. We conclude that metabolic engineering provides a highly effective strategy by which to discover drug-like compounds and new NPs in the genomic era.

Final Technical Report; NUCLEAR ENGINEERING RECRUITMENT EFFORT

Energy Technology Data Exchange (ETDEWEB)

Kerrick, Sharon S.; Vincent, Charles D.

2007-07-02

This report provides the summary of a project whose purpose was to support the costs of developing a nuclear engineering awareness program, an instruction program for teachers to integrate lessons on nuclear science and technology into their existing curricula, and web sites for the exchange of nuclear engineering career information and classroom materials. The specific objectives of the program were as follows: OBJECTIVE 1: INCREASE AWARENESS AND INTEREST OF NUCLEAR ENGINEERING; OBJECTIVE 2: INSTRUCT TEACHERS ON NUCLEAR TOPICS; OBJECTIVE 3: NUCLEAR EDUCATION PROGRAMS WEB-SITE; OBJECTIVE 4: SUPPORT TO UNIVERSITY/INDUSTRY MATCHING GRANTS AND REACTOR SHARING; OBJECTIVE 5: PILOT PROJECT; OBJECTIVE 6: NUCLEAR ENGINEERING ENROLLMENT SURVEY AT UNIVERSITIES

Development of biosensors and their application in metabolic engineering

DEFF Research Database (Denmark)

Zhang, Jie; Jensen, Michael Krogh; Keasling, Jay

2015-01-01

and ease of implementation with high-throughput analysis. Here we describe recent progress in biosensor development and their applications in a metabolic engineering context. We also highlight examples of how biosensors can be integrated with synthetic circuits to exert feedback regulation...... for the desired phenotypes. However, methods available for microbial genome diversification far exceed our ability to screen and select for those variants with optimal performance. Genetically encoded biosensors have shown the potential to address this gap, given their ability to respond to small molecule binding...

Systematic metabolic engineering of Methylomicrobium alcaliphilum 20Z for 2,3-butanediol production from methane.

Science.gov (United States)

Nguyen, Anh Duc; Hwang, In Yeub; Lee, Ok Kyung; Kim, Donghyuk; Kalyuzhnaya, Marina G; Mariyana, Rina; Hadiyati, Susila; Kim, Min Sik; Lee, Eun Yeol

2018-04-16

Methane is considered a next-generation feedstock, and methanotrophic cell-based biorefinery is attractive for production of a variety of high-value compounds from methane. In this work, we have metabolically engineered Methylomicrobium alcaliphilum 20Z for 2,3-butanediol (2,3-BDO) production from methane. The engineered strain 20Z/pBudK.p, harboring the 2,3-BDO synthesis gene cluster (budABC) from Klebsiella pneumoniae, accumulated 2,3-BDO in methane-fed shake flask cultures with a titer of 35.66 mg/L. Expression of the most efficient gene cluster was optimized using selection of promoters, translation initiation rates (TIR), and the combination of 2,3-BDO synthesis genes from different sources. A higher 2,3-BDO titer of 57.7 mg/L was measured in the 20Z/pNBM-Re strain with budA of K. pneumoniae and budB of Bacillus subtilis under the control of the Tac promoter. The genome-scale metabolic network reconstruction of M. alcaliphilum 20Z enabled in silico gene knockout predictions using an evolutionary programming method to couple growth and 2,3-BDO production. The ldh, ack, and mdh genes in M. alcaliphilum 20Z were identified as potential knockout targets. Pursuing these targets, a triple-mutant strain ∆ldh ∆ack ∆mdh was constructed, resulting in a further increase of the 2,3-BDO titer to 68.8 mg/L. The productivity of this optimized strain was then tested in a fed-batch stirred tank bioreactor, where final product concentrations of up to 86.2 mg/L with a yield of 0.0318 g-(2,3-BDO) /g-CH 4 were obtained under O 2 -limited conditions. This study first demonstrates the strategy of in silico simulation-guided metabolic engineering and represents a proof-of-concept for the production of value-added compounds using systematic approaches from engineered methanotrophs. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Natural and modified promoters for tailored metabolic engineering of the yeast Saccharomyces cerevisiae

NARCIS (Netherlands)

Hubmann, Georg; Thevelein, Johan M; Nevoigt, Elke

2014-01-01

The ease of highly sophisticated genetic manipulations in the yeast Saccharomyces cerevisiae has initiated numerous initiatives towards development of metabolically engineered strains for novel applications beyond its traditional use in brewing, baking, and wine making. In fact, baker's yeast has

Metabolic and process engineering for biodesulfurization in Gram-negative bacteria.

Science.gov (United States)

Martínez, I; El-Said Mohamed, M; Santos, V E; García, J L; García-Ochoa, F; Díaz, E

2017-11-20

Microbial desulfurization or biodesulfurization (BDS) is an attractive low-cost and environmentally friendly complementary technology to the hydrotreating chemical process based on the potential of certain bacteria to specifically remove sulfur from S-heterocyclic compounds of crude fuels that are recalcitrant to the chemical treatments. The 4S or Dsz sulfur specific pathway for dibenzothiophene (DBT) and alkyl-substituted DBTs, widely used as model S-heterocyclic compounds, has been extensively studied at the physiological, biochemical and genetic levels mainly in Gram-positive bacteria. Nevertheless, several Gram-negative bacteria have been also used in BDS because they are endowed with some properties, e.g., broad metabolic versatility and easy genetic and genomic manipulation, that make them suitable chassis for systems metabolic engineering strategies. A high number of recombinant bacteria, many of which are Pseudomonas strains, have been constructed to overcome the major bottlenecks of the desulfurization process, i.e., expression of the dsz operon, activity of the Dsz enzymes, retro-inhibition of the Dsz pathway, availability of reducing power, uptake-secretion of substrate and intermediates, tolerance to organic solvents and metals, and other host-specific limitations. However, to attain a BDS process with industrial applicability, it is necessary to apply all the knowledge and advances achieved at the genetic and metabolic levels to the process engineering level, i.e., kinetic modelling, scale-up of biphasic systems, enhancing mass transfer rates, biocatalyst separation, etc. The production of high-added value products derived from the organosulfur material present in oil can be regarded also as an economically viable process that has barely begun to be explored. Copyright © 2017 Elsevier B.V. All rights reserved.

Expanding beyond canonical metabolism: Interfacing alternative elements, synthetic biology, and metabolic engineering

Directory of Open Access Journals (Sweden)

Kevin B. Reed

2018-03-01

Full Text Available Metabolic engineering offers an exquisite capacity to produce new molecules in a renewable manner. However, most industrial applications have focused on only a small subset of elements from the periodic table, centered around carbon biochemistry. This review aims to illustrate the expanse of chemical elements that can currently (and potentially be integrated into useful products using cellular systems. Specifically, we describe recent advances in expanding the cellular scope to include the halogens, selenium and the metalloids, and a variety of metal incorporations. These examples range from small molecules, heteroatom-linked uncommon elements, and natural products to biomining and nanotechnology applications. Collectively, this review covers the promise of an expanded range of elemental incorporations and the future impacts it may have on biotechnology.

Metabolic interpretation of ventilatory parameters during maximal effort test and their applicability to sports

Directory of Open Access Journals (Sweden)

Luis Eduardo Barreto Martins

2007-09-01

Full Text Available One important tool for producing specifi c and individualized training intensities is to determine ventilatory threshold (VT, respiratory compensation point (RCP and maximal oxygen uptake (VO2max by means of maximum effort testing. However, in order to be able to interpret these data in a wide-ranging manner, it is also important to understand the metabolic responses that occur during the test as the systems transporting and utilizing O2 and producing CO2 adjust. This review article presents an overview of the metabolic responses that take place during a hypothetical maximum effort test, and the applicability of the fi gures thus obtained to the training of athletes. ABSTRACT A determinação das velocidades atingidas no limiar ventilatório (LV, ponto de compensação respiratório (PCR e consumo máximo de O2 (VO2max através de um teste de esforço máximo, é uma ferramenta importante para a aplicação de intensidades de treinamento específicas e individualizadas. Mas para poder interpretar os dados de uma forma abrangente, também é importante o entendimento das respostas metabólicas presentes no ajuste dos sistemas de transporte e utilização de O2 e produção de CO2 durante a realização do teste. Esta revisão apresenta um panorama das respostas metabólicas que acontecem durante a realização de um teste de esforço máximo hipotético, e a aplicabilidade dos valores obtidos no treinamento de atletas.

OptFlux: an open-source software platform for in silico metabolic engineering.

Science.gov (United States)

Rocha, Isabel; Maia, Paulo; Evangelista, Pedro; Vilaça, Paulo; Soares, Simão; Pinto, José P; Nielsen, Jens; Patil, Kiran R; Ferreira, Eugénio C; Rocha, Miguel

2010-04-19

Over the last few years a number of methods have been proposed for the phenotype simulation of microorganisms under different environmental and genetic conditions. These have been used as the basis to support the discovery of successful genetic modifications of the microbial metabolism to address industrial goals. However, the use of these methods has been restricted to bioinformaticians or other expert researchers. The main aim of this work is, therefore, to provide a user-friendly computational tool for Metabolic Engineering applications. OptFlux is an open-source and modular software aimed at being the reference computational application in the field. It is the first tool to incorporate strain optimization tasks, i.e., the identification of Metabolic Engineering targets, using Evolutionary Algorithms/Simulated Annealing metaheuristics or the previously proposed OptKnock algorithm. It also allows the use of stoichiometric metabolic models for (i) phenotype simulation of both wild-type and mutant organisms, using the methods of Flux Balance Analysis, Minimization of Metabolic Adjustment or Regulatory on/off Minimization of Metabolic flux changes, (ii) Metabolic Flux Analysis, computing the admissible flux space given a set of measured fluxes, and (iii) pathway analysis through the calculation of Elementary Flux Modes. OptFlux also contemplates several methods for model simplification and other pre-processing operations aimed at reducing the search space for optimization algorithms. The software supports importing/exporting to several flat file formats and it is compatible with the SBML standard. OptFlux has a visualization module that allows the analysis of the model structure that is compatible with the layout information of Cell Designer, allowing the superimposition of simulation results with the model graph. The OptFlux software is freely available, together with documentation and other resources, thus bridging the gap from research in strain optimization

Extremely Thermophilic Microorganisms as Metabolic Engineering Platforms for Production of Fuels and Industrial Chemicals

Directory of Open Access Journals (Sweden)

Benjamin M Zeldes

2015-11-01

Full Text Available Enzymes from extremely thermophilic microorganisms have been of technological interest for some time because of their ability to catalyze reactions of industrial significance at elevated temperatures. Thermophilic enzymes are now routinely produced in recombinant mesophilic hosts for use as discrete biocatalysts. Genome and metagenome sequence data for extreme thermophiles provide useful information for putative biocatalysts for a wide range of biotransformations, albeit involving at most a few enzymatic steps. However, in the past several years, unprecedented progress has been made in establishing molecular genetics tools for extreme thermophiles to the point that the use of these microorganisms as metabolic engineering platforms has become possible. While in its early days, complex metabolic pathways have been altered or engineered into recombinant extreme thermophiles, such that the production of fuels and chemicals at elevated temperatures has become possible. Not only does this expand the thermal range for industrial biotechnology, it also potentially provides biodiverse options for specific biotransformations unique to these microorganisms. The list of extreme thermophiles growing optimally between 70 and 100°C with genetic toolkits currently available includes archaea and bacteria, aerobes and anaerobes, coming from genera such as Caldicellulosiruptor, Sulfolobus, Thermotoga, Thermococcus and Pyrococcus. These organisms exhibit unusual and potentially useful native metabolic capabilities, including cellulose degradation, metal solubilization, and RuBisCO-free carbon fixation. Those looking to design a thermal bioprocess now have a host of potential candidates to choose from, each with its own advantages and challenges that will influence its appropriateness for specific applications. Here, the issues and opportunities for extremely thermophilic metabolic engineering platforms are considered with an eye towards potential technological

Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals

Science.gov (United States)

Zeldes, Benjamin M.; Keller, Matthew W.; Loder, Andrew J.; Straub, Christopher T.; Adams, Michael W. W.; Kelly, Robert M.

2015-01-01

Enzymes from extremely thermophilic microorganisms have been of technological interest for some time because of their ability to catalyze reactions of industrial significance at elevated temperatures. Thermophilic enzymes are now routinely produced in recombinant mesophilic hosts for use as discrete biocatalysts. Genome and metagenome sequence data for extreme thermophiles provide useful information for putative biocatalysts for a wide range of biotransformations, albeit involving at most a few enzymatic steps. However, in the past several years, unprecedented progress has been made in establishing molecular genetics tools for extreme thermophiles to the point that the use of these microorganisms as metabolic engineering platforms has become possible. While in its early days, complex metabolic pathways have been altered or engineered into recombinant extreme thermophiles, such that the production of fuels and chemicals at elevated temperatures has become possible. Not only does this expand the thermal range for industrial biotechnology, it also potentially provides biodiverse options for specific biotransformations unique to these microorganisms. The list of extreme thermophiles growing optimally between 70 and 100°C with genetic toolkits currently available includes archaea and bacteria, aerobes and anaerobes, coming from genera such as Caldicellulosiruptor, Sulfolobus, Thermotoga, Thermococcus, and Pyrococcus. These organisms exhibit unusual and potentially useful native metabolic capabilities, including cellulose degradation, metal solubilization, and RuBisCO-free carbon fixation. Those looking to design a thermal bioprocess now have a host of potential candidates to choose from, each with its own advantages and challenges that will influence its appropriateness for specific applications. Here, the issues and opportunities for extremely thermophilic metabolic engineering platforms are considered with an eye toward potential technological advantages for high

Application of a controllable degron strategy for metabolic engineering

DEFF Research Database (Denmark)

Knuf, Christoph; Maury, Jerome; Jacobsen, Simo Abdessamad

2014-01-01

In numerous cases of metabolic engineering, metabolite pools have to be increased in order to obtain flux into heterologous pathways. A simple tool for this would be the deletion of genes that would practically lead to a block of the natural pathway, so that the carbon can flow into the heterolog...... of intermediates of the mevalonate pathway around 2,3-oxidosqualene, which is the precursor for triterpenoids. Many triterpenoids are pharmaceutically relevant compounds which nowadays need to be extracted from plant material through an intricate and resource consuming process....

Validation of RetroPath, a computer-aided design tool for metabolic pathway engineering.

Science.gov (United States)

Fehér, Tamás; Planson, Anne-Gaëlle; Carbonell, Pablo; Fernández-Castané, Alfred; Grigoras, Ioana; Dariy, Ekaterina; Perret, Alain; Faulon, Jean-Loup

2014-11-01

Metabolic engineering has succeeded in biosynthesis of numerous commodity or high value compounds. However, the choice of pathways and enzymes used for production was many times made ad hoc, or required expert knowledge of the specific biochemical reactions. In order to rationalize the process of engineering producer strains, we developed the computer-aided design (CAD) tool RetroPath that explores and enumerates metabolic pathways connecting the endogenous metabolites of a chassis cell to the target compound. To experimentally validate our tool, we constructed 12 top-ranked enzyme combinations producing the flavonoid pinocembrin, four of which displayed significant yields. Namely, our tool queried the enzymes found in metabolic databases based on their annotated and predicted activities. Next, it ranked pathways based on the predicted efficiency of the available enzymes, the toxicity of the intermediate metabolites and the calculated maximum product flux. To implement the top-ranking pathway, our procedure narrowed down a list of nine million possible enzyme combinations to 12, a number easily assembled and tested. One round of metabolic network optimization based on RetroPath output further increased pinocembrin titers 17-fold. In total, 12 out of the 13 enzymes tested in this work displayed a relative performance that was in accordance with its predicted score. These results validate the ranking function of our CAD tool, and open the way to its utilization in the biosynthesis of novel compounds. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production.

Science.gov (United States)

Alonso-Gutierrez, Jorge; Chan, Rossana; Batth, Tanveer S; Adams, Paul D; Keasling, Jay D; Petzold, Christopher J; Lee, Taek Soon

2013-09-01

Limonene is a valuable monoterpene used in the production of several commodity chemicals and medicinal compounds. Among them, perillyl alcohol (POH) is a promising anti-cancer agent that can be produced by hydroxylation of limonene. We engineered E. coli with a heterologous mevalonate pathway and limonene synthase for production of limonene followed by coupling with a cytochrome P450, which specifically hydroxylates limonene to produce POH. A strain containing all mevalonate pathway genes in a single plasmid produced limonene at titers over 400mg/L from glucose, substantially higher than has been achieved in the past. Incorporation of a cytochrome P450 to hydroxylate limonene yielded approximately 100mg/L of POH. Further metabolic engineering of the pathway and in situ product recovery using anion exchange resins would make this engineered E. coli a potential production platform for any valuable limonene derivative. © 2013 Elsevier Inc. All rights reserved.

Integrating biocompatible chemistry and manipulating cofactor partitioning in metabolically engineeredLactococcus lactisfor fermentative production of (3S)-acetoin

DEFF Research Database (Denmark)

Liu, Jianming; Solem, Christian; Jensen, Peter Ruhdal

2016-01-01

Biocompatible chemistry (BC), i.e. non-enzymatic chemical reactions compatible with living organisms, is increasingly used in conjunction with metabolically engineered microorganisms for producing compounds that do not usually occur naturally. Here we report production of one such compound, (3S......)-acetoin, a valuable precursor for chiral synthesis, using a metabolically engineered Lactococcus lactis strain growing under respiratory conditions with ferric iron serving as a BC component. The strain used has all competing product pathways inactivated, and an appropriate cofactor balance is achieved by fine...

Metabolic cartography: experimental quantification of metabolic fluxes from isotopic labelling studies.

Science.gov (United States)

O'Grady, John; Schwender, Jörg; Shachar-Hill, Yair; Morgan, John A

2012-03-01

For the past decade, flux maps have provided researchers with an in-depth perspective on plant metabolism. As a rapidly developing field, significant headway has been made recently in computation, experimentation, and overall understanding of metabolic flux analysis. These advances are particularly applicable to the study of plant metabolism. New dynamic computational methods such as non-stationary metabolic flux analysis are finding their place in the toolbox of metabolic engineering, allowing more organisms to be studied and decreasing the time necessary for experimentation, thereby opening new avenues by which to explore the vast diversity of plant metabolism. Also, improved methods of metabolite detection and measurement have been developed, enabling increasingly greater resolution of flux measurements and the analysis of a greater number of the multitude of plant metabolic pathways. Methods to deconvolute organelle-specific metabolism are employed with increasing effectiveness, elucidating the compartmental specificity inherent in plant metabolism. Advances in metabolite measurements have also enabled new types of experiments, such as the calculation of metabolic fluxes based on (13)CO(2) dynamic labelling data, and will continue to direct plant metabolic engineering. Newly calculated metabolic flux maps reveal surprising and useful information about plant metabolism, guiding future genetic engineering of crops to higher yields. Due to the significant level of complexity in plants, these methods in combination with other systems biology measurements are necessary to guide plant metabolic engineering in the future.

Design of an ectoine-responsive AraC mutant and its application in metabolic engineering of ectoine biosynthesis.

Science.gov (United States)

Chen, Wei; Zhang, Shan; Jiang, Peixia; Yao, Jun; He, Yongzhi; Chen, Lincai; Gui, Xiwu; Dong, Zhiyang; Tang, Shuang-Yan

2015-07-01

Advanced high-throughput screening methods for small molecules may have important applications in the metabolic engineering of the biosynthetic pathways of these molecules. Ectoine is an excellent osmoprotectant that has been widely used in cosmetics. In this study, the Escherichia coli regulatory protein AraC was engineered to recognize ectoine as its non-natural effector and to activate transcription upon ectoine binding. As an endogenous reporter of ectoine, the mutated AraC protein was successfully incorporated into high-throughput screening of ectoine hyper-producing strains. The ectoine biosynthetic cluster from Halomonas elongata was cloned into E. coli. By engineering the rate-limiting enzyme L-2,4-diaminobutyric acid (DABA) aminotransferase (EctB), ectoine production and the specific activity of the EctB mutant were increased. Thus, these results demonstrated the effectiveness of engineering regulatory proteins into sensitive and rapid screening tools for small molecules and highlighted the importance and efficacy of directed evolution strategies applied to the engineering of genetic components for yield improvement in the biosynthesis of small molecules. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Metabolic engineering of riboflavin production in Ashbya gossypii through pathway optimization.

Science.gov (United States)

Ledesma-Amaro, Rodrigo; Serrano-Amatriain, Cristina; Jiménez, Alberto; Revuelta, José Luis

2015-10-14

The industrial production of riboflavin mostly relies on the microbial fermentation of flavinogenic microorganisms and Ashbya gossypii is the main industrial producer of the vitamin. Accordingly, bioengineering strategies aimed at increasing riboflavin production in A. gossypii are highly valuable for industry. We analyze the contribution of all the RIB genes to the production of riboflavin in A. gossypii. Two important metabolic rate-limiting steps that limit the overproduction of riboflavin have been found: first, low mRNA levels of the RIB genes hindered the overproduction of riboflavin; second, the competition of the AMP branch for purinogenic precursors also represents a limitation for riboflavin overproduction. Thus, overexpression of the RIB genes resulted in a significant increase in riboflavin yield. Moreover, both the inactivation and the underexpression of the ADE12 gene, which controls the first step of the AMP branch, also proved to have a positive effect on riboflavin production. Accordingly, a strain that combines both the overexpression of the RIB genes and the underexpression of the ADE12 gene was engineered. This strain produced 523 mg/L of riboflavin (5.4-fold higher than the wild-type), which is the highest titer of riboflavin obtained by metabolic engineering in A. gossypii so far. Riboflavin production in A. gossypii is limited by a low transcription activity of the RIB genes. Flux limitation towards AMP provides committed substrate GTP for riboflavin overproduction without detrimental effects on biomass formation. A multiple-engineered Ashbya strain that produces up to 523 mg/L of riboflavin was generated.

Biobased organic acids production by metabolically engineered microorganisms

DEFF Research Database (Denmark)

Chen, Yun; Nielsen, Jens

2016-01-01

Bio-based production of organic acids via microbial fermentation has been traditionally used in food industry. With the recent desire to develop more sustainable bioprocesses for production of fuels, chemicals and materials, the market for microbial production of organic acids has been further...... expanded as organic acids constitute a key group among top building block chemicals that can be produced from renewable resources. Here we review the current status for production of citric acid and lactic acid, and we highlight the use of modern metabolic engineering technologies to develop high...... performance microbes for production of succinic acid and 3-hydroxypropionic acid. Also, the key limitations and challenges in microbial organic acids production are discussed...

Expanding the chemical palate of cells by combining systems biology and metabolic engineering.

Science.gov (United States)

Curran, Kathleen A; Alper, Hal S

2012-07-01

The field of Metabolic Engineering has recently undergone a transformation that has led to a rapid expansion of the chemical palate of cells. Now, it is conceivable to produce nearly any organic molecule of interest using a cellular host. Significant advances have been made in the production of biofuels, biopolymers and precursors, pharmaceuticals and nutraceuticals, and commodity and specialty chemicals. Much of this rapid expansion in the field has been, in part, due to synergies and advances in the area of systems biology. Specifically, the availability of functional genomics, metabolomics and transcriptomics data has resulted in the potential to produce a wealth of new products, both natural and non-natural, in cellular factories. The sheer amount and diversity of this data however, means that uncovering and unlocking novel chemistries and insights is a non-obvious exercise. To address this issue, a number of computational tools and experimental approaches have been developed to help expedite the design process to create new cellular factories. This review will highlight many of the systems biology enabling technologies that have reduced the design cycle for engineered hosts, highlight major advances in the expanded diversity of products that can be synthesized, and conclude with future prospects in the field of metabolic engineering. Copyright © 2012 Elsevier Inc. All rights reserved.

Improving production of ?-lactam antibiotics by Penicillium chrysogenum : Metabolic engineering based on transcriptome analysis

NARCIS (Netherlands)

Veiga, T.

2012-01-01

In Chapters 2-5 of this thesis, the applicability of transcriptome analysis to guide metabolic engineering strategies in P. chrysogenum is explored by investigating four cellular processes that are of potential relevance for industrial production of ?-lactam antibiotics: - Regulation of secondary

Spatial separation of photosynthesis and ethanol production by cell type-specific metabolic engineering of filamentous cyanobacteria.

Science.gov (United States)

Ehira, Shigeki; Takeuchi, Takuto; Higo, Akiyoshi

2018-02-01

Cyanobacteria, which perform oxygenic photosynthesis, have drawn attention as hosts for the direct production of biofuels and commodity chemicals from CO 2 and H 2 O using light energy. Although cyanobacteria capable of producing diverse chemicals have been generated by metabolic engineering, anaerobic non-photosynthetic culture conditions are often necessary for their production. In this study, we conducted cell type-specific metabolic engineering of the filamentous cyanobacterium Anabaena sp. PCC 7120, which forms a terminally differentiated cell called a heterocyst with a semi-regular spacing of 10-15 cells. Because heterocysts are specialized cells for nitrogen fixation, the intracellular oxygen level of heterocysts is maintained very low even when adjacent cells perform oxygenic photosynthesis. Pyruvate decarboxylase of Zymomonas mobilis and alcohol dehydrogenase of Synechocystis sp. PCC 6803 were exclusively expressed in heterocysts. Ethanol production was concomitant with nitrogen fixation in genetically engineered Anabaena sp. PCC 7120. Engineering of carbon metabolism in heterocysts improved ethanol production, and strain ET14, with an extra copy of the invB gene expressed from a heterocyst-specific promoter, produced 130.9 mg L -1 of ethanol after 9 days. ET14 produced 1681.9 mg L -1 of ethanol by increasing the CO 2 supply. Ethanol production per heterocyst cell was approximately threefold higher than that per cell of unicellular cyanobacterium. This study demonstrates the potential of heterocysts for anaerobic production of biofuels and commodity chemicals under oxygenic photosynthetic conditions.

Recent advances in the metabolic engineering of Corynebacterium glutamicum for the production of lactate and succinate from renewable resources.

Science.gov (United States)

Tsuge, Yota; Hasunuma, Tomohisa; Kondo, Akihiko

2015-03-01

Recent increasing attention to environmental issues and the shortage of oil resources have spurred political and industrial interest in the development of environmental friendly and cost-effective processes for the production of bio-based chemicals from renewable resources. Thus, microbial production of commercially important chemicals is viewed as a desirable way to replace current petrochemical production. Corynebacterium glutamicum, a Gram-positive soil bacterium, is one of the most important industrial microorganisms as a platform for the production of various amino acids. Recent research has explored the use of C. glutamicum as a potential cell factory for producing organic acids such as lactate and succinate, both of which are commercially important bulk chemicals. Here, we summarize current understanding in this field and recent metabolic engineering efforts to develop C. glutamicum strains that efficiently produce L- and D-lactate, and succinate from renewable resources.

Computational metabolic engineering strategies for growth-coupled biofuel production by Synechocystis

Directory of Open Access Journals (Sweden)

Kiyan Shabestary

2016-12-01

Full Text Available Chemical and fuel production by photosynthetic cyanobacteria is a promising technology but to date has not reached competitive rates and titers. Genome-scale metabolic modeling can reveal limitations in cyanobacteria metabolism and guide genetic engineering strategies to increase chemical production. Here, we used constraint-based modeling and optimization algorithms on a genome-scale model of Synechocystis PCC6803 to find ways to improve productivity of fermentative, fatty-acid, and terpene-derived fuels. OptGene and MOMA were used to find heuristics for knockout strategies that could increase biofuel productivity. OptKnock was used to find a set of knockouts that led to coupling between biofuel and growth. Our results show that high productivity of fermentation or reversed beta-oxidation derived alcohols such as 1-butanol requires elimination of NADH sinks, while terpenes and fatty-acid based fuels require creating imbalances in intracellular ATP and NADPH production and consumption. The FBA-predicted productivities of these fuels are at least 10-fold higher than those reported so far in the literature. We also discuss the physiological and practical feasibility of implementing these knockouts. This work gives insight into how cyanobacteria could be engineered to reach competitive biofuel productivities. Keywords: Cyanobacteria, Modeling, Flux balance analysis, Biofuel, MOMA, OptFlux, OptKnock

Engineering oilseeds for sustainable production of industrial and nutritional feedstocks: solving bottlenecks in fatty acid flux.

Science.gov (United States)

Cahoon, Edgar B; Shockey, Jay M; Dietrich, Charles R; Gidda, Satinder K; Mullen, Robert T; Dyer, John M

2007-06-01

Oilseeds provide a unique platform for the production of high-value fatty acids that can replace non-sustainable petroleum and oceanic sources of specialty chemicals and aquaculture feed. However, recent efforts to engineer the seeds of crop and model plant species to produce new types of fatty acids, including hydroxy and conjugated fatty acids for industrial uses and long-chain omega-3 polyunsaturated fatty acids for farmed fish feed, have met with only modest success. The collective results from these studies point to metabolic 'bottlenecks' in the engineered plant seeds that substantially limit the efficient or selective flux of unusual fatty acids between different substrate pools and ultimately into storage triacylglycerol. Evidence is emerging that diacylglycerol acyltransferase 2, which catalyzes the final step in triacylglycerol assembly, is an important contributor to the synthesis of unusual fatty acid-containing oils, and is likely to be a key target for future oilseed metabolic engineering efforts.

Engineering fatty acid biosynthesis in microalgae for sustainable biodiesel.

Science.gov (United States)

Blatti, Jillian L; Michaud, Jennifer; Burkart, Michael D

2013-06-01

Microalgae are a promising feedstock for biodiesel and other liquid fuels due to their fast growth rate, high lipid yields, and ability to grow in a broad range of environments. However, many microalgae achieve maximal lipid yields only under stress conditions hindering growth and providing compositions not ideal for biofuel applications. Metabolic engineering of algal fatty acid biosynthesis promises to create strains capable of economically producing fungible and sustainable biofuels. The algal fatty acid biosynthetic pathway has been deduced by homology to bacterial and plant systems, and much of our understanding is gleaned from basic studies in these systems. However, successful engineering of lipid metabolism in algae will necessitate a thorough characterization of the algal fatty acid synthase (FAS) including protein-protein interactions and regulation. This review describes recent efforts to engineer fatty acid biosynthesis toward optimizing microalgae as a biodiesel feedstock. Copyright © 2013 Elsevier Ltd. All rights reserved.

Construction of expression vectors for metabolic engineering of the vanillin-producing actinomycete Amycolatopsis sp. ATCC 39116.

Science.gov (United States)

Fleige, Christian; Steinbüchel, Alexander

2014-01-01

Amycolatopsis sp. ATCC 39116 is able to synthesize the important flavoring agent vanillin from cheap natural substrates. The bacterium is therefore of great interest for the industry and used for the fermentative production of vanillin. In order to improve the production of natural vanillin with Amycolatopsis sp. ATCC 39116, the strain has been genetically engineered to optimize the metabolic flux towards the desired product. Extensive metabolic engineering was hitherto hampered, due to the lack of genetic tools like functional promoters and expression vectors. In this study, we report the establishment of a plasmid-based gene expression system for Amycolatopsis sp. ATCC 39116 that allows a further manipulation of the genotype. Four new Escherichia coli-Amycolatopsis shuttle vectors harboring different promoter elements were constructed, and the functionality of these regulatory elements was proven by the expression of the reporter gene gusA, encoding a β-glucuronidase. Glucuronidase activity was detected in all plasmid-harboring strains, and remarkable differences in the expression strength of the reporter gene depending on the used promoter were observed. The new expression vectors will promote the further genetic engineering of Amycolatopsis sp. ATCC 39116 to get insight into the metabolic network and to improve the strain for a more efficient industrial use.

Engineering 1-Alkene Biosynthesis and Secretion by Dynamic Regulation in Yeast

DEFF Research Database (Denmark)

Zhou, Yongjin J.; Hu, Yating; Zhu, Zhiwei

2018-01-01

strategy to control the expression of membrane enzyme and 1-alkene production and cell growth by relieving the possible toxicity of overexpressed membrane proteins. With these efforts, the engineered yeast cell factory produced 35.3 mg/L 1-alkenes with more than 80% being secreted. This represents a 10...... product secretion. Here, we engineered the budding yeast Saccharomyces cerevisiae to produce and secrete 1-alkenes by manipulation of the fatty acid metabolism, enzyme selection, engineering the electron transfer system and expressing a transporter. Furthermore, we implemented a dynamic regulation...

[Advances in metabolic engineering for the microbial production of naturally occurring terpenes-limonene and bisabolene: a mini review].

Science.gov (United States)

Pang, Yaru; Hu, Zhihui; Xiao, Dongguang; Yu, Aiqun

2018-01-25

Limonene (C₁₀H₁₆) and bisabolene (C₁₅H₂₄) are both naturally occurring terpenes in plants. Depending on the number of C₅ units, limonene and bisabolene are recognized as representative monoterpenes and sesquiterpenes, respectively. Limonene and bisabolene are important pharmaceutical and nutraceutical products used in the prevention and treatment of cancer and many other diseases. In addition, they can be used as starting materials to produce a range of commercially valuable products, such as pharmaceuticals, nutraceuticals, cosmetics, and biofuels. The low abundance or yield of limonene and bisabolene in plants renders their isolation from plant sources non-economically viable. Isolation of limonene and bisabolene from plants also suffers from low efficiency and often requires harsh reaction conditions, prolonged reaction times, and expensive equipment cost. Recently, the rapid developments in metabolic engineering of microbes provide a promising alternative route for producing these plant natural products. Therefore, producing limonene and bisabolene by engineering microbial cells into microbial factories is becoming an attractive alternative approach that can overcome the bottlenecks, making it more sustainable, environmentally friendly and economically competitive. Here, we reviewed the status of metabolic engineering of microbes that produce limonene and bisabolene including microbial hosts, key enzymes, metabolic pathways and engineering of limonene/bisabolene biosynthesis. Furthermore, key challenges and future perspectives were discussed.

Synthetic biology for engineering acetyl coenzyme a metabolism in yeast

DEFF Research Database (Denmark)

Nielsen, Jens

2014-01-01

The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels, chemicals, and pharmaceuticals. The use of this cell factory for cost-efficient production of novel fuels and chemicals requires high yields and low by-product production. Many industrially interesting...... chemicals are biosynthesized from acetyl coenzyme A (acetyl-CoA), which serves as a central precursor metabolite in yeast. To ensure high yields in production of these chemicals, it is necessary to engineer the central carbon metabolism so that ethanol production is minimized (or eliminated) and acetyl...

Engineering microbial electrocatalysis for chemical and fuel production.

Science.gov (United States)

Rosenbaum, Miriam A; Henrich, Alexander W

2014-10-01

In many biotechnological areas, metabolic engineering and synthetic biology have become core technologies for biocatalyst development. Microbial electrocatalysis for biochemical and fuel production is still in its infancy and reactions rates and the product spectrum are currently very low. Therefore, molecular engineering strategies will be crucial for the advancement and realization of many new bioproduction routes using electroactive microorganisms. The complex and unresolved biochemistry and physiology of extracellular electron transfer and the lack of molecular tools for these new non-model hosts for genetic engineering constitute the major challenges for this effort. This review is providing an insight into the current status, challenges and promising approaches of pathway engineering for microbial electrocatalysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Engineering yeast metabolism for production of terpenoids for use as perfume ingredients, pharmaceuticals and biofuels.

Science.gov (United States)

Zhang, Yueping; Nielsen, Jens; Liu, Zihe

2017-12-01

Terpenoids represent a large class of natural products with significant commercial applications. These chemicals are currently mainly obtained through extraction from plants and microbes or through chemical synthesis. However, these sources often face challenges of unsustainability and low productivity. In order to address these issues, Escherichia coli and yeast have been metabolic engineered to produce non-native terpenoids. With recent reports of engineering yeast metabolism to produce several terpenoids at high yields, it has become possible to establish commercial yeast production of terpenoids that find applications as perfume ingredients, pharmaceuticals and advanced biofuels. In this review, we describe the strategies to rewire the yeast pathway for terpenoid biosynthesis. Recent advances will be discussed together with challenges and perspectives of yeast as a cell factory to produce different terpenoids. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Advances in metabolic engineering of yeast Saccharomyces cerevisiae for production of chemicals.

Science.gov (United States)

Borodina, Irina; Nielsen, Jens

2014-05-01

Yeast Saccharomyces cerevisiae is an important industrial host for production of enzymes, pharmaceutical and nutraceutical ingredients and recently also commodity chemicals and biofuels. Here, we review the advances in modeling and synthetic biology tools and how these tools can speed up the development of yeast cell factories. We also present an overview of metabolic engineering strategies for developing yeast strains for production of polymer monomers: lactic, succinic, and cis,cis-muconic acids. S. cerevisiae has already firmly established itself as a cell factory in industrial biotechnology and the advances in yeast strain engineering will stimulate development of novel yeast-based processes for chemicals production. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enabling tools for high-throughput detection of metabolites: Metabolic engineering and directed evolution applications.

Science.gov (United States)

Lin, Jyun-Liang; Wagner, James M; Alper, Hal S

2017-12-01

Within the Design-Build-Test Cycle for strain engineering, rapid product detection and selection strategies remain challenging and limit overall throughput. Here we summarize a wide variety of modalities that transduce chemical concentrations into easily measured absorbance, luminescence, and fluorescence signals. Specifically, we cover protein-based biosensors (including transcription factors), nucleic acid-based biosensors, coupled enzyme reactions, bioorthogonal chemistry, and fluorescent and chromogenic dyes and substrates as modalities for detection. We focus on the use of these methods for strain engineering and enzyme discovery and conclude with remarks on the current and future state of biosensor development for application in the metabolic engineering field. Copyright © 2017 Elsevier Inc. All rights reserved.

Metabolic engineering of carbon overflow metabolism of Bacillus subtilis for improved N-acetyl-glucosamine production.

Science.gov (United States)

Ma, Wenlong; Liu, Yanfeng; Shin, Hyun-Dong; Li, Jianghua; Chen, Jian; Du, Guocheng; Liu, Long

2018-02-01

Bacillus subtilis is widely used as cell factories for the production of important industrial biochemicals. Although many studies have demonstrated the effects of organic acidic byproducts, such as acetate, on microbial fermentation, little is known about the effects of blocking the neutral byproduct overflow, such as acetoin, on bioproduction. In this study, we focused on the influences of modulating overflow metabolism on the production of N-acetyl-d-glucosamine (GlcNAc) in engineered B. subtilis. We found that acetoin overflow competes with GlcNAc production, and blocking acetoin overflow increased GlcNAc titer and yield by 1.38- and 1.39-fold, reaching 48.9 g/L and 0.32 g GlcNAc/g glucose, respectively. Further blocking acetate overflow inhibited cell growth and GlcNAc production may be induced by inhibiting glucose uptake. Taken together, our results show that blocking acetoin overflow is a promising strategy for enhancing GlcNAc production. The strategies developed in this work may be useful for engineering strains of B. subtilis for producing other important biochemicals. Copyright © 2017. Published by Elsevier Ltd.

Promiscuous activities of heterologous enzymes lead to unintended metabolic rerouting in Saccharomyces cerevisiae engineered to assimilate various sugars from renewable biomass.

Science.gov (United States)

Yun, Eun Ju; Oh, Eun Joong; Liu, Jing-Jing; Yu, Sora; Kim, Dong Hyun; Kwak, Suryang; Kim, Kyoung Heon; Jin, Yong-Su

2018-01-01

Understanding the global metabolic network, significantly perturbed upon promiscuous activities of foreign enzymes and different carbon sources, is crucial for systematic optimization of metabolic engineering of yeast Saccharomyces cerevisiae . Here, we studied the effects of promiscuous activities of overexpressed enzymes encoded by foreign genes on rerouting of metabolic fluxes of an engineered yeast capable of assimilating sugars from renewable biomass by profiling intracellular and extracellular metabolites. Unbiased metabolite profiling of the engineered S. cerevisiae strain EJ4 revealed promiscuous enzymatic activities of xylose reductase and xylitol dehydrogenase on galactose and galactitol, respectively, resulting in accumulation of galactitol and tagatose during galactose fermentation. Moreover, during glucose fermentation, a trisaccharide consisting of glucose accumulated outside of the cells probably owing to the promiscuous and transglycosylation activity of β-glucosidase expressed for hydrolyzing cellobiose. Meanwhile, higher accumulation of fatty acids and secondary metabolites was observed during xylose and cellobiose fermentations, respectively. The heterologous enzymes functionally expressed in S. cerevisiae showed promiscuous activities that led to unintended metabolic rerouting in strain EJ4. Such metabolic rerouting could result in a low yield and productivity of a final product due to the formation of unexpected metabolites. Furthermore, the global metabolic network can be significantly regulated by carbon sources, thus yielding different patterns of metabolite production. This metabolomic study can provide useful information for yeast strain improvement and systematic optimization of yeast metabolism to manufacture bio-based products.

Flux Balance Analysis Inspired Bioprocess Upgrading for Lycopene Production by a Metabolically Engineered Strain of Yarrowia lipolytica

Directory of Open Access Journals (Sweden)

Komi Nambou

2015-12-01

Full Text Available Genome-scale metabolic models embody a significant advantage of systems biology since their applications as metabolic flux simulation models enable predictions for the production of industrially-interesting metabolites. The biotechnological production of lycopene from Yarrowia lipolytica is an emerging scope that has not been fully scrutinized, especially for what concerns cultivation conditions of newly generated engineered strains. In this study, by combining flux balance analysis (FBA and Plackett-Burman design, we screened chemicals for lycopene production from a metabolically engineered strain of Y. lipolytica. Lycopene concentrations of 126 and 242 mg/L were achieved correspondingly from the FBA-independent and the FBA-assisted designed media in fed-batch cultivation mode. Transcriptional studies revealed upregulations of heterologous genes in media designed according to FBA, thus implying the efficiency of model predictions. Our study will potentially support upgraded lycopene and other terpenoids production from existing or prospect bioengineered strains of Y. lipolytica and/or closely related yeast species.

Recent advances in engineering propionyl-CoA metabolism for microbial production of value-added chemicals and biofuels.

Science.gov (United States)

Srirangan, Kajan; Bruder, Mark; Akawi, Lamees; Miscevic, Dragan; Kilpatrick, Shane; Moo-Young, Murray; Chou, C Perry

2017-09-01

Diminishing fossil fuel reserves and mounting environmental concerns associated with petrochemical manufacturing practices have generated significant interests in developing whole-cell biocatalytic systems for the production of value-added chemicals and biofuels. Although acetyl-CoA is a common natural biogenic precursor for the biosynthesis of numerous metabolites, propionyl-CoA is unpopular and non-native to most organisms. Nevertheless, with its C3-acyl moiety as a discrete building block, propionyl-CoA can serve as another key biogenic precursor to several biological products of industrial importance. As a result, engineering propionyl-CoA metabolism, particularly in genetically tractable hosts with the use of inexpensive feedstocks, has paved an avenue for novel biomanufacturing. Herein, we present a systematic review on manipulation of propionyl-CoA metabolism as well as relevant genetic and metabolic engineering strategies for microbial production of value-added chemicals and biofuels, including odd-chain alcohols and organic acids, bio(co)polymers and polyketides. [Formula: see text].

Lessons learned from metabolic engineering of cyanogenic glucosides

DEFF Research Database (Denmark)

Morant, Anne Vinther; Jørgensen, Kirsten; Jørgensen, Bodil

2007-01-01

Plants produce a plethora of secondary metabolites which constitute a wealth of potential pharmaceuticals, pro-vitamins, flavours, fragrances, colorants and toxins as well as a source of natural pesticides. Many of these valuable compounds are only synthesized in exotic plant species or in concen......Plants produce a plethora of secondary metabolites which constitute a wealth of potential pharmaceuticals, pro-vitamins, flavours, fragrances, colorants and toxins as well as a source of natural pesticides. Many of these valuable compounds are only synthesized in exotic plant species...... or in concentrations too low to facilitate commercialization. In some cases their presence constitutes a health hazard and renders the crops unsuitable for consumption. Metabolic engineering is a powerful tool to alter and ameliorate the secondary metabolite composition of crop plants and gain new desired traits...

Terpene metabolic engineering via nuclear or chloroplast genomes profoundly and globally impacts off-target pathways through metabolite signalling.

Science.gov (United States)

Pasoreck, Elise K; Su, Jin; Silverman, Ian M; Gosai, Sager J; Gregory, Brian D; Yuan, Joshua S; Daniell, Henry

2016-09-01

The impact of metabolic engineering on nontarget pathways and outcomes of metabolic engineering from different genomes are poorly understood questions. Therefore, squalene biosynthesis genes FARNESYL DIPHOSPHATE SYNTHASE (FPS) and SQUALENE SYNTHASE (SQS) were engineered via the Nicotiana tabacum chloroplast (C), nuclear (N) or both (CN) genomes to promote squalene biosynthesis. SQS levels were ~4300-fold higher in C and CN lines than in N, but all accumulated ~150-fold higher squalene due to substrate or storage limitations. Abnormal leaf and flower phenotypes, including lower pollen production and reduced fertility, were observed regardless of the compartment or level of transgene expression. Substantial changes in metabolomes of all lines were observed: levels of 65-120 unrelated metabolites, including the toxic alkaloid nicotine, changed by as much as 32-fold. Profound effects of transgenesis on nontarget gene expression included changes in the abundance of 19 076 transcripts by up to 2000-fold in CN; 7784 transcripts by up to 1400-fold in N; and 5224 transcripts by as much as 2200-fold in C. Transporter-related transcripts were induced, and cell cycle-associated transcripts were disproportionally repressed in all three lines. Transcriptome changes were validated by qRT-PCR. The mechanism underlying these large changes likely involves metabolite-mediated anterograde and/or retrograde signalling irrespective of the level of transgene expression or end product, due to imbalance of metabolic pools, offering new insight into both anticipated and unanticipated consequences of metabolic engineering. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Patchoulol Production with Metabolically Engineered Corynebacterium glutamicum

Directory of Open Access Journals (Sweden)

Nadja A. Henke

2018-04-01

Full Text Available Patchoulol is a sesquiterpene alcohol and an important natural product for the perfume industry. Corynebacterium glutamicum is the prominent host for the fermentative production of amino acids with an average annual production volume of ~6 million tons. Due to its robustness and well established large-scale fermentation, C. glutamicum has been engineered for the production of a number of value-added compounds including terpenoids. Both C40 and C50 carotenoids, including the industrially relevant astaxanthin, and short-chain terpenes such as the sesquiterpene valencene can be produced with this organism. In this study, systematic metabolic engineering enabled construction of a patchoulol producing C. glutamicum strain by applying the following strategies: (i construction of a farnesyl pyrophosphate-producing platform strain by combining genomic deletions with heterologous expression of ispA from Escherichia coli; (ii prevention of carotenoid-like byproduct formation; (iii overproduction of limiting enzymes from the 2-c-methyl-d-erythritol 4-phosphate (MEP-pathway to increase precursor supply; and (iv heterologous expression of the plant patchoulol synthase gene PcPS from Pogostemon cablin. Additionally, a proof of principle liter-scale fermentation with a two-phase organic overlay-culture medium system for terpenoid capture was performed. To the best of our knowledge, the patchoulol titers demonstrated here are the highest reported to date with up to 60 mg L−1 and volumetric productivities of up to 18 mg L−1 d−1.

Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae▿

Science.gov (United States)

Tokuhiro, Kenro; Muramatsu, Masayoshi; Ohto, Chikara; Kawaguchi, Toshiya; Obata, Shusei; Muramoto, Nobuhiko; Hirai, Masana; Takahashi, Haruo; Kondo, Akihiko; Sakuradani, Eiji; Shimizu, Sakayu

2009-01-01

(E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter−1) rather than GGOH (0.2 mg liter−1) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter−1 GGOH and 6.5 mg liter−1 squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter−1 GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering. PMID:19592534

Biotechnology and synthetic biology approaches for metabolic engineering of bioenergy crops.

Science.gov (United States)

Shih, Patrick M; Liang, Yan; Loqué, Dominique

2016-07-01

The Green Revolution has fuelled an exponential growth in human population since the mid-20th century. Due to population growth, food and energy demands will soon surpass supply capabilities. To overcome these impending problems, significant improvements in genetic engineering will be needed to complement breeding efforts in order to accelerate the improvement of agronomical traits. The new field of plant synthetic biology has emerged in recent years and is expected to support rapid, precise, and robust engineering of plants. In this review, we present recent advances made in the field of plant synthetic biology, specifically in genome editing, transgene expression regulation, and bioenergy crop engineering, with a focus on traits related to lignocellulose, oil, and soluble sugars. Ultimately, progress and innovation in these fields may facilitate the development of beneficial traits in crop plants to meet society's bioenergy needs. © 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

Engineering synergy in biotechnology

DEFF Research Database (Denmark)

Nielsen, Jens; Fussenegger, Martin; Keasling, Jay

2014-01-01

In this article, the author focuses on approaches in metabolic engineering and synthetic biology for the creation of efficient cell factories, which can be bused to convert biomass and other feedstocks for the generation of chemicals. Topics discussed include development of restriction enzymes......, engineering plasmids and recyclable markers, production of 1,3-propanediol using a metabolically engineered Escherichia coli and production of isobutanol by using metabolically engineered yeast....

Metabolic engineering of deinococcus radiodurans based on computational analysis and functional genomics

Energy Technology Data Exchange (ETDEWEB)

Edwards, Jeremy, S.

2005-02-02

The objective of our work is to develop novel computational tools to analyze the Deinococcus radiodurans DNA repair pathways and the influence of the metabolic flux distribution on DNA repair. These tools will be applied to provide insights for metabolic engineering of strains capable of growing under nutrient poor conditions similar to those found in mixed contaminant sites of interest to the DOE. Over the entire grant period we accomplished all our specific aims and were also able to pursue new directions of research. Below, I will list the major accomplishments over the previous 3 years. (1) Performed Monte Carlo Simulations of RecA Mediated Pairing of Homologous DNA Molecules. (2) Developed a statistical approach to study the gene expression data from D. radiodurans. We have been studying the data from John Batista's. (3) Developed an expression profiling technology to generate very accurate and precise expression data. We followed up on results from John Batista's group using this approach. (4) Developed and put online a database for metabolic reconstructions. (5) We have developed and applied new Monte Carlo algorithms that are optimized for studying biological systems. (6) We developed a flux balance model for the D. radiodurans metabolic network

L-Cysteine Production in Escherichia coli Based on Rational Metabolic Engineering and Modular Strategy.

Science.gov (United States)

Liu, Han; Fang, Guochen; Wu, Hui; Li, Zhimin; Ye, Qin

2018-05-01

L-cysteine is an amino acid with important physiological functions and has a wide range of applications in medicine, food, animal feed, and cosmetics industry. In this study, the L-cysteine synthesis in Escherichia coliEscherichia coli is divided into four modules: the transport module, sulfur module, precursor module, and degradation module. The engineered strain LH03 (overexpression of the feedback-insensitive cysE and the exporter ydeD in JM109) accumulated 45.8 mg L -1 of L-cysteine in 48 hr with yield of 0.4% g/g glucose. Further modifications of strains and culture conditions which based on the rational metabolic engineering and modular strategy improved the L-cysteine biosynthesis significantly. The engineered strain LH06 (with additional overexpression of serA, serC, and serB and double mutant of tnaA and sdaA in LH03) produced 620.9 mg L -1 of L-cysteine with yield of 6.0% g/g glucose, which increased the production by 12 times and the yield by 14 times more than those of LH03 in the original condition. In fed-batch fermentation performed in a 5-L reactor, the concentration of L-cysteine achieved 5.1 g L -1 in 32 hr. This work demonstrates that the combination of rational metabolic engineering and module strategy is a promising approach for increasing the L-cysteine production in E. coli. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In vitro metabolic engineering for the salvage synthesis of NAD(.).

Science.gov (United States)

Honda, Kohsuke; Hara, Naoya; Cheng, Maria; Nakamura, Anna; Mandai, Komako; Okano, Kenji; Ohtake, Hisao

2016-05-01

Excellent thermal and operational stabilities of thermophilic enzymes can greatly increase the applicability of biocatalysis in various industrial fields. However, thermophilic enzymes are generally incompatible with thermo-labile substrates, products, and cofactors, since they show the maximal activities at high temperatures. Despite their pivotal roles in a wide range of enzymatic redox reactions, NAD(P)(+) and NAD(P)H exhibit relatively low stabilities at high temperatures, tending to be a major obstacle in the long-term operation of biocatalytic chemical manufacturing with thermophilic enzymes. In this study, we constructed an in vitro artificial metabolic pathway for the salvage synthesis of NAD(+) from its degradation products by the combination of eight thermophilic enzymes. The enzymes were heterologously produced in recombinant Escherichia coli and the heat-treated crude extracts of the recombinant cells were directly used as enzyme solutions. When incubated with experimentally optimized concentrations of the enzymes at 60°C, the NAD(+) concentration could be kept almost constant for 15h. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Biosynthesis and metabolic engineering of palmitoleate production, an important contributor to human health and sustainable industry.

Science.gov (United States)

Wu, Yongmei; Li, Runzhi; Hildebrand, David F

2012-10-01

Palmitoleate (cis-Δ9-16:1) shows numerous health benefits such as increased cell membrane fluidity, reduced inflammation, protection of the cardiovascular system, and inhibition of oncogenesis. Plant oils containing this unusual fatty acid can also be sustainable feedstocks for producing industrially important and high-demand 1-octene. Vegetable oils rich in palmitoleate are the ideal candidates for biodiesel production. Several wild plants are known that can synthesize high levels of palmitoleate in seeds. However, low yields and poor agronomic characteristics of these plants limit their commercialization. Metabolic engineering has been developed to create oilseed crops that accumulate high levels of palmitoleate or other unusual fatty acids, and significant advances have been made recently in this field, particularly using the model plant Arabidopsis as the host. The engineered targets for enhancing palmitoleate synthesis include overexpression of Δ9 desaturase from mammals, yeast, fungi, and plants, down-regulating KASII, coexpression of an ACP-Δ9 desaturase in plastids and CoA-Δ9 desaturase in endoplasmic reticulum (ER), and optimizing the metabolic flux into triacylglycerols (TAGs). This review will mainly describe the recent progress towards producing palmitoleate in transgenic plants by metabolic engineering along with our current understanding of palmitoleate biosynthesis and its regulation, as well as highlighting the bottlenecks that require additional investigation by combining lipidomics, transgenics and other "-omics" tools. A brief review of reported health benefits and non-food uses of palmitoleate will also be covered. Copyright © 2012. Published by Elsevier Ltd.

Systems-wide metabolic pathway engineering in Corynebacterium glutamicum for bio-based production of diaminopentane.

Science.gov (United States)

Kind, Stefanie; Jeong, Weol Kyu; Schröder, Hartwig; Wittmann, Christoph

2010-07-01

In the present work the Gram-positive bacterium Corynebacterium glutamicum was engineered into an efficient, tailor-made production strain for diaminopentane (cadaverine), a highly attractive building block for bio-based polyamides. The engineering comprised expression of lysine decarboxylase (ldcC) from Escherichia coli, catalyzing the conversion of lysine into diaminopentane, and systems-wide metabolic engineering of central supporting pathways. Substantially re-designing the metabolism yielded superior strains with desirable properties such as (i) the release from unwanted feedback regulation at the level of aspartokinase and pyruvate carboxylase by introducing the point mutations lysC311 and pycA458, (ii) an optimized supply of the key precursor oxaloacetate by amplifying the anaplerotic enzyme, pyruvate carboxylase, and deleting phosphoenolpyruvate carboxykinase which otherwise removes oxaloacetate, (iii) enhanced biosynthetic flux via combined amplification of aspartokinase, dihydrodipicolinate reductase, diaminopimelate dehydrogenase and diaminopimelate decarboxylase, and (iv) attenuated flux into the threonine pathway competing with production by the leaky mutation hom59 in the homoserine dehydrogenase gene. Lysine decarboxylase proved to be a bottleneck for efficient production, since its in vitro activity and in vivo flux were closely correlated. To achieve an optimal strain having only stable genomic modifications, the combination of the strong constitutive C. glutamicum tuf promoter and optimized codon usage allowed efficient genome-based ldcC expression and resulted in a high diaminopentane yield of 200 mmol mol(-1). By supplementing the medium with 1 mgL(-1) pyridoxal, the cofactor of lysine decarboxylase, the yield was increased to 300 mmol mol(-1). In the production strain obtained, lysine secretion was almost completely abolished. Metabolic analysis, however, revealed substantial formation of an as yet unknown by-product. It was identified as an

Production of the sesquiterpenoid (+)-nootkatone by metabolic engineering of Pichia pastoris.

Science.gov (United States)

Wriessnegger, Tamara; Augustin, Peter; Engleder, Matthias; Leitner, Erich; Müller, Monika; Kaluzna, Iwona; Schürmann, Martin; Mink, Daniel; Zellnig, Günther; Schwab, Helmut; Pichler, Harald

2014-07-01

The sesquiterpenoid (+)-nootkatone is a highly demanded and highly valued aroma compound naturally found in grapefruit, pummelo or Nootka cypress tree. Extraction of (+)-nootkatone from plant material or its production by chemical synthesis suffers from low yields and the use of environmentally harmful methods, respectively. Lately, major attention has been paid to biotechnological approaches, using cell extracts or whole-cell systems for the production of (+)-nootkatone. In our study, the yeast Pichia pastoris initially was applied as whole-cell biocatalyst for the production of (+)-nootkatone from (+)-valencene, the abundant aroma compound of oranges. Therefore, we generated a strain co-expressing the premnaspirodiene oxygenase of Hyoscyamus muticus (HPO) and the Arabidopsis thaliana cytochrome P450 reductase (CPR) that hydroxylated extracellularly added (+)-valencene. Intracellular production of (+)-valencene by co-expression of valencene synthase from Callitropsis nootkatensis resolved the phase-transfer issues of (+)-valencene. Bi-phasic cultivations of P. pastoris resulted in the production of trans-nootkatol, which was oxidized to (+)-nootkatone by an intrinsic P. pastoris activity. Additional overexpression of a P. pastoris alcohol dehydrogenase and truncated hydroxy-methylglutaryl-CoA reductase (tHmg1p) significantly enhanced the (+)-nootkatone yield to 208mg L(-1) cell culture in bioreactor cultivations. Thus, metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of (+)-nootkatone from simple carbon sources. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Metabolic engineering of β-carotene in orange fruit increases its in vivo antioxidant properties.

Science.gov (United States)

Pons, Elsa; Alquézar, Berta; Rodríguez, Ana; Martorell, Patricia; Genovés, Salvador; Ramón, Daniel; Rodrigo, María Jesús; Zacarías, Lorenzo; Peña, Leandro

2014-01-01

Orange is a major crop and an important source of health-promoting bioactive compounds. Increasing the levels of specific antioxidants in orange fruit through metabolic engineering could strengthen the fruit's health benefits. In this work, we have afforded enhancing the β-carotene content of orange fruit through blocking by RNA interference the expression of an endogenous β-carotene hydroxylase gene (Csβ-CHX) that is involved in the conversion of β-carotene into xanthophylls. Additionally, we have simultaneously overexpressed a key regulator gene of flowering transition, the FLOWERING LOCUS T from sweet orange (CsFT), in the transgenic juvenile plants, which allowed us to obtain fruit in an extremely short period of time. Silencing the Csβ-CHX gene resulted in oranges with a deep yellow ('golden') phenotype and significant increases (up to 36-fold) in β-carotene content in the pulp. The capacity of β-carotene-enriched oranges for protection against oxidative stress in vivo was assessed using Caenorhabditis elegans as experimental animal model. Golden oranges induced a 20% higher antioxidant effect than the isogenic control. This is the first example of the successful metabolic engineering of the β-carotene content (or the content of any other phytonutrient) in oranges and demonstrates the potential of genetic engineering for the nutritional enhancement of fruit tree crops. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Metabolic engineering of a haploid strain derived from a triploid industrial yeast for producing cellulosic ethanol.

Science.gov (United States)

Kim, Soo Rin; Skerker, Jeffrey M; Kong, In Iok; Kim, Heejin; Maurer, Matthew J; Zhang, Guo-Chang; Peng, Dairong; Wei, Na; Arkin, Adam P; Jin, Yong-Su

2017-03-01

Many desired phenotypes for producing cellulosic biofuels are often observed in industrial Saccharomyces cerevisiae strains. However, many industrial yeast strains are polyploid and have low spore viability, making it difficult to use these strains for metabolic engineering applications. We selected the polyploid industrial strain S. cerevisiae ATCC 4124 exhibiting rapid glucose fermentation capability, high ethanol productivity, strong heat and inhibitor tolerance in order to construct an optimal yeast strain for producing cellulosic ethanol. Here, we focused on developing a general approach and high-throughput screening method to isolate stable haploid segregants derived from a polyploid parent, such as triploid ATCC 4124 with a poor spore viability. Specifically, we deleted the HO genes, performed random sporulation, and screened the resulting segregants based on growth rate, mating type, and ploidy. Only one stable haploid derivative (4124-S60) was isolated, while 14 other segregants with a stable mating type were aneuploid. The 4124-S60 strain inherited only a subset of desirable traits present in the parent strain, same as other aneuploids, suggesting that glucose fermentation and specific ethanol productivity are likely to be genetically complex traits and/or they might depend on ploidy. Nonetheless, the 4124-60 strain did inherit the ability to tolerate fermentation inhibitors. When additional genetic perturbations known to improve xylose fermentation were introduced into the 4124-60 strain, the resulting engineered strain (IIK1) was able to ferment a Miscanthus hydrolysate better than a previously engineered laboratory strain (SR8), built by making the same genetic changes. However, the IIK1 strain showed higher glycerol and xylitol yields than the SR8 strain. In order to decrease glycerol and xylitol production, an NADH-dependent acetate reduction pathway was introduced into the IIK1 strain. By consuming 2.4g/L of acetate, the resulting strain (IIK1A

Introduction and expression of genes for metabolic engineering applications in Saccharomyces cerevisiae.

Science.gov (United States)

Da Silva, Nancy A; Srikrishnan, Sneha

2012-03-01

Metabolic pathway engineering in the yeast Saccharomyces cerevisiae leads to improved production of a wide range of compounds, ranging from ethanol (from biomass) to natural products such as sesquiterpenes. The introduction of multienzyme pathways requires precise control over the level and timing of expression of the associated genes. Gene number and promoter strength/regulation are two critical control points, and multiple studies have focused on modulating these in yeast. This MiniReview focuses on methods for introducing genes and controlling their copy number and on the many promoters (both constitutive and inducible) that have been successfully employed. The advantages and disadvantages of the methods will be presented, and applications to pathway engineering will be highlighted. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Metabolic engineering of Saccharomyces cerevisiae for production of germacrene A, a precursor of beta-elemene

DEFF Research Database (Denmark)

Hu, Yating; Zhou, Yongjin J.; Bao, Jichen

2017-01-01

inefficient and suffers from limited natural resources. Here, we engineered a yeast cell factory for the sustainable production of germacrene A, which can be transformed to beta-elemene by a one-step chemical reaction in vitro. Two heterologous germacrene A synthases (GASs) converting farnesyl pyrophosphate...... (FPP) to germacrene A were evaluated in yeast for their ability to produce germacrene A. Thereafter, several metabolic engineering strategies were used to improve the production level. Overexpression of truncated 3-hydroxyl-3-methylglutaryl-CoA reductase and fusion of FPP synthase with GAS, led...

Metabolic engineering of Escherichia coli for production of mixed-acid fermentation end products

Directory of Open Access Journals (Sweden)

Andreas Hartmut Förster

2014-05-01

Full Text Available Mixed-acid fermentation end products have numerous applications in biotechnology. This is probably the main driving force for the development of multiple strains that are supposed to produce individual end products with high yields. The process of engineering Escherichia coli strains for applied production of ethanol, lactate, succinate, or acetate was initiated several decades ago and is still ongoing. This review follows the path of strain development from the general characteristics of aerobic versus anaerobic metabolism over the regulatory machinery that enables the different metabolic routes. Thereafter, major improvements for broadening the substrate spectrum of Escherichia coli towards cheap carbon sources like molasses or lignocellulose are highlighted before major routes of strain development for the production of ethanol, acetate, lactate and succinate are presented.

Combination of traditional mutation and metabolic engineering to enhance ansamitocin P-3 production in Actinosynnema pretiosum.

Science.gov (United States)

Du, Zhi-Qiang; Zhang, Yuan; Qian, Zhi-Gang; Xiao, Han; Zhong, Jian-Jiang

2017-12-01

Ansamitocin P-3 (AP-3) is a maytansinoid with its most compelling antitumor activity, however, the low production titer of AP-3 greatly restricts its wide commercial application. In this work, a combinatorial approach including random mutation and metabolic engineering was conducted to enhance AP-3 biosynthesis in Actinosynnema pretiosum. First, a mutant strain M was isolated by N-methyl-N'-nitro-N-nitrosoguanidine mutation, which could produce AP-3 almost threefold that of wild type (WT) in 48 deep-well plates. Then, by overexpressing key biosynthetic genes asmUdpg and asm13-17 in the M strain, a further 60% increase of AP-3 production in 250-ml shake flasks was achieved in the engineered strain M-asmUdpg:asm13-17 compared to the M strain, and its maximum AP-3 production reached 582.7 mg/L, which is the highest as ever reported. Both the gene transcription levels and intracellular intermediate concentrations in AP-3 biosynthesis pathway were significantly increased in the M and M-asmUdpg:asm13-17 during fermentation compared to the WT. The good fermentation performance of the engineered strain was also confirmed in a lab-scale bioreactor. This work demonstrated that combination of random mutation and metabolic engineering could promote AP-3 biosynthesis and might be helpful for increasing the production of other industrially important secondary metabolites. © 2017 Wiley Periodicals, Inc.

Glycerol positive promoters for tailored metabolic engineering of the yeast Saccharomyces cerevisiae.

Science.gov (United States)

Ho, Ping-Wei; Klein, Mathias; Futschik, Matthias; Nevoigt, Elke

2018-05-01

Glycerol offers several advantages as a substrate for biotechnological applications. An important step toward using the popular production host Saccharomyces cerevisiae for glycerol-based bioprocesses has been the fact that in recent studies commonly used S. cerevisiae strains were engineered to grow in synthetic medium containing glycerol as the sole carbon source. For metabolic engineering projects of S. cerevisiae growing on glycerol, characterized promoters are missing. In the current study, we used transcriptome analysis and a yECitrine-based fluorescence reporter assay to select and characterize 25 useful promoters. The promoters of the genes ALD4 and ADH2 showed 4.2-fold and 3-fold higher activities compared to the well-known strong TEF1 promoter. Moreover, the collection contains promoters with graded activities in synthetic glycerol medium and different degrees of glucose repression. To demonstrate the general applicability of the promoter collection, we successfully used a subset of the characterized promoters with graded activities in order to optimize growth on glycerol in an engineered derivative of CEN.PK, in which glycerol catabolism exclusively occurs via a non-native DHA pathway.

Validated Predictions of Metabolic Energy Consumption for Submaximal Effort Movement

OpenAIRE

Tsianos, George A.; MacFadden, Lisa N.

2016-01-01

Author Summary Muscles consume metabolic energy to generate movement. Performing a movement over a long period of time or at a high intensity strains the respiratory and cardiovascular systems that need to replenish the energy reserves in muscle. Furthermore, consuming and replenishing metabolic energy involves biochemical reactions with byproducts that cause muscle fatigue. These biochemical reactions also produce heat that increases body temperature, potentially causing central fatigue. A m...

Increasing galactose consumption by Saccharomyces cerevisiae through metabolic engineering of the GAL gene regulatory network

DEFF Research Database (Denmark)

Østergaard, Simon; Olsson, Lisbeth; Johnston, M.

2000-01-01

Increasing the flux through central carbon metabolism is difficult because of rigidity in regulatory structures, at both the genetic and the enzymatic levels. Here we describe metabolic engineering of a regulatory network to obtain a balanced increase in the activity of all the enzymes in the pat...... media. The improved galactose consumption of the gal mutants did not favor biomass formation, but rather caused excessive respiro-fermentative metabolism, with the ethanol production rate increasing linearly with glycolytic flux....... by eliminating three known negative regulators of the GAL system: Gale, Gal80, and Mig1. This led to a 41% increase in flux through the galactose utilization pathway compared with the wild-type strain. This is of significant interest within the field of biotechnology since galactose is present in many industrial...

Metabolic engineering of Escherichia coli for the production of riboflavin

Science.gov (United States)

2014-01-01

Background Riboflavin (vitamin B2), the precursor of the flavin cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), is used commercially as an animal feed supplement and food colorant. E. coli is a robust host for various genetic manipulations and has been employed for efficient production of biofuels, polymers, amino acids, and bulk chemicals. Thus, the aim of this study was to understand the metabolic capacity of E. coli for the riboflavin production by modification of central metabolism, riboflavin biosynthesis pathway and optimization of the fermentation conditions. Results The basic producer RF01S, in which the riboflavin biosynthesis genes ribABDEC from E. coli were overexpressed under the control of the inducible trc promoter, could accumulate 229.1 mg/L of riboflavin. Further engineering was performed by examining the impact of expression of zwf (encodes glucose 6-phosphate dehydrogenase) and gnd (encodes 6-phosphogluconate dehydrogenase) from Corynebacterium glutamicum and pgl (encodes 6-phosphogluconolactonase) from E. coli on riboflavin production. Deleting pgi (encodes glucose-6-phosphate isomerase) and genes of Entner-Doudoroff (ED) pathway successfully redirected the carbon flux into the oxidative pentose phosphate pathway, and overexpressing the acs (encodes acetyl-CoA synthetase) reduced the acetate accumulation. These modifications increased riboflavin production to 585.2 mg/L. By further modulating the expression of ribF (encodes riboflavin kinase) for reducing the conversion of riboflavin to FMN in RF05S, the final engineering strain RF05S-M40 could produce 1036.1 mg/L riboflavin in LB medium at 37°C. After optimizing the fermentation conditions, strain RF05S-M40 produced 2702.8 mg/L riboflavin in the optimized semi-defined medium, which was a value nearly 12-fold higher than that of RF01S, with a yield of 137.5 mg riboflavin/g glucose. Conclusions The engineered strain RF05S-M40 has the highest yield among all

Metabolic engineering of Escherichia coli for the production of riboflavin.

Science.gov (United States)

Lin, Zhenquan; Xu, Zhibo; Li, Yifan; Wang, Zhiwen; Chen, Tao; Zhao, Xueming

2014-07-16

Riboflavin (vitamin B2), the precursor of the flavin cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), is used commercially as an animal feed supplement and food colorant. E. coli is a robust host for various genetic manipulations and has been employed for efficient production of biofuels, polymers, amino acids, and bulk chemicals. Thus, the aim of this study was to understand the metabolic capacity of E. coli for the riboflavin production by modification of central metabolism, riboflavin biosynthesis pathway and optimization of the fermentation conditions. The basic producer RF01S, in which the riboflavin biosynthesis genes ribABDEC from E. coli were overexpressed under the control of the inducible trc promoter, could accumulate 229.1 mg/L of riboflavin. Further engineering was performed by examining the impact of expression of zwf (encodes glucose 6-phosphate dehydrogenase) and gnd (encodes 6-phosphogluconate dehydrogenase) from Corynebacterium glutamicum and pgl (encodes 6-phosphogluconolactonase) from E. coli on riboflavin production. Deleting pgi (encodes glucose-6-phosphate isomerase) and genes of Entner-Doudoroff (ED) pathway successfully redirected the carbon flux into the oxidative pentose phosphate pathway, and overexpressing the acs (encodes acetyl-CoA synthetase) reduced the acetate accumulation. These modifications increased riboflavin production to 585.2 mg/L. By further modulating the expression of ribF (encodes riboflavin kinase) for reducing the conversion of riboflavin to FMN in RF05S, the final engineering strain RF05S-M40 could produce 1036.1 mg/L riboflavin in LB medium at 37°C. After optimizing the fermentation conditions, strain RF05S-M40 produced 2702.8 mg/L riboflavin in the optimized semi-defined medium, which was a value nearly 12-fold higher than that of RF01S, with a yield of 137.5 mg riboflavin/g glucose. The engineered strain RF05S-M40 has the highest yield among all reported riboflavin production

Final Technical Report and management: NUCLEAR ENGINEERING RECRUITMENT EFFORT

International Nuclear Information System (INIS)

Kerrick, Sharon S.; Vincent, Charles D.

2007-01-01

This report provides the summary of a project whose purpose was to support the costs of developing a nuclear engineering awareness program, an instruction program for teachers to integrate lessons on nuclear science and technology into their existing curricula, and web sites for the exchange of nuclear engineering career information and classroom materials. The specific objectives of the program were as follows: Objective 1--Increase awareness and interest of nuclear engineering; Objective 2--Instruct Teachers on nuclear topics; Objective 3--Nuclear education programs web-site; Objective 4--Support to university/industry matching grants and reactor sharing; Objective 5--Pilot project; and Objective 6--Nuclear engineering enrollment survey at universities

Combining metabolic engineering and biocompatible chemistry for high-yield production of homo-diacetyl and homo-(S,S)-2,3-butanediol.

Science.gov (United States)

Liu, Jianming; Chan, Siu Hung Joshua; Brock-Nannestad, Theis; Chen, Jun; Lee, Sang Yup; Solem, Christian; Jensen, Peter Ruhdal

2016-07-01

Biocompatible chemistry is gaining increasing attention because of its potential within biotechnology for expanding the repertoire of biological transformations carried out by enzymes. Here we demonstrate how biocompatible chemistry can be used for synthesizing valuable compounds as well as for linking metabolic pathways to achieve redox balance and rescued growth. By comprehensive rerouting of metabolism, activation of respiration, and finally metal ion catalysis, we successfully managed to convert the homolactic bacterium Lactococcus lactis into a homo-diacetyl producer with high titer (95mM or 8.2g/L) and high yield (87% of the theoretical maximum). Subsequently, the pathway was extended to (S,S)-2,3-butanediol (S-BDO) through efficiently linking two metabolic pathways via chemical catalysis. This resulted in efficient homo-S-BDO production with a titer of 74mM (6.7g/L) S-BDO and a yield of 82%. The diacetyl and S-BDO production rates and yields obtained are the highest ever reported, demonstrating the promising combination of metabolic engineering and biocompatible chemistry as well as the great potential of L. lactis as a new production platform. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Engineering isoprene synthesis in cyanobacteria.

Science.gov (United States)

Chaves, Julie E; Melis, Anastasios

2018-04-24

The renewable production of isoprene (Isp) hydrocarbons, to serve as fuel and synthetic chemistry feedstock, has attracted interest in the field recently. Isp (C 5 H 8 ) is naturally produced from sunlight, CO 2 and H 2 O photosynthetically in terrestrial plant chloroplasts via the terpenoid biosynthetic pathway and emitted in the atmosphere as a response to heat stress. Efforts to institute a high capacity continuous and renewable process have included heterologous expression of the Isp synthesis pathway in photosynthetic microorganisms. This review examines the premise and promise emanating from this relatively new research effort. Also examined are the metabolic engineering approaches applied in the quest of renewable Isp hydrocarbons production, the progress achieved so far, and barriers encountered along the way. © 2018 Federation of European Biochemical Societies.

ASME Code Efforts Supporting HTGRs

Energy Technology Data Exchange (ETDEWEB)

D.K. Morton

2012-09-01

In 1999, an international collaborative initiative for the development of advanced (Generation IV) reactors was started. The idea behind this effort was to bring nuclear energy closer to the needs of sustainability, to increase proliferation resistance, and to support concepts able to produce energy (both electricity and process heat) at competitive costs. The U.S. Department of Energy has supported this effort by pursuing the development of the Next Generation Nuclear Plant, a high temperature gas-cooled reactor. This support has included research and development of pertinent data, initial regulatory discussions, and engineering support of various codes and standards development. This report discusses the various applicable American Society of Mechanical Engineers (ASME) codes and standards that are being developed to support these high temperature gascooled reactors during construction and operation. ASME is aggressively pursuing these codes and standards to support an international effort to build the next generation of advanced reactors so that all can benefit.

ASME Code Efforts Supporting HTGRs

Energy Technology Data Exchange (ETDEWEB)

D.K. Morton

2011-09-01

In 1999, an international collaborative initiative for the development of advanced (Generation IV) reactors was started. The idea behind this effort was to bring nuclear energy closer to the needs of sustainability, to increase proliferation resistance, and to support concepts able to produce energy (both electricity and process heat) at competitive costs. The U.S. Department of Energy has supported this effort by pursuing the development of the Next Generation Nuclear Plant, a high temperature gas-cooled reactor. This support has included research and development of pertinent data, initial regulatory discussions, and engineering support of various codes and standards development. This report discusses the various applicable American Society of Mechanical Engineers (ASME) codes and standards that are being developed to support these high temperature gascooled reactors during construction and operation. ASME is aggressively pursuing these codes and standards to support an international effort to build the next generation of advanced reactors so that all can benefit.

Medicine is not health care, food is health care: plant metabolic engineering, diet and human health.

Science.gov (United States)

Martin, Cathie; Li, Jie

2017-11-01

Contents 699 I. 699 II. 700 III. 700 IV. 706 V. 707 VI. 714 714 References 714 SUMMARY: Plants make substantial contributions to our health through our diets, providing macronutrients for energy and growth as well as essential vitamins and phytonutrients that protect us from chronic diseases. Imbalances in our food can lead to deficiency diseases or obesity and associated metabolic disorders, increased risk of cardiovascular diseases and cancer. Nutritional security is now a global challenge which can be addressed, at least in part, through plant metabolic engineering for nutritional improvement of foods that are accessible to and eaten by many. We review the progress that has been made in nutritional enhancement of foods, both improvements through breeding and through biotechnology and the engineering principles on which increased phytonutrient levels are based. We also consider the evidence, where available, that such foods do enhance health and protect against chronic diseases. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Development of Renewable Biofuels Technology by Transcriptomic Analysis and Metabolic Engineering of Diatoms

Energy Technology Data Exchange (ETDEWEB)

Hildebrand, Mark [Univ. of California, San Diego, CA (United States)

2013-11-18

There is enormous interest in developing renewable sources of liquid fuels because of depletion of fossil fuel reserves, dependence on foreign sources, and increasing atmospheric CO₂ levels. Algae produce neutral lipids that are readily converted into liquid fuels such as biodiesel or JP-8 equivalent, and are attractive sources because they are far more productive than plants (yielding 10 -100’s of time more lipid per land area), and can be grown on non-cultivatable land with non-potable (brackish or salt) water sources. Unicellular algae known as diatoms were the most thoroughly characterized species in the National Renewable Energy Laboratory’s Aquatic Species Program, whose goal was to develop microalgae as renewable fuel sources. Lipid accumulation in microalgae is generally induced by nutrient limitation, which involves a change in environmental conditions. Intrinsic variability in cellular response to environmental changes prevents a high degree of control over the process. Nutrient limitation also inhibits biomass accumulation; therefore a tradeoff between high biomass and lipid production occurs. The goal of this project was to develop metabolic engineering approaches for diatoms to enable induction of lipid accumulation by controllable manipulation of intracellular processes rather than from external environmental conditions, and to manipulate carbon partitioning within the cell between lipid and carbohydrate synthesis to enable both abundant biomass and lipid accumulation. There were two specific objectives for this project; Objective 1:To perform comparative transcriptomic analysis in T. pseudonana and C. cryptica of lipid accumulation resulting from silicon and nitrogen limitation, to identify common and key regulatory steps involved in controlling lipid accumulation and carbon partitioning; and Objective 2: To metabolically engineer the cell to alter carbon partitioning to either trigger lipid induction without the need for nutrient

Engineering education research: Impacts of an international network of female engineers on the persistence of Liberian undergraduate women studying engineering

Science.gov (United States)

Rimer, Sara; Reddivari, Sahithya; Cotel, Aline

2015-11-01

As international efforts to educate and empower women continue to rise, engineering educators are in a unique position to be a part of these efforts by encouraging and supporting women across the world at the university level through STEM education and outreach. For the past two years, the University of Michigan has been a part of a grassroots effort to encourage and support the persistence of engineering female students at University of Liberia. This effort has led to the implementation of a leadership camp this past August for Liberian engineering undergraduate women, meant to: (i) to empower engineering students with the skills, support, and inspiration necessary to become successful and well-rounded engineering professionals in a global engineering market; and (ii) to strengthen the community of Liberian female engineers by building cross-cultural partnerships among students resulting in a international network of women engineers. This session will present qualitative research findings on the impact of this grassroots effort on Liberian female students? persistence in engineering, and the future directions of this work.

Controlling cell-free metabolism through physiochemical perturbations.

Science.gov (United States)

Karim, Ashty S; Heggestad, Jacob T; Crowe, Samantha A; Jewett, Michael C

2018-01-01

physiochemical perturbations and suggests the need for a more controllable, multi-step, separated cell-free framework for future pathway prototyping and enzyme discovery efforts. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Engineering of metabolic control

Science.gov (United States)

Liao, James C.

2004-03-16

The invention features a method of producing heterologous molecules in cells under the regulatory control of a metabolite and metabolic flux. The method can enhance the synthesis of heterologous polypeptides and metabolites.

Deletion of acetyl-CoA synthetases I and II increases production of 3-hydroxypropionate by the metabolically-engineered hyperthermophile Pyrococcus furiosus.

Science.gov (United States)

Thorgersen, Michael P; Lipscomb, Gina L; Schut, Gerrit J; Kelly, Robert M; Adams, Michael W W

2014-03-01

The heterotrophic, hyperthermophilic archaeon Pyrococcus furiosus is a new addition to the growing list of genetically-tractable microorganisms suitable for metabolic engineering to produce liquid fuels and industrial chemicals. P. furiosus was recently engineered to generate 3-hydroxypropionate (3-HP) from CO₂ and acetyl-CoA by the heterologous-expression of three enzymes from the CO₂ fixation cycle of the thermoacidophilic archaeon Metallosphaera sedula using a thermally-triggered induction system. The acetyl-CoA for this pathway is generated from glucose catabolism that in wild-type P. furiosus is converted to acetate with concurrent ATP production by the heterotetrameric (α₂β₂) acetyl-CoA synthetase (ACS). Hence ACS in the engineered 3-HP production strain (MW56) competes with the heterologous pathway for acetyl-CoA. Herein we show that strains of MW56 lacking the α-subunit of either of the two ACSs previously characterized from P. furiosus (ACSI and ACSII) exhibit a three-fold increase in specific 3-HP production. The ΔACSIα strain displayed only a minor defect in growth on either maltose or peptides, while no growth defect on these substrates was observed with the ΔACSIIα strain. Deletion of individual and multiple ACS subunits was also shown to decrease CoA release activity for several different CoA ester substrates in addition to acetyl-CoA, information that will be extremely useful for future metabolic engineering endeavors in P. furiosus. Copyright © 2014 International Metabolic Engineering Society. All rights reserved.

OptFlux: an open-source software platform for in silico metabolic engineering

DEFF Research Database (Denmark)

Rocha, I.; Maia, P.; Evangelista, P.

2010-01-01

to address industrial goals. However, the use of these methods has been restricted to bioinformaticians or other expert researchers. The main aim of this work is, therefore, to provide a user-friendly computational tool for Metabolic Engineering applications. Results: OptFlux is an open-source and modular...... available a number of useful tools. Its open-source nature invites contributions by all those interested in making their methods available for the community. Given its plug-in based architecture it can be extended with new functionalities. Currently, several plug-ins are being developed, including network...

Metabolic engineering of Saccharomyces cerevisiae for overproduction of triacylglycerols

DEFF Research Database (Denmark)

Ferreira, Raphael; Teixeira, Paulo Goncalves; Gossing, Michael

2018-01-01

Triacylglycerols (TAGs) are valuable versatile compounds that can be used as metabolites for nutrition and health, as well as feedstocks for biofuel production. Although Saccharomyces cerevisiae is the favored microbial cell factory for industrial production of biochemicals, it does not produce...... large amounts of lipids and TAGs comprise only ~1% of its cell dry weight. Here, we engineered S. cerevisiae to reorient its metabolism for overproduction of TAGs, by regulating lipid droplet associated-proteins involved in TAG synthesis and hydrolysis. We implemented a push-and-pull strategy...... PXA1 led to accumulation of  254 mg∙gCDW−1. The TAG levels achieved here are the highest titer reported in S. cerevisiae, reaching 27.4% of the maximum theoretical yield in minimal medium with 2% glucose. This work shows the potential of using an industrially established and robust yeast species...

Metabolic network model guided engineering ethylmalonyl-CoA pathway to improve ascomycin production in Streptomyces hygroscopicus var. ascomyceticus.

Science.gov (United States)

Wang, Junhua; Wang, Cheng; Song, Kejing; Wen, Jianping

2017-10-03

Ascomycin is a 23-membered polyketide macrolide with high immunosuppressant and antifungal activity. As the lower production in bio-fermentation, global metabolic analysis is required to further explore its biosynthetic network and determine the key limiting steps for rationally engineering. To achieve this goal, an engineering approach guided by a metabolic network model was implemented to better understand ascomycin biosynthesis and improve its production. The metabolic conservation of Streptomyces species was first investigated by comparing the metabolic enzymes of Streptomyces coelicolor A3(2) with those of 31 Streptomyces strains, the results showed that more than 72% of the examined proteins had high sequence similarity with counterparts in every surveyed strain. And it was found that metabolic reactions are more highly conserved than the enzymes themselves because of its lower diversity of metabolic functions than that of genes. The main source of the observed metabolic differences was from the diversity of secondary metabolism. According to the high conservation of primary metabolic reactions in Streptomyces species, the metabolic network model of Streptomyces hygroscopicus var. ascomyceticus was constructed based on the latest reported metabolic model of S. coelicolor A3(2) and validated experimentally. By coupling with flux balance analysis and using minimization of metabolic adjustment algorithm, potential targets for ascomycin overproduction were predicted. Since several of the preferred targets were highly associated with ethylmalonyl-CoA biosynthesis, two target genes hcd (encoding 3-hydroxybutyryl-CoA dehydrogenase) and ccr (encoding crotonyl-CoA carboxylase/reductase) were selected for overexpression in S. hygroscopicus var. ascomyceticus FS35. Both the mutants HA-Hcd and HA-Ccr showed higher ascomycin titer, which was consistent with the model predictions. Furthermore, the combined effects of the two genes were evaluated and the strain HA

Simple glycolipids of microbes: Chemistry, biological activity and metabolic engineering

Directory of Open Access Journals (Sweden)

Ahmad Mohammad Abdel-Mawgoud

2018-03-01

Full Text Available Glycosylated lipids (GLs are added-value lipid derivatives of great potential. Besides their interesting surface activities that qualify many of them to act as excellent ecological detergents, they have diverse biological activities with promising biomedical and cosmeceutical applications. Glycolipids, especially those of microbial origin, have interesting antimicrobial, anticancer, antiparasitic as well as immunomodulatory activities. Nonetheless, GLs are hardly accessing the market because of their high cost of production. We believe that experience of metabolic engineering (ME of microbial lipids for biofuel production can now be harnessed towards a successful synthesis of microbial GLs for biomedical and other applications. This review presents chemical groups of bacterial and fungal GLs, their biological activities, their general biosynthetic pathways and an insight on ME strategies for their production.

The growth of tissue engineering.

Science.gov (United States)

Lysaght, M J; Reyes, J

2001-10-01

This report draws upon data from a variety of sources to estimate the size, scope, and growth rate of the contemporary tissue engineering enterprise. At the beginning of 2001, tissue engineering research and development was being pursued by 3,300 scientists and support staff in more than 70 startup companies or business units with a combined annual expenditure of over $600 million. Spending by tissue engineering firms has been growing at a compound annual rate of 16%, and the aggregate investment since 1990 now exceeds $3.5 billion. At the beginning of 2001, the net capital value of the 16 publicly traded tissue engineering startups had reached $2.6 billion. Firms focusing on structural applications (skin, cartilage, bone, cardiac prosthesis, and the like) comprise the fastest growing segment. In contrast, efforts in biohybrid organs and other metabolic applications have contracted over the past few years. The number of companies involved in stem cells and regenerative medicine is rapidly increasing, and this area represents the most likely nidus of future growth for tissue engineering. A notable recent trend has been the emergence of a strong commercial activity in tissue engineering outside the United States, with at least 16 European or Australian companies (22% of total) now active.

Weedy lignocellulosic feedstock and microbial metabolic engineering. Advancing the generation of 'Biofuel'

Energy Technology Data Exchange (ETDEWEB)

Chandel, Anuj K. [Jawaharlal Nehru Technological Univ., Hyderabad (India). Centre of Biotechnology; Singh, Om V. [Pittsburgh Univ., Bradford, PA (United States). Div. of Biological and Health Sciences

2011-03-15

Lignocellulosic materials are the most abundant renewable organic resources ({proportional_to}200 billion tons annually) on earth that are readily available for conversion to ethanol and other value-added products, but they have not yet been tapped for the commercial production of fuel ethanol. The lignocellulosic substrates include woody substrates such as hardwood (birch and aspen, etc.) and softwood (spruce and pine, etc.), agro residues (wheat straw, sugarcane bagasse, corn stover, etc.), dedicated energy crops (switch grass, and Miscanthus etc.), weedy materials (Eicchornia crassipes, Lantana camara etc.), and municipal solid waste (food and kitchen waste, etc.). Despite the success achieved in the laboratory, there are limitations to success with lignocellulosic substrates on a commercial scale. The future of lignocellulosics is expected to lie in improvements of plant biomass, metabolic engineering of ethanol, and cellulolytic enzyme-producing microorganisms, fullest exploitation of weed materials, and process integration of the individual steps involved in bioethanol production. Issues related to the chemical composition of various weedy raw substrates for bioethanol formation, including chemical composition-based structural hydrolysis of the substrate, need special attention. This area could be opened up further by exploring genetically modified metabolic engineering routes in weedy materials and in biocatalysts that would make the production of bioethanol more efficient. (orig.)

Production of L-carnitine by secondary metabolism of bacteria

Directory of Open Access Journals (Sweden)

Iborra José L

2007-10-01

Full Text Available Abstract The increasing commercial demand for L-carnitine has led to a multiplication of efforts to improve its production with bacteria. The use of different cell environments, such as growing, resting, permeabilized, dried, osmotically stressed, freely suspended and immobilized cells, to maintain enzymes sufficiently active for L-carnitine production is discussed in the text. The different cell states of enterobacteria, such as Escherichia coli and Proteus sp., which can be used to produce L-carnitine from crotonobetaine or D-carnitine as substrate, are analyzed. Moreover, the combined application of both bioprocess and metabolic engineering has allowed a deeper understanding of the main factors controlling the production process, such as energy depletion and the alteration of the acetyl-CoA/CoA ratio which are coupled to the end of the biotransformation. Furthermore, the profiles of key central metabolic activities such as the TCA cycle, the glyoxylate shunt and the acetate metabolism are seen to be closely interrelated and affect the biotransformation efficiency. Although genetically modified strains have been obtained, new strain improvement strategies are still needed, especially in Escherichia coli as a model organism for molecular biology studies. This review aims to summarize and update the state of the art in L-carnitine production using E. coli and Proteus sp, emphasizing the importance of proper reactor design and operation strategies, together with metabolic engineering aspects and the need for feed-back between wet and in silico work to optimize this biotransformation.

Enhancement of Naringenin Biosynthesis from Tyrosine by Metabolic Engineering of Saccharomyces cerevisiae.

Science.gov (United States)

Lyu, Xiaomei; Ng, Kuan Rei; Lee, Jie Lin; Mark, Rita; Chen, Wei Ning

2017-08-09

Flavonoids are an important class of plant polyphenols that possess a variety of health benefits. In this work, S. cerevisiae was metabolically engineered to produce the flavonoid naringenin, using tyrosine as the precursor. Our strategy to improve naringenin production comprised three modules. In module 1, we employed a modified GAL system to overexpress the genes of the naringenin biosynthesis pathway and investigated their synergistic action. In module 2, we simultaneously up-regulated acetyl-CoA production and down-regulated fatty acid biosynthesis in order to increase the precursor supply, malonyl-CoA. In module 3, we engineered the tyrosine biosynthetic pathway to eliminate the feedback inhibition of tyrosine and also down-regulated competing pathways. It was found that modules 1 and 3 played important roles in improving naringenin production. We succeeded in producing up to ∼90 mg/L of naringenin in our final strain, which is a 20-fold increase as compared to the parental strain.

An extensive case study of hairy-root cultures for enhanced secondary-metabolite production through metabolic-pathway engineering.

Science.gov (United States)

Mehrotra, Shakti; Rahman, Laiq Ur; Kukreja, Arun Kumar

2010-08-23

An intrinsic improvement is taking place in the methodologies for the development of culture systems with first-rate production of plant-based molecules. The blending of HR (hairy root) cultures with ME (metabolic engineering) approaches offers new insights into, and possibilities for, improving the system productivity for known and/or novel high-value plant-derived active compounds. The introduction and expression of foreign genes in plants results in improvement of cellular activities by manipulating enzymatic, regulatory and transport function of the cell. The rational amendments in the rate-limiting steps of a biosynthetic pathway as well as inactivating the inefficient pathway(s) for by-product formation can be accomplished either through single-step engineering or through the multi-step engineering. The hierarchical control of any metabolic process can lead the engineer to apply the ME ideas and principles to any of the strata, including transcriptional, moving on to translational and enzymatic activity. The HR culture systems offer a remarkable potential for commercial production of a number of low-volume, but high-value, secondary metabolites. Taking HR as a model system, in the present review, we discuss engineering principles and perceptions to exploit secondary-metabolite pathways for the production of important bioactive compounds. We also talk about requisites and possible challenges that occur during ME, with emphasis on examples of various HR systems. Furthermore, it also highlights the utilization of global information obtained from '-omic' platforms in order to explore pathway architecture, structural and functional aspects of important enzymes and genes that can support the design of sets of engineering, resulting in the generation of wide-ranging views of DNA sequence-to-metabolite passageway networking and their control to obtain desired results.

Metabolic engineering of Escherichia coli for biotechnological production of high-value organic acids and alcohols

Energy Technology Data Exchange (ETDEWEB)

Yu, Chao; Cao, Yujin; Zou, Huibin; Xian, Mo [Chinese Academy of Sciences, Qingdao (China). Key Lab. of Biofuels

2011-02-15

Confronted with the gradual and inescapable exhaustion of the earth's fossil energy resources, the bio-based process to produce platform chemicals from renewable carbohydrates is attracting growing interest. Escherichia coli has been chosen as a workhouse for the production of many valuable chemicals due to its clear genetic background, convenient to be genetically modified and good growth properties with low nutrient requirements. Rational strain development of E. coli achieved by metabolic engineering strategies has provided new processes for efficiently biotechnological production of various high-value chemical building blocks. Compared to previous reviews, this review focuses on recent advances in metabolic engineering of the industrial model bacteria E. coli that lead to efficient recombinant biocatalysts for the production of high-value organic acids like succinic acid, lactic acid, 3-hydroxypropanoic acid and glucaric acid as well as alcohols like 1,3-propanediol, xylitol, mannitol, and glycerol with the discussion of the future research in this area. Besides, this review also discusses several platform chemicals, including fumaric acid, aspartic acid, glutamic acid, sorbitol, itaconic acid, and 2,5-furan dicarboxylic acid, which have not been produced by E. coli until now. (orig.)

Engineering microbial consortia for controllable outputs

Energy Technology Data Exchange (ETDEWEB)

Lindemann, Stephen R.; Bernstein, Hans C.; Song, Hyun-Seob; Fredrickson, Jim K.; Fields, Matthew W.; Shou, Wenying; Johnson, David R.; Beliaev, Alexander S.

2016-03-11

Much research has been invested into engineering microorganisms to perform desired biotransformations; nonetheless, these efforts frequently fall short of expected results due to the unforeseen effects of biofeedback regulation and functional incompatibility. In nature, metabolic function is compartmentalized into diverse organisms assembled into resilient consortia, in which the division of labor is thought to lead to increased community efficiency and productivity. Here, we consider whether and how consortia can be designed to perform bioprocesses of interest beyond the metabolic flexibility limitations of a single organism. Advances in post-genomic analysis of microbial consortia and application of high-resolution global measurements now offer the promise of systems-level understanding of how microbial consortia adapt to changes in environmental variables and inputs of carbon and energy. We argue that when combined with appropriate modeling framework that predictive knowledge generates testable hypotheses and orthogonal synthetic biology tools, such understanding can dramatically improve our ability to control the fate and functioning of consortia. In this article, we articulate our collective perspective on the current and future state of microbial community engineering and control while placing specific emphasis on ecological principles that promote control over community function and emergent properties.

Genome-scale modeling enables metabolic engineering of Saccharomyces cerevisiae for succinic acid production.

Science.gov (United States)

Agren, Rasmus; Otero, José Manuel; Nielsen, Jens

2013-07-01

In this work, we describe the application of a genome-scale metabolic model and flux balance analysis for the prediction of succinic acid overproduction strategies in Saccharomyces cerevisiae. The top three single gene deletion strategies, Δmdh1, Δoac1, and Δdic1, were tested using knock-out strains cultivated anaerobically on glucose, coupled with physiological and DNA microarray characterization. While Δmdh1 and Δoac1 strains failed to produce succinate, Δdic1 produced 0.02 C-mol/C-mol glucose, in close agreement with model predictions (0.03 C-mol/C-mol glucose). Transcriptional profiling suggests that succinate formation is coupled to mitochondrial redox balancing, and more specifically, reductive TCA cycle activity. While far from industrial titers, this proof-of-concept suggests that in silico predictions coupled with experimental validation can be used to identify novel and non-intuitive metabolic engineering strategies.

Combining CRISPR and CRISPRi Systems for Metabolic Engineering of E. coli and 1,4-BDO Biosynthesis.

Science.gov (United States)

Wu, Meng-Ying; Sung, Li-Yu; Li, Hung; Huang, Chun-Hung; Hu, Yu-Chen

2017-12-15

Biosynthesis of 1,4-butanediol (1,4-BDO) in E. coli requires an artificial pathway that involves six genes and time-consuming, iterative genome engineering. CRISPR is an effective gene editing tool, while CRISPR interference (CRISPRi) is repurposed for programmable gene suppression. This study aimed to combine both CRISPR and CRISPRi for metabolic engineering of E. coli and 1,4-BDO production. We first exploited CRISPR to perform point mutation of gltA, replacement of native lpdA with heterologous lpdA, knockout of sad and knock-in of two large (6.0 and 6.3 kb in length) gene cassettes encoding the six genes (cat1, sucD, 4hbd, cat2, bld, bdh) in the 1,4-BDO biosynthesis pathway. The successive E. coli engineering enabled production of 1,4-BDO to a titer of 0.9 g/L in 48 h. By combining the CRISPRi system to simultaneously suppress competing genes that divert the flux from the 1,4-BDO biosynthesis pathway (gabD, ybgC and tesB) for >85%, we further enhanced the 1,4-BDO titer for 100% to 1.8 g/L while reducing the titers of byproducts gamma-butyrolactone and succinate for 55% and 83%, respectively. These data demonstrate the potential of combining CRISPR and CRISPRi for genome engineering and metabolic flux regulation in microorganisms such as E. coli and production of chemicals (e.g., 1,4-BDO).

Synthetic biology and metabolic engineering for marine carotenoids: new opportunities and future prospects.

Science.gov (United States)

Wang, Chonglong; Kim, Jung-Hun; Kim, Seon-Won

2014-09-17

Carotenoids are a class of diverse pigments with important biological roles such as light capture and antioxidative activities. Many novel carotenoids have been isolated from marine organisms to date and have shown various utilizations as nutraceuticals and pharmaceuticals. In this review, we summarize the pathways and enzymes of carotenoid synthesis and discuss various modifications of marine carotenoids. The advances in metabolic engineering and synthetic biology for carotenoid production are also reviewed, in hopes that this review will promote the exploration of marine carotenoid for their utilizations.

ITs in Engineering Education: Joining Efforts Between SPEE and IGIP

Directory of Open Access Journals (Sweden)

Alberto Cardoso

2012-01-01

Full Text Available The International Society for Engineering Education (IGIP and The Portuguese Society for Engineering Education (SPEE, the first being the oldest European Society for Engineering Education in Europe and the second the very young Society for Engineering Education in Portugal, have been intensifying the collaboration between the two societies as well as the exchange and dissemination of information about their relevant activities, whilst promoting understanding and cooperation between their respective members. One possible way is to create joint working groups, open to the members of both societies, on common topics of interest. In fact, both societies already kicked off this activity. The first initiative happened during the 1st World Engineering Education Flash Week (WEE, Lisbon, 2011. The SPEE-IGIP Flash Moment was a one day event integrated in the main Conference, which was dedicated to “Information & Communication Technologies in Engineering Education”.
ITs allow the development of different teaching strategies which contribute to enhance the learning outcomes of students. ITs are also particularly suited to develop Life Long Learning tools, in a broad range of Engineering subjects, either open to the general market or oriented to a very specific public.
Examples of teaching strategies involving ITs have been addressed during the Flash Moment SPEE-IGIP which took place during WEE, and some are described in detail in the present work.

Production of L-lactic acid from metabolically engineered strain of Enterobacter aerogenes ATCC 29007.

Science.gov (United States)

Thapa, Laxmi Prasad; Lee, Sang Jun; Park, Chulhwan; Kim, Seung Wook

2017-07-01

In this study, L-lactic acid production was investigated from metabolically engineered strain of E. aerogenes ATCC 29007. The engineered strain E. aerogenes SUMI01 (Δpta) was generated by the deletion of phosphate acetyltransferase (pta) gene from the chromosome of E. aerogenes ATCC 29007 and deletion was confirmed by colony PCR. Under the optimized fermentation conditions, at 37°C and pH 6 for 84h, the L-lactic acid produced by engineered strain E. aerogenes SUMI01 (Δpta) in flask fermentation using 100g/L mannitol as the carbon source was 40.05g/L as compared to that of the wild type counterpart 20.70g/L. At the end of the batch fermentation in bioreactor the production of L-lactic acid reached to 46.02g/L and yield was 0.41g/g by utilizing 112.32g/L mannitol. This is the first report regarding the production of L-lactic acid from Enterobacter species. We believe that this result may provide valuable guidelines for further engineering Enterobacter strain for the improvement of L-lactic acid production. Copyright © 2017 Elsevier Inc. All rights reserved.

Phytohormones and their metabolic engineering for abiotic stress tolerance in crop plants

Directory of Open Access Journals (Sweden)

Shabir H. Wani

2016-06-01

Full Text Available Abiotic stresses including drought, salinity, heat, cold, flooding, and ultraviolet radiation causes crop losses worldwide. In recent times, preventing these crop losses and producing more food and feed to meet the demands of ever-increasing human populations have gained unprecedented importance. However, the proportion of agricultural lands facing multiple abiotic stresses is expected only to rise under a changing global climate fueled by anthropogenic activities. Identifying the mechanisms developed and deployed by plants to counteract abiotic stresses and maintain their growth and survival under harsh conditions thus holds great significance. Recent investigations have shown that phytohormones, including the classical auxins, cytokinins, ethylene, and gibberellins, and newer members including brassinosteroids, jasmonates, and strigolactones may prove to be important metabolic engineering targets for producing abiotic stress-tolerant crop plants. In this review, we summarize and critically assess the roles that phytohormones play in plant growth and development and abiotic stress tolerance, besides their engineering for conferring abiotic stress tolerance in transgenic crops. We also describe recent successes in identifying the roles of phytohormones under stressful conditions. We conclude by describing the recent progress and future prospects including limitations and challenges of phytohormone engineering for inducing abiotic stress tolerance in crop plants.

Metabolome analysis-based design and engineering of a metabolic pathway in Corynebacterium glutamicum to match rates of simultaneous utilization of D-glucose and L-arabinose.

Science.gov (United States)

Kawaguchi, Hideo; Yoshihara, Kumiko; Hara, Kiyotaka Y; Hasunuma, Tomohisa; Ogino, Chiaki; Kondo, Akihiko

2018-05-17

L-Arabinose is the second most abundant component of hemicellulose in lignocellulosic biomass, next to D-xylose. However, few microorganisms are capable of utilizing pentoses, and catabolic genes and operons enabling bacterial utilization of pentoses are typically subject to carbon catabolite repression by more-preferred carbon sources, such as D-glucose, leading to a preferential utilization of D-glucose over pentoses. In order to simultaneously utilize both D-glucose and L-arabinose at the same rate, a modified metabolic pathway was rationally designed based on metabolome analysis. Corynebacterium glutamicum ATCC 31831 utilized D-glucose and L-arabinose simultaneously at a low concentration (3.6 g/L each) but preferentially utilized D-glucose over L-arabinose at a high concentration (15 g/L each), although L-arabinose and D-glucose were consumed at comparable rates in the absence of the second carbon source. Metabolome analysis revealed that phosphofructokinase and pyruvate kinase were major bottlenecks for D-glucose and L-arabinose metabolism, respectively. Based on the results of metabolome analysis, a metabolic pathway was engineered by overexpressing pyruvate kinase in combination with deletion of araR, which encodes a repressor of L-arabinose uptake and catabolism. The recombinant strain utilized high concentrations of D-glucose and L-arabinose (15 g/L each) at the same consumption rate. During simultaneous utilization of both carbon sources at high concentrations, intracellular levels of phosphoenolpyruvate declined and acetyl-CoA levels increased significantly as compared with the wild-type strain that preferentially utilized D-glucose. These results suggest that overexpression of pyruvate kinase in the araR deletion strain increased the specific consumption rate of L-arabinose and that citrate synthase activity becomes a new bottleneck in the engineered pathway during the simultaneous utilization of D-glucose and L-arabinose. Metabolome analysis

The flexible feedstock concept in Industrial Biotechnology: Metabolic engineering of Escherichia coli, Corynebacterium glutamicum, Pseudomonas, Bacillus and yeast strains for access to alternative carbon sources.

Science.gov (United States)

Wendisch, Volker F; Brito, Luciana Fernandes; Gil Lopez, Marina; Hennig, Guido; Pfeifenschneider, Johannes; Sgobba, Elvira; Veldmann, Kareen H

2016-09-20

Most biotechnological processes are based on glucose that is either present in molasses or generated from starch by enzymatic hydrolysis. At the very high, million-ton scale production volumes, for instance for fermentative production of the biofuel ethanol or of commodity chemicals such as organic acids and amino acids, competing uses of carbon sources e.g. in human and animal nutrition have to be taken into account. Thus, the biotechnological production hosts E. coli, C. glutamicum, pseudomonads, bacilli and Baker's yeast used in these large scale processes have been engineered for efficient utilization of alternative carbon sources. This flexible feedstock concept is central to the use of non-glucose second and third generation feedstocks in the emerging bioeconomy. The metabolic engineering efforts to broaden the substrate scope of E. coli, C. glutamicum, pseudomonads, B. subtilis and yeasts to include non-native carbon sources will be reviewed. Strategies to enable simultaneous consumption of mixtures of native and non-native carbon sources present in biomass hydrolysates will be summarized and a perspective on how to further increase feedstock flexibility for the realization of biorefinery processes will be given. Copyright © 2016 Elsevier B.V. All rights reserved.

Synthetic Biology and Metabolic Engineering for Marine Carotenoids: New Opportunities and Future Prospects

Directory of Open Access Journals (Sweden)

Chonglong Wang

2014-09-01

Full Text Available Carotenoids are a class of diverse pigments with important biological roles such as light capture and antioxidative activities. Many novel carotenoids have been isolated from marine organisms to date and have shown various utilizations as nutraceuticals and pharmaceuticals. In this review, we summarize the pathways and enzymes of carotenoid synthesis and discuss various modifications of marine carotenoids. The advances in metabolic engineering and synthetic biology for carotenoid production are also reviewed, in hopes that this review will promote the exploration of marine carotenoid for their utilizations.

Synthetic Biology and Metabolic Engineering for Marine Carotenoids: New Opportunities and Future Prospects

Science.gov (United States)

Wang, Chonglong; Kim, Jung-Hun; Kim, Seon-Won

2014-01-01

Carotenoids are a class of diverse pigments with important biological roles such as light capture and antioxidative activities. Many novel carotenoids have been isolated from marine organisms to date and have shown various utilizations as nutraceuticals and pharmaceuticals. In this review, we summarize the pathways and enzymes of carotenoid synthesis and discuss various modifications of marine carotenoids. The advances in metabolic engineering and synthetic biology for carotenoid production are also reviewed, in hopes that this review will promote the exploration of marine carotenoid for their utilizations. PMID:25233369

Engineering youth service system infrastructure: Hawaii's continued efforts at large-scale implementation through knowledge management strategies.

Science.gov (United States)

Nakamura, Brad J; Mueller, Charles W; Higa-McMillan, Charmaine; Okamura, Kelsie H; Chang, Jaime P; Slavin, Lesley; Shimabukuro, Scott

2014-01-01

Hawaii's Child and Adolescent Mental Health Division provides a unique illustration of a youth public mental health system with a long and successful history of large-scale quality improvement initiatives. Many advances are linked to flexibly organizing and applying knowledge gained from the scientific literature and move beyond installing a limited number of brand-named treatment approaches that might be directly relevant only to a small handful of system youth. This article takes a knowledge-to-action perspective and outlines five knowledge management strategies currently under way in Hawaii. Each strategy represents one component of a larger coordinated effort at engineering a service system focused on delivering both brand-named treatment approaches and complimentary strategies informed by the evidence base. The five knowledge management examples are (a) a set of modular-based professional training activities for currently practicing therapists, (b) an outreach initiative for supporting youth evidence-based practices training at Hawaii's mental health-related professional programs, (c) an effort to increase consumer knowledge of and demand for youth evidence-based practices, (d) a practice and progress agency performance feedback system, and (e) a sampling of system-level research studies focused on understanding treatment as usual. We end by outlining a small set of lessons learned and a longer term vision for embedding these efforts into the system's infrastructure.

Metabolic Regulation of a Bacterial Cell System with Emphasis on Escherichia coli Metabolism

Science.gov (United States)

Shimizu, Kazuyuki

2013-01-01

It is quite important to understand the overall metabolic regulation mechanism of bacterial cells such as Escherichia coli from both science (such as biochemistry) and engineering (such as metabolic engineering) points of view. Here, an attempt was made to clarify the overall metabolic regulation mechanism by focusing on the roles of global regulators which detect the culture or growth condition and manipulate a set of metabolic pathways by modulating the related gene expressions. For this, it was considered how the cell responds to a variety of culture environments such as carbon (catabolite regulation), nitrogen, and phosphate limitations, as well as the effects of oxygen level, pH (acid shock), temperature (heat shock), and nutrient starvation. PMID:25937963

Enhanced isoprenoid production from xylose by engineered Saccharomyces cerevisiae.

Science.gov (United States)

Kwak, Suryang; Kim, Soo Rin; Xu, Haiqing; Zhang, Guo-Chang; Lane, Stephan; Kim, Heejin; Jin, Yong-Su

2017-11-01

Saccharomyces cerevisiae has limited capabilities for producing fuels and chemicals derived from acetyl-CoA, such as isoprenoids, due to a rigid flux partition toward ethanol during glucose metabolism. Despite numerous efforts, xylose fermentation by engineered yeast harboring heterologous xylose metabolic pathways was not as efficient as glucose fermentation for producing ethanol. Therefore, we hypothesized that xylose metabolism by engineered yeast might be a better fit for producing non-ethanol metabolites. We indeed found that engineered S. cerevisiae on xylose showed higher expression levels of the enzymes involved in ethanol assimilation and cytosolic acetyl-CoA synthesis than on glucose. When genetic perturbations necessary for overproducing squalene and amorphadiene were introduced into engineered S. cerevisiae capable of fermenting xylose, we observed higher titers and yields of isoprenoids under xylose than glucose conditions. Specifically, co-overexpression of a truncated HMG1 (tHMG1) and ERG10 led to substantially higher squalene accumulation under xylose than glucose conditions. In contrast to glucose utilization producing massive amounts of ethanol regardless of aeration, xylose utilization allowed much less amounts of ethanol accumulation, indicating ethanol is simultaneously re-assimilated with xylose consumption and utilized for the biosynthesis of cytosolic acetyl-CoA. In addition, xylose utilization by engineered yeast with overexpression of tHMG1, ERG10, and ADS coding for amorphadiene synthase, and the down-regulation of ERG9 resulted in enhanced amorphadiene production as compared to glucose utilization. These results suggest that the problem of the rigid flux partition toward ethanol production in yeast during the production of isoprenoids and other acetyl-CoA derived chemicals can be bypassed by using xylose instead of glucose as a carbon source. Biotechnol. Bioeng. 2017;114: 2581-2591. © 2017 Wiley Periodicals, Inc. © 2017 Wiley

Metabolic engineering of Corynebacterium glutamicum to produce GDP-L-fucose from glucose and mannose.

Science.gov (United States)

Chin, Young-Wook; Park, Jin-Byung; Park, Yong-Cheol; Kim, Kyoung Heon; Seo, Jin-Ho

2013-06-01

Wild-type Corynebacterium glutamicum was metabolically engineered to convert glucose and mannose into guanosine 5'-diphosphate (GDP)-L-fucose, a precursor of fucosyl-oligosaccharides, which are involved in various biological and pathological functions. This was done by introducing the gmd and wcaG genes of Escherichia coli encoding GDP-D-mannose-4,6-dehydratase and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase, respectively, which are known as key enzymes in the production of GDP-L-fucose from GDP-D-mannose. Coexpression of the genes allowed the recombinant C. glutamicum cells to produce GDP-L-fucose in a minimal medium containing glucose and mannose as carbon sources. The specific product formation rate was much higher during growth on mannose than on glucose. In addition, the specific product formation rate was further increased by coexpressing the endogenous phosphomanno-mutase gene (manB) and GTP-mannose-1-phosphate guanylyl-transferase gene (manC), which are involved in the conversion of mannose-6-phosphate into GDP-D-mannose. However, the overexpression of manA encoding mannose-6-phosphate isomerase, catalyzing interconversion of mannose-6-phosphate and fructose-6-phosphate showed a negative effect on formation of the target product. Overall, coexpression of gmd, wcaG, manB and manC in C. glutamicum enabled production of GDP-L-fucose at the specific rate of 0.11 mg g cell(-1) h(-1). The specific GDP-L-fucose content reached 5.5 mg g cell(-1), which is a 2.4-fold higher than that of the recombinant E. coli overexpressing gmd, wcaG, manB and manC under comparable conditions. Well-established metabolic engineering tools may permit optimization of the carbon and cofactor metabolisms of C. glutamicum to further improve their production capacity.

Efficient odd straight medium chain free fatty acid production by metabolically engineered Escherichia coli.

Science.gov (United States)

Wu, Hui; San, Ka-Yiu

2014-11-01

Free fatty acids (FFAs) can be used as precursors for the production of biofuels or chemicals. Different composition of FFAs will be useful for further modification of the biofuel/biochemical quality. Microbial biosynthesis of even chain FFAs can be achieved by introducing an acyl-acyl carrier protein thioesterase gene into E. coli. In this study, odd straight medium chain FFAs production was investigated by using metabolic engineered E. coli carrying acyl-ACP thioesterase (TE, Ricinus communis), propionyl-CoA synthase (Salmonella enterica), and β-ketoacyl-acyl carrier protein synthase III (four different sources) with supplement of extracellular propionate. By using these metabolically engineered E. coli, significant quantity of C13 and C15 odd straight-chain FFAs could be produced from glucose and propionate. The highest concentration of total odd straight chain FFAs attained was 1205 mg/L by the strain HWK201 (pXZ18, pBHE2), and 85% of the odd straight chain FFAs was C15. However, the highest percentage of odd straight chain FFAs was achieved by the strain HWK201 (pXZ18, pBHE3) of 83.2% at 48 h. This strategy was also applied successfully in strains carrying different TE, such as the medium length acyl-ACP thioesterase gene from Umbellularia californica. C11 and C13 became the major odd straight-chain FFAs. © 2014 Wiley Periodicals, Inc.

Selection Finder (SelFi: A computational metabolic engineering tool to enable directed evolution of enzymes

Directory of Open Access Journals (Sweden)

Neda Hassanpour

2017-06-01

Full Text Available Directed evolution of enzymes consists of an iterative process of creating mutant libraries and choosing desired phenotypes through screening or selection until the enzymatic activity reaches a desired goal. The biggest challenge in directed enzyme evolution is identifying high-throughput screens or selections to isolate the variant(s with the desired property. We present in this paper a computational metabolic engineering framework, Selection Finder (SelFi, to construct a selection pathway from a desired enzymatic product to a cellular host and to couple the pathway with cell survival. We applied SelFi to construct selection pathways for four enzymes and their desired enzymatic products xylitol, D-ribulose-1,5-bisphosphate, methanol, and aniline. Two of the selection pathways identified by SelFi were previously experimentally validated for engineering Xylose Reductase and RuBisCO. Importantly, SelFi advances directed evolution of enzymes as there is currently no known generalized strategies or computational techniques for identifying high-throughput selections for engineering enzymes.

Genome-scale consequences of cofactor balancing in engineered pentose utilization pathways in Saccharomyces cerevisiae.

Directory of Open Access Journals (Sweden)

Amit Ghosh

Full Text Available Biofuels derived from lignocellulosic biomass offer promising alternative renewable energy sources for transportation fuels. Significant effort has been made to engineer Saccharomyces cerevisiae to efficiently ferment pentose sugars such as D-xylose and L-arabinose into biofuels such as ethanol through heterologous expression of the fungal D-xylose and L-arabinose pathways. However, one of the major bottlenecks in these fungal pathways is that the cofactors are not balanced, which contributes to inefficient utilization of pentose sugars. We utilized a genome-scale model of S. cerevisiae to predict the maximal achievable growth rate for cofactor balanced and imbalanced D-xylose and L-arabinose utilization pathways. Dynamic flux balance analysis (DFBA was used to simulate batch fermentation of glucose, D-xylose, and L-arabinose. The dynamic models and experimental results are in good agreement for the wild type and for the engineered D-xylose utilization pathway. Cofactor balancing the engineered D-xylose and L-arabinose utilization pathways simulated an increase in ethanol batch production of 24.7% while simultaneously reducing the predicted substrate utilization time by 70%. Furthermore, the effects of cofactor balancing the engineered pentose utilization pathways were evaluated throughout the genome-scale metabolic network. This work not only provides new insights to the global network effects of cofactor balancing but also provides useful guidelines for engineering a recombinant yeast strain with cofactor balanced engineered pathways that efficiently co-utilizes pentose and hexose sugars for biofuels production. Experimental switching of cofactor usage in enzymes has been demonstrated, but is a time-consuming effort. Therefore, systems biology models that can predict the likely outcome of such strain engineering efforts are highly useful for motivating which efforts are likely to be worth the significant time investment.

Shikimic acid production in Escherichia coli: From classical metabolic engineering strategies to omics applied to improve its production

Directory of Open Access Journals (Sweden)

Juan Andrés Martínez

2015-09-01

Full Text Available Shikimic acid (SA is an intermediate of the SA pathway that is present in bacteria and plants. SA has gained great interest because it is a precursor in the synthesis of the drug oseltamivir phosphate (OSF, an efficient inhibitor of the neuraminidase enzyme of diverse seasonal influenza viruses, the avian influenza virus H5N1, and the human influenza virus H1N1. For the purposes of OSF production, SA is extracted from the pods of Chinese star anise plants (Illicium spp., yielding up to 17% of SA (dry basis content. The high demand for OSF necessary to manage a major influenza outbreak is not adequately met by industrial production using SA from plants sources. As the SA pathway is present in the model bacteria Escherichia coli, several intuitive metabolically engineered strains have been applied for its successful overproduction by biotechnological processes, resulting in strains producing up to 71 g/L of SA, with high conversion yields of up to 0.42 (mol SA/mol Glc, in both batch and fed-batch cultures using complex fermentation broths, including glucose as a carbon source and yeast extract. Global transcriptomic analyses have been performed in SA producing strains, resulting in the identification of possible key target genes for the design of a rational strain improvement strategy. Because possible target genes are involved in the transport, catabolism and interconversion of different carbon sources and metabolic intermediates outside the central carbon metabolism and SA pathways, as genes involved in diverse cellular stress responses, the development of rational cellular strain improvement strategies based on omics data constitutes a challenging task to improve SA production in currently overproducing engineered strains. In this review, we discuss the main metabolic engineering strategies that have been applied for the development of efficient SA producing strains, as the perspective of omics analysis has focused on further strain improvement

Key Process Conditions for Production of C4 Dicarboxylic Acids in Bioreactor Batch Cultures of an Engineered Saccharomyces cerevisiae Strain

NARCIS (Netherlands)

Zelle, R.M.; De Hulster, E.; Kloezen, W.; Pronk, J.T.; Van Maris, A.J.A.

2010-01-01

A recent effort to improve malic acid production by Saccharomyces cerevisiae by means of metabolic engineering resulted in a strain that produced up to 59 g liter(-1) of malate at a yield of 0.42 mol (mol glucose)(-1) in calcium carbonate-buffered shake flask cultures. With shake flasks, process

Carotenoid Metabolism in Plants: The Role of Plastids.

Science.gov (United States)

Sun, Tianhu; Yuan, Hui; Cao, Hongbo; Yazdani, Mohammad; Tadmor, Yaakov; Li, Li

2018-01-08

Carotenoids are indispensable to plants and critical in human diets. Plastids are the organelles for carotenoid biosynthesis and storage in plant cells. They exist in various types, which include proplastids, etioplasts, chloroplasts, amyloplasts, and chromoplasts. These plastids have dramatic differences in their capacity to synthesize and sequester carotenoids. Clearly, plastids play a central role in governing carotenogenic activity, carotenoid stability, and pigment diversity. Understanding of carotenoid metabolism and accumulation in various plastids expands our view on the multifaceted regulation of carotenogenesis and facilitates our efforts toward developing nutrient-enriched food crops. In this review, we provide a comprehensive overview of the impact of various types of plastids on carotenoid biosynthesis and accumulation, and discuss recent advances in our understanding of the regulatory control of carotenogenesis and metabolic engineering of carotenoids in light of plastid types in plants. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

Metabolic Engineering of the Shikimate Pathway for Production of Aromatics and Derived Compounds—Present and Future Strain Construction Strategies

Directory of Open Access Journals (Sweden)

Nils J. H. Averesch

2018-03-01

Full Text Available The aromatic nature of shikimate pathway intermediates gives rise to a wealth of potential bio-replacements for commonly fossil fuel-derived aromatics, as well as naturally produced secondary metabolites. Through metabolic engineering, the abundance of certain intermediates may be increased, while draining flux from other branches off the pathway. Often targets for genetic engineering lie beyond the shikimate pathway, altering flux deep in central metabolism. This has been extensively used to develop microbial production systems for a variety of compounds valuable in chemical industry, including aromatic and non-aromatic acids like muconic acid, para-hydroxybenzoic acid, and para-coumaric acid, as well as aminobenzoic acids and aromatic α-amino acids. Further, many natural products and secondary metabolites that are valuable in food- and pharma-industry are formed outgoing from shikimate pathway intermediates. (Reconstruction of such routes has been shown by de novo production of resveratrol, reticuline, opioids, and vanillin. In this review, strain construction strategies are compared across organisms and put into perspective with requirements by industry for commercial viability. Focus is put on enhancing flux to and through shikimate pathway, and engineering strategies are assessed in order to provide a guideline for future optimizations.

CHO On A Detox: Removing By-Product Formation Through Cell Engineering

DEFF Research Database (Denmark)

Pereira, Sara; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

Chinese Hamster Ovary (CHO) cells are the preferred hosts for the production of therapeutic glycoproteins. However, there is a need for improvement of the bioprocesses towards increased cell growth and higher productivities without compromising the product quality. Efforts to obtain tailor-made p......-made products with the desired properties that meet the requirements of regulatory authorities are continuously being made. Of equal relevance is to develop methods to engineer cell lines with improved by-product metabolism....

Dedicated Industrial Oilseed Crops as Metabolic Engineering Platforms for Sustainable Industrial Feedstock Production.

Science.gov (United States)

Zhu, Li-Hua; Krens, Frans; Smith, Mark A; Li, Xueyuan; Qi, Weicong; van Loo, Eibertus N; Iven, Tim; Feussner, Ivo; Nazarenus, Tara J; Huai, Dongxin; Taylor, David C; Zhou, Xue-Rong; Green, Allan G; Shockey, Jay; Klasson, K Thomas; Mullen, Robert T; Huang, Bangquan; Dyer, John M; Cahoon, Edgar B

2016-02-26

Feedstocks for industrial applications ranging from polymers to lubricants are largely derived from petroleum, a non-renewable resource. Vegetable oils with fatty acid structures and storage forms tailored for specific industrial uses offer renewable and potentially sustainable sources of petrochemical-type functionalities. A wide array of industrial vegetable oils can be generated through biotechnology, but will likely require non-commodity oilseed platforms dedicated to specialty oil production for commercial acceptance. Here we show the feasibility of three Brassicaceae oilseeds crambe, camelina, and carinata, none of which are widely cultivated for food use, as hosts for complex metabolic engineering of wax esters for lubricant applications. Lines producing wax esters >20% of total seed oil were generated for each crop and further improved for high temperature oxidative stability by down-regulation of fatty acid polyunsaturation. Field cultivation of optimized wax ester-producing crambe demonstrated commercial utility of these engineered crops and a path for sustainable production of other industrial oils in dedicated specialty oilseeds.

Genome-scale metabolic analysis of Clostridium thermocellum for bioethanol production

Directory of Open Access Journals (Sweden)

Brooks J Paul

2010-03-01

Full Text Available Abstract Background Microorganisms possess diverse metabolic capabilities that can potentially be leveraged for efficient production of biofuels. Clostridium thermocellum (ATCC 27405 is a thermophilic anaerobe that is both cellulolytic and ethanologenic, meaning that it can directly use the plant sugar, cellulose, and biochemically convert it to ethanol. A major challenge in using microorganisms for chemical production is the need to modify the organism to increase production efficiency. The process of properly engineering an organism is typically arduous. Results Here we present a genome-scale model of C. thermocellum metabolism, iSR432, for the purpose of establishing a computational tool to study the metabolic network of C. thermocellum and facilitate efforts to engineer C. thermocellum for biofuel production. The model consists of 577 reactions involving 525 intracellular metabolites, 432 genes, and a proteomic-based representation of a cellulosome. The process of constructing this metabolic model led to suggested annotation refinements for 27 genes and identification of areas of metabolism requiring further study. The accuracy of the iSR432 model was tested using experimental growth and by-product secretion data for growth on cellobiose and fructose. Analysis using this model captures the relationship between the reduction-oxidation state of the cell and ethanol secretion and allowed for prediction of gene deletions and environmental conditions that would increase ethanol production. Conclusions By incorporating genomic sequence data, network topology, and experimental measurements of enzyme activities and metabolite fluxes, we have generated a model that is reasonably accurate at predicting the cellular phenotype of C. thermocellum and establish a strong foundation for rational strain design. In addition, we are able to draw some important conclusions regarding the underlying metabolic mechanisms for observed behaviors of C. thermocellum

Fuel Saving Strategy in Spark Ignition Engine Using Fuzzy Logic Engine Torque Control

OpenAIRE

Aris Triwiyatno; Sumardi

2012-01-01

In the case of injection gasoline engine, or better known as spark ignition engines, an effort to improve engine performance as well as to reduce fuel consumption is a fairly complex problem. Generally, engine performance improvement efforts will lead to increase in fuel consumption. However, this problem can be solved by implementing engine torque control based on intelligent regulation such as the fuzzy logic inference system. In this study, fuzzy logic engine torque regulation is used to c...

Simulation Modeling to Compare High-Throughput, Low-Iteration Optimization Strategies for Metabolic Engineering.

Science.gov (United States)

Heinsch, Stephen C; Das, Siba R; Smanski, Michael J

2018-01-01

Increasing the final titer of a multi-gene metabolic pathway can be viewed as a multivariate optimization problem. While numerous multivariate optimization algorithms exist, few are specifically designed to accommodate the constraints posed by genetic engineering workflows. We present a strategy for optimizing expression levels across an arbitrary number of genes that requires few design-build-test iterations. We compare the performance of several optimization algorithms on a series of simulated expression landscapes. We show that optimal experimental design parameters depend on the degree of landscape ruggedness. This work provides a theoretical framework for designing and executing numerical optimization on multi-gene systems.

Systems metabolic engineering as an enabling technology in accomplishing sustainable development goals.

Science.gov (United States)

Yang, Dongsoo; Cho, Jae Sung; Choi, Kyeong Rok; Kim, Hyun Uk; Lee, Sang Yup

2017-09-01

With pressing issues arising in recent years, the United Nations proposed 17 Sustainable Development Goals (SDGs) as an agenda urging international cooperations for sustainable development. In this perspective, we examine the roles of systems metabolic engineering (SysME) and its contribution to improving the quality of life and protecting our environment, presenting how this field of study offers resolutions to the SDGs with relevant examples. We conclude with offering our opinion on the current state of SysME and the direction it should move forward in the generations to come, explicitly focusing on addressing the SDGs. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Engineering Mycorrhizal Symbioses to Alter Plant Metabolism and Improve Crop Health

Directory of Open Access Journals (Sweden)

Katherine E. French

2017-07-01

Full Text Available Creating sustainable bioeconomies for the 21st century relies on optimizing the use of biological resources to improve agricultural productivity and create new products. Arbuscular mycorrhizae (phylum Glomeromycota form symbiotic relationships with over 80% of vascular plants. In return for carbon, these fungi improve plant health and tolerance to environmental stress. This symbiosis is over 400 million years old and there are currently over 200 known arbuscular mycorrhizae, with dozens of new species described annually. Metagenomic sequencing of native soil communities, from species-rich meadows to mangroves, suggests biologically diverse habitats support a variety of mycorrhizal species with potential agricultural, medical, and biotechnological applications. This review looks at the effect of mycorrhizae on plant metabolism and how we can harness this symbiosis to improve crop health. I will first describe the mechanisms that underlie this symbiosis and what physiological, metabolic, and environmental factors trigger these plant-fungal relationships. These include mycorrhizal manipulation of host genetic expression, host mitochondrial and plastid proliferation, and increased production of terpenoids and jasmonic acid by the host plant. I will then discuss the effects of mycorrhizae on plant root and foliar secondary metabolism. I subsequently outline how mycorrhizae induce three key benefits in crops: defense against pathogen and herbivore attack, drought resistance, and heavy metal tolerance. I conclude with an overview of current efforts to harness mycorrhizal diversity to improve crop health through customized inoculum. I argue future research should embrace synthetic biology to create mycorrhizal chasses with improved symbiotic abilities and potentially novel functions to improve plant health. As the effects of climate change and anthropogenic disturbance increase, the global diversity of arbuscular mycorrhizal fungi should be monitored

Terminal alkenes as versatile chemical reporter groups for metabolic oligosaccharide engineering.

Science.gov (United States)

Späte, Anne-Katrin; Schart, Verena F; Schöllkopf, Sophie; Niederwieser, Andrea; Wittmann, Valentin

2014-12-08

The Diels-Alder reaction with inverse electron demand (DAinv reaction) of 1,2,4,5-tetrazines with electron rich or strained alkenes was proven to be a bioorthogonal ligation reaction that proceeds fast and with high yields. An important application of the DAinv reaction is metabolic oligosaccharide engineering (MOE) which allows the visualization of glycoconjugates in living cells. In this approach, a sugar derivative bearing a chemical reporter group is metabolically incorporated into cellular glycoconjugates and subsequently derivatized with a probe by means of a bioorthogonal ligation reaction. Here, we investigated a series of new mannosamine and glucosamine derivatives with carbamate-linked side chains of varying length terminated by alkene groups and their suitability for labeling cell-surface glycans. Kinetic investigations showed that the reactivity of the alkenes in DAinv reactions increases with growing chain length. When applied to MOE, one of the compounds, peracetylated N-butenyloxycarbonylmannosamine, was especially well suited for labeling cell-surface glycans. Obviously, the length of its side chain represents the optimal balance between incorporation efficiency and speed of the labeling reaction. Sialidase treatment of the cells before the bioorthogonal labeling reaction showed that this sugar derivative is attached to the glycans in form of the corresponding sialic acid derivative and not epimerized to another hexosamine derivative to a considerable extent. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhancing Carbon Fixation by Metabolic Engineering: A Model System of Complex Network Modulation

Energy Technology Data Exchange (ETDEWEB)

Dr. Gregory Stephanopoulos

2008-04-10

In the first two years of this research we focused on the development of a DNA microarray for transcriptional studies in the photosynthetic organism Synechocystis and the elucidation of the metabolic pathway for biopolymer synthesis in this organism. In addition we also advanced the molecular biological tools for metabolic engineering of biopolymer synthesis in Synechocystis and initiated a series of physiological studies for the elucidation of the carbon fixing pathways and basic central carbon metabolism of these organisms. During the last two-year period we focused our attention on the continuation and completion of the last task, namely, the development of tools for basic investigations of the physiology of these cells through, primarily, the determination of their metabolic fluxes. The reason for this decision lies in the importance of fluxes as key indicators of physiology and the high level of information content they carry in terms of identifying rate limiting steps in a metabolic pathway. While flux determination is a well-advanced subject for heterotrophic organisms, for the case of autotrophic bacteria, like Synechocystis, some special challenges had to be overcome. These challenges stem mostly from the fact that if one uses {sup 13}C labeled CO{sub 2} for flux determination, the {sup 13}C label will mark, at steady state, all carbon atoms of all cellular metabolites, thus eliminating the necessary differentiation required for flux determination. This peculiarity of autotrophic organisms makes it imperative to carry out flux determination under transient conditions, something that had not been accomplished before. We are pleased to report that we have solved this problem and we are now able to determine fluxes in photosynthetic organisms from stable isotope labeling experiments followed by measurements of label enrichment in cellular metabolites using Gas Chromatography-Mass Spectrometry. We have conducted extensive simulations to test the method and

Developing a set of strong intronic promoters for robust metabolic engineering in oleaginous Rhodotorula (Rhodosporidium) yeast species.

Science.gov (United States)

Liu, Yanbin; Yap, Sihui Amy; Koh, Chong Mei John; Ji, Lianghui

2016-11-25

Red yeast species in the Rhodotorula/Rhodosporidium genus are outstanding producers of triacylglyceride and cell biomass. Metabolic engineering is expected to further enhance the productivity and versatility of these hosts for the production of biobased chemicals and fuels. Promoters with strong activity during oil-accumulation stage are critical tools for metabolic engineering of these oleaginous yeasts. The upstream DNA sequences of 6 genes involved in lipid biosynthesis or accumulation in Rhodotorula toruloides were studied by luciferase reporter assay. The promoter of perilipin/lipid droplet protein 1 gene (LDP1) displayed much stronger activity (4-11 folds) than that of glyceraldehyde-3-phosphate dehydrogenase gene (GPD1), one of the strongest promoters known in yeasts. Depending on the stage of cultivation, promoter of acetyl-CoA carboxylase gene (ACC1) and fatty acid synthase β subunit gene (FAS1) exhibited intermediate strength, displaying 50-160 and 20-90% levels of GPD1 promoter, respectively. Interestingly, introns significantly modulated promoter strength at high frequency. The incorporation of intron 1 and 2 of LDP1 (LDP1in promoter) enhanced its promoter activity by 1.6-3.0 folds. Similarly, the strength of ACC1 promoter was enhanced by 1.5-3.2 folds if containing intron 1. The intron 1 sequences of ACL1 and FAS1 also played significant regulatory roles. When driven by the intronic promoters of ACC1 and LDP1 (ACC1in and LDP1in promoter, respectively), the reporter gene expression were up-regulated by nitrogen starvation, independent of de novo oil biosynthesis and accumulation. As a proof of principle, overexpression of the endogenous acyl-CoA-dependent diacylglycerol acyltransferase 1 gene (DGA1) by LDP1in promoter was significantly more efficient than GPD1 promoter in enhancing lipid accumulation. Intronic sequences play an important role in regulating gene expression in R. toruloides. Three intronic promoters, LDP1in, ACC1in and FAS1in, are

Metabolic engineering of β-oxidation in Penicillium chrysogenum for improved semi-synthetic cephalosporin biosynthesis.

Science.gov (United States)

Veiga, Tânia; Gombert, Andreas K; Landes, Nils; Verhoeven, Maarten D; Kiel, Jan A K W; Krikken, Arjen M; Nijland, Jeroen G; Touw, Hesselien; Luttik, Marijke A H; van der Toorn, John C; Driessen, Arnold J M; Bovenberg, Roel A L; van den Berg, Marco A; van der Klei, Ida J; Pronk, Jack T; Daran, Jean-Marc

2012-07-01

Industrial production of semi-synthetic cephalosporins by Penicillium chrysogenum requires supplementation of the growth media with the side-chain precursor adipic acid. In glucose-limited chemostat cultures of P. chrysogenum, up to 88% of the consumed adipic acid was not recovered in cephalosporin-related products, but used as an additional carbon and energy source for growth. This low efficiency of side-chain precursor incorporation provides an economic incentive for studying and engineering the metabolism of adipic acid in P. chrysogenum. Chemostat-based transcriptome analysis in the presence and absence of adipic acid confirmed that adipic acid metabolism in this fungus occurs via β-oxidation. A set of 52 adipate-responsive genes included six putative genes for acyl-CoA oxidases and dehydrogenases, enzymes responsible for the first step of β-oxidation. Subcellular localization of the differentially expressed acyl-CoA oxidases and dehydrogenases revealed that the oxidases were exclusively targeted to peroxisomes, while the dehydrogenases were found either in peroxisomes or in mitochondria. Deletion of the genes encoding the peroxisomal acyl-CoA oxidase Pc20g01800 and the mitochondrial acyl-CoA dehydrogenase Pc20g07920 resulted in a 1.6- and 3.7-fold increase in the production of the semi-synthetic cephalosporin intermediate adipoyl-6-APA, respectively. The deletion strains also showed reduced adipate consumption compared to the reference strain, indicating that engineering of the first step of β-oxidation successfully redirected a larger fraction of adipic acid towards cephalosporin biosynthesis. Copyright © 2012 Elsevier Inc. All rights reserved.

Metabolic Engineering of the Actinomycete Amycolatopsis sp. Strain ATCC 39116 towards Enhanced Production of Natural Vanillin

OpenAIRE

Fleige, Christian; Meyer, Florian; Steinbüchel, Alexander

2016-01-01

The Gram-positive bacterium Amycolatopsis sp. ATCC 39116 is used for the fermentative production of natural vanillin from ferulic acid on an industrial scale. The strain is known for its outstanding tolerance to this toxic product. In order to improve the productivity of the fermentation process, the strain's metabolism was engineered for higher final concentrations and molar yields. Degradation of vanillin could be decreased by more than 90% through deletion of the vdh gene, which codes for ...

Bioethanol a Microbial Biofuel Metabolite; New Insights of Yeasts Metabolic Engineering

Directory of Open Access Journals (Sweden)

Khaled A. Selim

2018-03-01

Full Text Available Scarcity of the non-renewable energy sources, global warming, environmental pollution, and raising the cost of petroleum are the motive for the development of renewable, eco-friendly fuels production with low costs. Bioethanol production is one of the promising materials that can subrogate the petroleum oil, and it is considered recently as a clean liquid fuel or a neutral carbon. Diverse microorganisms such as yeasts and bacteria are able to produce bioethanol on a large scale, which can satisfy our daily needs with cheap and applicable methods. Saccharomyces cerevisiae and Pichia stipitis are two of the pioneer yeasts in ethanol production due to their abilities to produce a high amount of ethanol. The recent focus is directed towards lignocellulosic biomass that contains 30–50% cellulose and 20–40% hemicellulose, and can be transformed into glucose and fundamentally xylose after enzymatic hydrolysis. For this purpose, a number of various approaches have been used to engineer different pathways for improving the bioethanol production with simultaneous fermentation of pentose and hexoses sugars in the yeasts. These approaches include metabolic and flux analysis, modeling and expression analysis, followed by targeted deletions or the overexpression of key genes. In this review, we highlight and discuss the current status of yeasts genetic engineering for enhancing bioethanol production, and the conditions that influence bioethanol production.

The National Air Intelligence Center Software Process Improvement Effort (NAIC SPI)

National Research Council Canada - National Science Library

Blankenship, Donald

2001-01-01

...) Software Process Improvements effort. The objective of this effort was for the contractor to provide engineering and software process improvement for NAIC/SCD to reach SEI's CMM Level 2 in process maturity...

Saccharomyces cerevisiae engineered for xylose metabolism requires gluconeogenesis and the oxidative branch of the pentose phosphate pathway for aerobic xylose assimilation

Science.gov (United States)

Saccharomyces strains engineered to ferment xylose using Scheffersomyces stipitis xylose reductase (XR) and xylitol dehydrogenase (XDH) genes appear to be limited by metabolic imbalances due to differing cofactor specificities of XR and XDH. The S. stipitis XR, which uses nicotinamide adenine dinucl...

Sustainable source of omega-3 eicosapentaenoic acid from metabolically engineered Yarrowia lipolytica: from fundamental research to commercial production.

Science.gov (United States)

Xie, Dongming; Jackson, Ethel N; Zhu, Quinn

2015-02-01

The omega-3 fatty acids, cis-5, 8, 11, 14, and 17-eicosapentaenoic acid (C20:5; EPA) and cis-4, 7, 10, 13, 16, and 19-docosahexaenoic acid (C22:6; DHA), have wide-ranging benefits in improving heart health, immune function, mental health, and infant cognitive development. Currently, the major source for EPA and DHA is from fish oil, and a minor source of DHA is from microalgae. With the increased demand for EPA and DHA, DuPont has developed a clean and sustainable source of the omega-3 fatty acid EPA through fermentation using metabolically engineered strains of Yarrowia lipolytica. In this mini-review, we will focus on DuPont's technology for EPA production. Specifically, EPA biosynthetic and supporting pathways have been introduced into the oleaginous yeast to synthesize and accumulate EPA under fermentation conditions. This Yarrowia platform can also produce tailored omega-3 (EPA, DHA) and/or omega-6 (ARA, GLA) fatty acid mixtures in the cellular lipid profiles. Fundamental research such as metabolic engineering for strain construction, high-throughput screening for strain selection, fermentation process development, and process scale-up were all needed to achieve the high levels of EPA titer, rate, and yield required for commercial application. Here, we summarize how we have combined the fundamental bioscience and the industrial engineering skills to achieve large-scale production of Yarrowia biomass containing high amounts of EPA, which led to two commercial products, New Harvest™ EPA oil and Verlasso® salmon.

Compartmentalized Metabolic Engineering for Artemisinin Biosynthesis and Effective Malaria Treatment by Oral Delivery of Plant Cells.

Science.gov (United States)

Malhotra, Karan; Subramaniyan, Mayavan; Rawat, Khushboo; Kalamuddin, Md; Qureshi, M Irfan; Malhotra, Pawan; Mohmmed, Asif; Cornish, Katrina; Daniell, Henry; Kumar, Shashi

2016-11-07

Artemisinin is highly effective against drug-resistant malarial parasites, which affects nearly half of the global population and kills >500 000 people each year. The primary cost of artemisinin is the very expensive process used to extract and purify the drug from Artemisia annua. Elimination of this apparently unnecessary step will make this potent antimalarial drug affordable to the global population living in endemic regions. Here we reported the oral delivery of a non-protein drug artemisinin biosynthesized (∼0.8 mg/g dry weight) at clinically meaningful levels in tobacco by engineering two metabolic pathways targeted to three different cellular compartments (chloroplast, nucleus, and mitochondria). The doubly transgenic lines showed a three-fold enhancement of isopentenyl pyrophosphate, and targeting AACPR, DBR2, and CYP71AV1 to chloroplasts resulted in higher expression and an efficient photo-oxidation of dihydroartemisinic acid to artemisinin. Partially purified extracts from the leaves of transgenic tobacco plants inhibited in vitro growth progression of Plasmodium falciparum-infected red blood cells. Oral feeding of whole intact plant cells bioencapsulating the artemisinin reduced the parasitemia levels in challenged mice in comparison with commercial drug. Such novel synergistic approaches should facilitate low-cost production and delivery of artemisinin and other drugs through metabolic engineering of edible plants. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Plasmid-encoded biosynthetic genes alleviate metabolic disadvantages while increasing glucose conversion to shikimate in an engineered Escherichia coli strain.

Science.gov (United States)

Rodriguez, Alberto; Martínez, Juan A; Millard, Pierre; Gosset, Guillermo; Portais, Jean-Charles; Létisse, Fabien; Bolivar, Francisco

2017-06-01

Metabolic engineering strategies applied over the last two decades to produce shikimate (SA) in Escherichia coli have resulted in a battery of strains bearing many expression systems. However, the effects that these systems have on the host physiology and how they impact the production of SA are still not well understood. In this work we utilized an engineered E. coli strain to determine the consequences of carrying a vector that promotes SA production from glucose with a high-yield but that is also expected to impose a significant cellular burden. Kinetic comparisons in fermentors showed that instead of exerting a negative effect, the sole presence of the plasmid increased glucose consumption without diminishing the growth rate. By constitutively expressing a biosynthetic operon from this vector, the more active glycolytic metabolism was exploited to redirect intermediates toward the production of SA, which further increased the glucose consumption rate and avoided excess acetate production. Fluxomics and metabolomics experiments revealed a global remodeling of the carbon and energy metabolism in the production strain, where the increased SA production reduced the carbon available for oxidative and fermentative pathways. Moreover, the results showed that the production of SA relies on a specific setup of the pentose phosphate pathway, where both its oxidative and non-oxidative branches are strongly activated to supply erythrose-4-phosphate and balance the NADPH requirements. This work improves our understanding of the metabolic reorganization observed in E. coli in response to the plasmid-based expression of the SA biosynthetic pathway. Biotechnol. Bioeng. 2017;114: 1319-1330. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Metabolic Engineering of Oleaginous Yeasts for Fatty Alcohol Production

Energy Technology Data Exchange (ETDEWEB)

Wang, Wei; Wei, Hui; Knoshaug, Eric; Van Wychen, Stefanie; Xu, Qi; Himmel, Michael E.; Zhang, Min

2016-04-25

To develop pathways for advanced biological upgrading of sugars to hydrocarbons, we are seeking biological approaches to produce high carbon efficiency intermediates amenable to separations and catalytic upgrading to hydrocarbon fuels. In this study, we successfully demonstrated fatty alcohol production by oleaginous yeasts Yarrowia lipolytica and Lipomyces starkeyi by expressing a bacteria-derived fatty acyl-CoA reductase (FAR). Moreover, we find higher extracellular distribution of fatty alcohols produced by FAR-expressing L. starkeyi strain as compared to Y. lipolytica strain, which would benefit the downstream product recovery process. In both oleaginous yeasts, long chain length saturated fatty alcohols were predominant, accounting for more than 85% of the total fatty alcohols produced. To the best of our knowledge, this is the first report of fatty alcohol production in L. starkeyi. Taken together, our work demonstrates that in addition to Y. lipolytica, L. starkeyi can also serve as a platform organism for production of fatty acid-derived biofuels and bioproducts via metabolic engineering. We believe strain and process development both will significantly contribute to our goal of producing scalable and cost-effective fatty alcohols from renewable biomass.

Metabolic engineering of Dunaliella salina for production of ketocarotenoids.

Science.gov (United States)

Anila, N; Simon, Daris P; Chandrashekar, Arun; Ravishankar, G A; Sarada, R

2016-03-01

Dunaliella is a commercially important marine alga producing high amount of β-carotene. The use of Dunaliella as a potential transgenic system for the production of recombinant proteins has been recently recognized. The present study reports for the first time the metabolic engineering of carotenoid biosynthesis in Dunaliella salina for ketocarotenoid production. The pathway modification included the introduction of a bkt gene from H. pluvialis encoding β-carotene ketolase (4,4'β-oxygenase) along with chloroplast targeting for the production of ketocarotenoids. The bkt under the control of Dunaliella Rubisco smaller subunit promoter along with its transit peptide sequence was introduced into the alga through standardized Agrobacterium-mediated transformation procedure. The selected transformants were confirmed using GFP and GUS expression, PCR and southern blot analysis. A notable upregulation of the endogenous hydroxylase level of transformants was observed where the BKT expression was higher in nutrient-limiting conditions. Carotenoid analysis of the transformants through HPLC and MS analysis showed the presence of astaxanthin and canthaxanthin with maximum content of 3.5 and 1.9 µg/g DW, respectively. The present study reports the feasibility of using D. salina for the production of ketocarotenoids including astaxanthin.

Harnessing the respiration machinery for high-yield production of chemicals in metabolically engineered Lactococcus lactis

DEFF Research Database (Denmark)

Liu, Jianming; Wang, Zhihao; Kandasamy, Vijayalakshmi

2017-01-01

on metabolically engineered Lactococcus lactis strains to optimize the production of acetoin and (R,R)−2,3-butanediol (R-BDO). In the absence of an external electron acceptor, a surplus of two NADH per acetoin molecule is produced. We found that a fully activated respiration was able to efficiently regenerate NAD......+, and a high titer of 371 mM (32 g/L) of acetoin was obtained with a yield of 82% of the theoretical maximum. Subsequently, we extended the metabolic pathway from acetoin to R-BDO by introducing the butanediol dehydrogenase gene from Bacillus subtilis. Since one mole of NADH is consumed when acetoin...... is converted into R-BDO per mole, only the excess of NADH needs to be oxidized via respiration. Either by fine-tuning the respiration capacity or by using a dual-phase fermentation approach involving a switch from fully respiratory to non-respiratory conditions, we obtained 361 mM (32 g/L) R-BDO with a yield...

System Engineering Concept Demonstration, Effort Summary. Volume 1

Science.gov (United States)

1992-12-01

involve only the system software, user frameworks and user tools. U •User Tool....s , Catalyst oExternal 00 Computer Framwork P OSystems • •~ Sysytem...analysis, synthesis, optimization, conceptual design of Catalyst. The paper discusses the definition, design, test, and evaluation; operational concept...This approach will allow system engineering The conceptual requirements for the Process Model practitioners to recognize and tailor the model. This

Computational design of auxotrophy-dependent microbial biosensors for combinatorial metabolic engineering experiments.

Science.gov (United States)

Tepper, Naama; Shlomi, Tomer

2011-01-21

Combinatorial approaches in metabolic engineering work by generating genetic diversity in a microbial population followed by screening for strains with improved phenotypes. One of the most common goals in this field is the generation of a high rate chemical producing strain. A major hurdle with this approach is that many chemicals do not have easy to recognize attributes, making their screening expensive and time consuming. To address this problem, it was previously suggested to use microbial biosensors to facilitate the detection and quantification of chemicals of interest. Here, we present novel computational methods to: (i) rationally design microbial biosensors for chemicals of interest based on substrate auxotrophy that would enable their high-throughput screening; (ii) predict engineering strategies for coupling the synthesis of a chemical of interest with the production of a proxy metabolite for which high-throughput screening is possible via a designed bio-sensor. The biosensor design method is validated based on known genetic modifications in an array of E. coli strains auxotrophic to various amino-acids. Predicted chemical production rates achievable via the biosensor-based approach are shown to potentially improve upon those predicted by current rational strain design approaches. (A Matlab implementation of the biosensor design method is available via http://www.cs.technion.ac.il/~tomersh/tools).

Advances in metabolic engineering of yeast Saccharomyces cerevisiae for production of chemicals

DEFF Research Database (Denmark)

Borodina, Irina; Nielsen, Jens

2014-01-01

Yeast Saccharomyces cerevisiae is an important industrial host for production of enzymes, pharmaceutical and nutraceutical ingredients and recently also commodity chemicals and biofuels. Here, we review the advances in modeling and synthetic biology tools and how these tools can speed up the deve......Yeast Saccharomyces cerevisiae is an important industrial host for production of enzymes, pharmaceutical and nutraceutical ingredients and recently also commodity chemicals and biofuels. Here, we review the advances in modeling and synthetic biology tools and how these tools can speed up...... the development of yeast cell factories. We also present an overview of metabolic engineering strategies for developing yeast strains for production of polymer monomers: lactic, succinic, and cis,cis-muconic acids. S. cerevisiae has already firmly established itself as a cell factory in industrial biotechnology...

A real-time control system of gene expression using ligand-bound nucleic acid aptamer for metabolic engineering.

Science.gov (United States)

Wang, Jing; Cui, Xun; Yang, Le; Zhang, Zhe; Lv, Liping; Wang, Haoyuan; Zhao, Zhenmin; Guan, Ningzi; Dong, Lichun; Chen, Rachel

2017-07-01

Artificial control of bio-functions through regulating gene expression is one of the most important and attractive technologies to build novel living systems that are useful in the areas of chemical synthesis, nanotechnology, pharmacology, cell biology. Here, we present a novel real-time control system of gene regulation that includes an enhancement element by introducing duplex DNA aptamers upstream promoter and a repression element by introducing a RNA aptamer upstream ribosome binding site. With the presence of ligands corresponding to the DNA aptamers, the expression of the target gene can be potentially enhanced at the transcriptional level by strengthening the recognition capability of RNAP to the recognition region and speeding up the separation efficiency of the unwinding region due to the induced DNA bubble around the thrombin-bound aptamers; while with the presence of RNA aptamer ligand, the gene expression can be repressed at the translational level by weakening the recognition capability of ribosome to RBS due to the shielding of RBS by the formed aptamer-ligand complex upstream RBS. The effectiveness and potential utility of the developed gene regulation system were demonstrated by regulating the expression of ecaA gene in the cell-free systems. The realistic metabolic engineering application of the system has also tested by regulating the expression of mgtC gene and thrombin cDNA in Escherichia coli JD1021 for controlling metabolic flux and improving thrombin production, verifying that the real-time control system of gene regulation is able to realize the dynamic regulation of gene expression with potential applications in bacterial physiology studies and metabolic engineering. Copyright © 2017. Published by Elsevier Inc.

Metabolic transcription analysis of engineered Escherichia coli strains that overproduce L-phenylalanine

Directory of Open Access Journals (Sweden)

Gosset Guillermo

2007-09-01

Full Text Available Abstract Background The rational design of L-phenylalanine (L-Phe overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS and overexpression of key genes (DAHP synthase, transketolase and chorismate mutase-prephenate dehydratase, reaching yields of 0.33 (g-Phe/g-Glc, which correspond to 60% of theoretical maximum. Although genetic modifications introduced into the cell for the generation of overproducing organisms are specifically targeted to a particular pathway, these can trigger unexpected transcriptional responses of several genes. In the current work, metabolic transcription analysis (MTA of both L-Phe overproducing and non-engineered strains using Real-Time PCR was performed, allowing the detection of transcriptional responses to PTS deletion and plasmid presence of genes related to central carbon metabolism. This MTA included 86 genes encoding enzymes of glycolysis, gluconeogenesis, pentoses phosphate, tricarboxylic acid cycle, fermentative and aromatic amino acid pathways. In addition, 30 genes encoding regulatory proteins and transporters for aromatic compounds and carbohydrates were also analyzed. Results MTA revealed that a set of genes encoding carbohydrate transporters (galP, mglB, gluconeogenic (ppsA, pckA and fermentative enzymes (ldhA were significantly induced, while some others were down-regulated such as ppc, pflB, pta and ackA, as a consequence of PTS inactivation. One of the most relevant findings was the coordinated up-regulation of several genes that are exclusively gluconeogenic (fbp, ppsA, pckA, maeB, sfcA, and glyoxylate shunt in the best PTS- L-Phe overproducing strain (PB12-ev2. Furthermore, it was noticeable that most of the TCA genes showed a strong up-regulation in the presence of multicopy plasmids by an unknown mechanism. A group of genes exhibited transcriptional responses to

Combinatorial metabolic engineering of industrial Gluconobacter oxydans DSM2343 for boosting 5-keto-D-gluconic acid accumulation.

Science.gov (United States)

Yuan, Jianfeng; Wu, Mianbin; Lin, Jianping; Yang, Lirong

2016-05-17

L-(+)-tartaric acid (L-TA) is an important organic acid, which is produced from the cream of tartar or stereospecific hydrolysis of the cis-epoxysuccinate. The former method is limited by the availability of raw material and the latter is dependent on the petrochemical material. Thus, new processes for the economical preparation of L-TA from carbohydrate or renewable resource would be much more attractive. Production of 5-keto-D-gluconate (5-KGA) from glucose by Gluconobacter oxydans is the first step to produce L-TA. The aim of this work is to enhance 5-KGA accumulation using combinatorial metabolic engineering strategies in G. oxydans. The sldAB gene, encoding sorbitol dehydrogenase, was overexpressed in an industrial strain G. oxydans ZJU2 under a carefully selected promoter, P0169. To enhance the efficiency of the oxidation by sldAB, the coenzyme pyrroloquinoline quinone (PQQ) and respiratory chain were engineered. Besides, the role in sldAB overexpression, coenzyme and respiratory chain engineering and their subsequent effects on 5-KGA production were investigated. An efficient, stable recombinant strain was constructed, whereas the 5-KGA production could be enhanced. By self-overexpressing the sldAB gene in G. oxydans ZJU2 under the constitutive promoter P0169, the resulting strain, G. oxydans ZJU3, produced 122.48 ± 0.41 g/L of 5-KGA. Furthermore, through the coenzyme and respiratory chain engineering, the titer and productivity of 5-KGA reached 144.52 ± 2.94 g/L and 2.26 g/(L · h), respectively, in a 15 L fermenter. It could be further improved the 5-KGA titer by 12.10 % through the fed-batch fermentation under the pH shift and dissolved oxygen tension (DOT) control condition, obtained 162 ± 2.12 g/L with the productivity of 2.53 g/(L · h) within 64 h. The 5-KGA production could be significantly enhanced with the combinatorial metabolic engineering strategy in Gluconobacter strain, including sldAB overexpression, coenzyme

Volatile terpenoids: multiple functions, biosynthesis, modulation and manipulation by genetic engineering.

Science.gov (United States)

Abbas, Farhat; Ke, Yanguo; Yu, Rangcai; Yue, Yuechong; Amanullah, Sikandar; Jahangir, Muhammad Muzammil; Fan, Yanping

2017-11-01

Terpenoids play several physiological and ecological functions in plant life through direct and indirect plant defenses and also in human society because of their enormous applications in the pharmaceutical, food and cosmetics industries. Through the aid of genetic engineering its role can by magnified to broad spectrum by improving genetic ability of crop plants, enhancing the aroma quality of fruits and flowers and the production of pharmaceutical terpenoids contents in medicinal plants. Terpenoids are structurally diverse and the most abundant plant secondary metabolites, playing an important role in plant life through direct and indirect plant defenses, by attracting pollinators and through different interactions between the plants and their environment. Terpenoids are also significant because of their enormous applications in the pharmaceutical, food and cosmetics industries. Due to their broad distribution and functional versatility, efforts are being made to decode the biosynthetic pathways and comprehend the regulatory mechanisms of terpenoids. This review summarizes the recent advances in biosynthetic pathways, including the spatiotemporal, transcriptional and post-transcriptional regulatory mechanisms. Moreover, we discuss the multiple functions of the terpene synthase genes (TPS), their interaction with the surrounding environment and the use of genetic engineering for terpenoid production in model plants. Here, we also provide an overview of the significance of terpenoid metabolic engineering in crop protection, plant reproduction and plant metabolic engineering approaches for pharmaceutical terpenoids production and future scenarios in agriculture, which call for sustainable production platforms by improving different plant traits.

Advances in metabolic engineering in the microbial production of fuels and chemicals from C1 gas.

Science.gov (United States)

Humphreys, Christopher M; Minton, Nigel P

2018-04-01

The future sustainable production of chemicals and fuels from non-petrochemical sources, while at the same time reducing greenhouse gas (GHG) emissions, represent two of society's greatest challenges. Microbial chassis able to grow on waste carbon monoxide (CO) and carbon dioxide (CO 2 ) can provide solutions to both. Ranging from the anaerobic acetogens, through the aerobic chemoautotrophs to the photoautotrophic cyanobacteria, they are able to convert C1 gases into a range of chemicals and fuels which may be enhanced and extended through appropriate metabolic engineering. The necessary improvements will be facilitated by the increasingly sophisticated gene tools that are beginning to emerge as part of the Synthetic Biology revolution. These tools, in combination with more accurate metabolic and genome scale models, will enable C1 chassis to deliver their full potential. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Metabolic engineering to expand the substrate spectrum of Pseudomonas putida toward sucrose.

Science.gov (United States)

Löwe, Hannes; Schmauder, Lukas; Hobmeier, Karina; Kremling, Andreas; Pflüger-Grau, Katharina

2017-08-01

Sucrose is an important disaccharide used as a substrate in many industrial applications. It is a major component of molasses, a cheap by-product of the sugar industry. Unfortunately, not all industrially relevant organisms, among them Pseudomonas putida, are capable of metabolizing sucrose. We chose a metabolic engineering approach to circumvent this blockage and equip P. putida with the activities necessary to consume sucrose. Therefore, we constructed a pair of broad-host range mini-transposons (pSST - sucrose splitting transposon), carrying either cscA, encoding an invertase able to split sucrose into glucose and fructose, or additionally cscB, encoding a sucrose permease. Introduction of cscA was sufficient to convey sucrose consumption and the additional presence of cscB had no further effect, though the sucrose permease was built and localized to the membrane. Sucrose was split extracellularly by the activity of the invertase CscA leaking out of the cell. The transposons were also used to confer sucrose consumption to Cupriavidus necator. Interestingly, in this strain, CscB acted as a glucose transporter, such that C. necator also gained the ability to grow on glucose. Thus, the pSST transposons are functional tools to extend the substrate spectrum of Gram-negative bacterial strains toward sucrose. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Engineering Background: Modern Formats and Challenges of Conceptual Engineering

Science.gov (United States)

Khamidullina, A. F.; Kuzmina, M. A.; Khusnutdinova, E. M.; Konakhina, I. A.

2017-09-01

This paper describes the analysis of problems and development perspectives of engineering education in our and other countries. Special attention is given to modern formats of education that motivate creative efforts of engineers-to-be as well as issues of conceptual engineering taking the challenges of modernity into account.

Production of 3-hydroxypropionic acid from glucose and xylose by metabolically engineered Saccharomyces cerevisiae

Directory of Open Access Journals (Sweden)

Kanchana R. Kildegaard

2015-12-01

Full Text Available Biomass, the most abundant carbon source on the planet, may in the future become the primary feedstock for production of fuels and chemicals, replacing fossil feedstocks. This will, however, require development of cell factories that can convert both C6 and C5 sugars present in lignocellulosic biomass into the products of interest. We engineered Saccharomyces cerevisiae for production of 3-hydroxypropionic acid (3HP, a potential building block for acrylates, from glucose and xylose. We introduced the 3HP biosynthetic pathways via malonyl-CoA or β-alanine intermediates into a xylose-consuming yeast. Using controlled fed-batch cultivation, we obtained 7.37±0.17 g 3HP L−1 in 120 hours with an overall yield of 29±1% Cmol 3HP Cmol−1 xylose. This study is the first demonstration of the potential of using S. cerevisiae for production of 3HP from the biomass sugar xylose. Keywords: Metabolic engineering, Biorefineries, 3-hydroxypropionic acid, Saccharomyces cerevisiae, Xylose utilization

Engineering microbes for efficient production of chemicals

Science.gov (United States)

Gong, Wei; Dole, Sudhanshu; Grabar, Tammy; Collard, Andrew Christopher; Pero, Janice G; Yocum, R Rogers

2015-04-28

This present invention relates to production of chemicals from microorganisms that have been genetically engineered and metabolically evolved. Improvements in chemical production have been established, and particular mutations that lead to those improvements have been identified. Specific examples are given in the identification of mutations that occurred during the metabolic evolution of a bacterial strain genetically engineered to produce succinic acid. This present invention also provides a method for evaluating the industrial applicability of mutations that were selected during the metabolic evolution for increased succinic acid production. This present invention further provides microorganisms engineered to have mutations that are selected during metabolic evolution and contribute to improved production of succinic acid, other organic acids and other chemicals of commercial interest.

Dynamic optimal metabolic control theory: a cybernetic approach for modelling of the central nitrogen metabolism of S. cerevisiae

NARCIS (Netherlands)

Riel, van N.A.W.; Giuseppin, M.L.F.; Verrips, C.T.

2000-01-01

The theory of dynamic optimal metabolic control (DOMC), as developed by Giuseppin and Van Riel (Metab. Eng., 2000), is applied to model the central nitrogen metabolism (CNM) in Saccharomyces cerevisiae. The CNM represents a typical system encountered in advanced metabolic engineering. The CNM is the

Artificial Promoters for Metabolic Optimization

DEFF Research Database (Denmark)

Jensen, Peter Ruhdal; Hammer, Karin

1998-01-01

In this article, we review some of the expression systems that are available for Metabolic Control Analysis and Metabolic Engineering, and examine their advantages and disadvantages in different contexts. In a recent approach, artificial promoters for modulating gene expression in micro-organisms...

Kinetic modeling of cell metabolism for microbial production.

Science.gov (United States)

Costa, Rafael S; Hartmann, Andras; Vinga, Susana

2016-02-10

Kinetic models of cellular metabolism are important tools for the rational design of metabolic engineering strategies and to explain properties of complex biological systems. The recent developments in high-throughput experimental data are leading to new computational approaches for building kinetic models of metabolism. Herein, we briefly survey the available databases, standards and software tools that can be applied for kinetic models of metabolism. In addition, we give an overview about recently developed ordinary differential equations (ODE)-based kinetic models of metabolism and some of the main applications of such models are illustrated in guiding metabolic engineering design. Finally, we review the kinetic modeling approaches of large-scale networks that are emerging, discussing their main advantages, challenges and limitations. Copyright © 2015 Elsevier B.V. All rights reserved.

Metabolic engineering of the phenylpropanoid pathway enhances the antioxidant capacity of Saussurea involucrata.

Directory of Open Access Journals (Sweden)

Jian Qiu

Full Text Available The rare wild species of snow lotus Saussurea involucrata is a commonly used medicinal herb with great pharmacological value for human health, resulting from its uniquely high level of phenylpropanoid compound production. To gain information on the phenylpropanid biosynthetic pathway genes in this critically important medicinal plant, global transcriptome sequencing was performed. It revealed that the phenylpropanoid pathway genes were well represented in S. involucrata. In addition, we introduced two key phenylpropanoid pathway inducing transcription factors (PAP1 and Lc into this medicinal plant. Transgenic S. involucrata co-expressing PAP1 and Lc exhibited purple pigments due to a massive accumulation of anthocyanins. The over-expression of PAP1 and Lc largely activated most of the phenylpropanoid pathway genes, and increased accumulation of several phenylpropanoid compounds significantly, including chlorogenic acid, syringin, cyanrine and rutin. Both ABTS (2,2'-azinobis-3-ethylbenzotiazo-line-6-sulfonic acid and FRAP (ferric reducing anti-oxidant power assays revealed that the antioxidant capacity of transgenic S. involucrata lines was greatly enhanced over controls. In addition to providing a deeper understanding of the molecular basis of phenylpropanoid metabolism, our results potentially enable an alternation of bioactive compound production in S. involucrata through metabolic engineering.

Metabolic engineering of Pseudomonas fluorescens for the production of vanillin from ferulic acid.

Science.gov (United States)

Di Gioia, Diana; Luziatelli, Francesca; Negroni, Andrea; Ficca, Anna Grazia; Fava, Fabio; Ruzzi, Maurizio

2011-12-20

Vanillin is one of the most important flavors in the food industry and there is great interest in its production through biotechnological processes starting from natural substrates such as ferulic acid. Among bacteria, recombinant Escherichia coli strains are the most efficient vanillin producers, whereas Pseudomonas spp. strains, although possessing a broader metabolic versatility, rapidly metabolize various phenolic compounds including vanillin. In order to develop a robust Pseudomonas strain that can produce vanillin in high yields and at high productivity, the vanillin dehydrogenase (vdh)-encoding gene of Pseudomonas fluorescens BF13 strain was inactivated via targeted mutagenesis. The results demonstrated that engineered derivatives of strain BF13 accumulate vanillin if inactivation of vdh is associated with concurrent expression of structural genes for feruloyl-CoA synthetase (fcs) and hydratase/aldolase (ech) from a low-copy plasmid. The conversion of ferulic acid to vanillin was enhanced by optimization of growth conditions, growth phase and parameters of the bioconversion process. The developed strain produced up to 8.41 mM vanillin, which is the highest final titer of vanillin produced by a Pseudomonas strain to date and opens new perspectives in the use of bacterial biocatalysts for biotechnological production of vanillin from agro-industrial wastes which contain ferulic acid. Copyright © 2011 Elsevier B.V. All rights reserved.

A mathematical framework for yield (vs. rate) optimization in constraint-based modeling and applications in metabolic engineering.

Science.gov (United States)

Klamt, Steffen; Müller, Stefan; Regensburger, Georg; Zanghellini, Jürgen

2018-02-07

example and demonstrate their relevance for metabolic engineering with realistic models of E. coli. We develop a comprehensive mathematical framework for yield optimization in metabolic models. Our theory is particularly useful for the study and rational modification of cell factories designed under given yield and/or rate requirements. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

System-level perturbations of cell metabolism using CRISPR/Cas9

DEFF Research Database (Denmark)

Jakociunas, Tadas; Jensen, Michael Krogh; Keasling, Jay

2017-01-01

CRISPR/Cas9 (clustered regularly interspaced palindromic repeats and the associated protein Cas9) techniques have made genome engineering and transcriptional reprogramming studies more advanced and cost-effective. For metabolic engineering purposes, the CRISPR-based tools have been applied...... previously possible. In this mini-review we highlight recent studies adopting CRISPR/Cas9 for systems-level perturbations and model-guided metabolic engineering....

Protein engineering approaches to chemical biotechnology.

Science.gov (United States)

Chen, Zhen; Zeng, An-Ping

2016-12-01

Protein engineering for the improvement of properties of biocatalysts and for the generation of novel metabolic pathways plays more and more important roles in chemical biotechnology aiming at the production of chemicals from biomass. Although widely used in single-enzyme catalysis process, protein engineering is only being increasingly explored in recent years to achieve more complex in vitro and in vivo biocatalytic processes. This review focuses on major contributions of protein engineering to chemical biotechnology in the field of multi-enzymatic cascade catalysis and metabolic engineering. Especially, we discuss and highlight recent strategies for combining pathway design and protein engineering for the production of novel products. Copyright © 2016. Published by Elsevier Ltd.

Transcription activator-like effector nucleases mediated metabolic engineering for enhanced fatty acids production in Saccharomyces cerevisiae

KAUST Repository

Aouida, Mustapha; Li, Lixin; Mahjoub, Ali; Alshareef, Sahar; Ali, Zahir; Piatek, Agnieszka Anna; Mahfouz, Magdy M.

2015-01-01

Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S.cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S.cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production. © 2015 The Society for Biotechnology, Japan.

Transcription activator-like effector nucleases mediated metabolic engineering for enhanced fatty acids production in Saccharomyces cerevisiae

KAUST Repository

Aouida, Mustapha

2015-04-01

Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S.cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S.cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production. © 2015 The Society for Biotechnology, Japan.

Advances in metabolic pathway and strain engineering paving the way for sustainable production of chemical building blocks

DEFF Research Database (Denmark)

Chen, Yun; Nielsen, Jens

2013-01-01

Bio-based production of chemical building blocks from renewable resources is an attractive alternative to petroleum-based platform chemicals. Metabolic pathway and strain engineering is the key element in constructing robust microbial chemical factories within the constraints of cost effective...... production. Here we discuss how the development of computational algorithms, novel modules and methods, omics-based techniques combined with modeling refinement are enabling reduction in development time and thus advance the field of industrial biotechnology. We further discuss how recent technological...

Molecular Cloning Designer Simulator (MCDS: All-in-one molecular cloning and genetic engineering design, simulation and management software for complex synthetic biology and metabolic engineering projects

Directory of Open Access Journals (Sweden)

Zhenyu Shi

2016-12-01

Full Text Available Molecular Cloning Designer Simulator (MCDS is a powerful new all-in-one cloning and genetic engineering design, simulation and management software platform developed for complex synthetic biology and metabolic engineering projects. In addition to standard functions, it has a number of features that are either unique, or are not found in combination in any one software package: (1 it has a novel interactive flow-chart user interface for complex multi-step processes, allowing an integrated overview of the whole project; (2 it can perform a user-defined workflow of cloning steps in a single execution of the software; (3 it can handle multiple types of genetic recombineering, a technique that is rapidly replacing classical cloning for many applications; (4 it includes experimental information to conveniently guide wet lab work; and (5 it can store results and comments to allow the tracking and management of the whole project in one platform. MCDS is freely available from https://mcds.codeplex.com. Keywords: BioCAD, Genetic engineering software, Molecular cloning software, Synthetic biology, Workflow simulation and management

Metabolic and Transcriptional Reprogramming in Developing Soybean (Glycine max Embryos

Directory of Open Access Journals (Sweden)

Ruth Grene

2013-05-01

Full Text Available Soybean (Glycine max seeds are an important source of seed storage compounds, including protein, oil, and sugar used for food, feed, chemical, and biofuel production. We assessed detailed temporal transcriptional and metabolic changes in developing soybean embryos to gain a systems biology view of developmental and metabolic changes and to identify potential targets for metabolic engineering. Two major developmental and metabolic transitions were captured enabling identification of potential metabolic engineering targets specific to seed filling and to desiccation. The first transition involved a switch between different types of metabolism in dividing and elongating cells. The second transition involved the onset of maturation and desiccation tolerance during seed filling and a switch from photoheterotrophic to heterotrophic metabolism. Clustering analyses of metabolite and transcript data revealed clusters of functionally related metabolites and transcripts active in these different developmental and metabolic programs. The gene clusters provide a resource to generate predictions about the associations and interactions of unknown regulators with their targets based on “guilt-by-association” relationships. The inferred regulators also represent potential targets for future metabolic engineering of relevant pathways and steps in central carbon and nitrogen metabolism in soybean embryos and drought and desiccation tolerance in plants.

MetaFluxNet: the management of metabolic reaction information and quantitative metabolic flux analysis.

Science.gov (United States)

Lee, Dong-Yup; Yun, Hongsoek; Park, Sunwon; Lee, Sang Yup

2003-11-01

MetaFluxNet is a program package for managing information on the metabolic reaction network and for quantitatively analyzing metabolic fluxes in an interactive and customized way. It allows users to interpret and examine metabolic behavior in response to genetic and/or environmental modifications. As a result, quantitative in silico simulations of metabolic pathways can be carried out to understand the metabolic status and to design the metabolic engineering strategies. The main features of the program include a well-developed model construction environment, user-friendly interface for metabolic flux analysis (MFA), comparative MFA of strains having different genotypes under various environmental conditions, and automated pathway layout creation. http://mbel.kaist.ac.kr/ A manual for MetaFluxNet is available as PDF file.

Engineering Robustness of Microbial Cell Factories.

Science.gov (United States)

Gong, Zhiwei; Nielsen, Jens; Zhou, Yongjin J

2017-10-01

Metabolic engineering and synthetic biology offer great prospects in developing microbial cell factories capable of converting renewable feedstocks into fuels, chemicals, food ingredients, and pharmaceuticals. However, prohibitively low production rate and mass concentration remain the major hurdles in industrial processes even though the biosynthetic pathways are comprehensively optimized. These limitations are caused by a variety of factors unamenable for host cell survival, such as harsh industrial conditions, fermentation inhibitors from biomass hydrolysates, and toxic compounds including metabolic intermediates and valuable target products. Therefore, engineered microbes with robust phenotypes is essential for achieving higher yield and productivity. In this review, the recent advances in engineering robustness and tolerance of cell factories is described to cope with these issues and briefly introduce novel strategies with great potential to enhance the robustness of cell factories, including metabolic pathway balancing, transporter engineering, and adaptive laboratory evolution. This review also highlights the integration of advanced systems and synthetic biology principles toward engineering the harmony of overall cell function, more than the specific pathways or enzymes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Improving polyglucan production in cyanobacteria and microalgae via cultivation design and metabolic engineering.

Science.gov (United States)

Aikawa, Shimpei; Ho, Shih-Hsin; Nakanishi, Akihito; Chang, Jo-Shu; Hasunuma, Tomohisa; Kondo, Akihiko

2015-06-01

Photosynthetic microorganisms, such as cyanobacteria and microalgae, are currently being investigated as alternative biomass resources for bioethanol production, owing to their benefits, including high-photosynthetic activity and whole-year cultivation without utilization of arable land. Polyglucans comprise the major carbohydrate content of these organisms. Polyglucans can be utilized as a carbon source for microbial fermentation. Although polyglucan production has so far been promoted by nutrient limitation, it must be further enhanced to accommodate market demand. This review focuses on the recent progress in the production of α-polyglucans such asglycogen and starch in cyanobacteria and green microalgae via cultivation design, including modifying the nutrient supply and replacing the growth medium. The control and manipulation of polyglucan metabolism necessitates the elucidation of the polyglucan production mechanism. We reviewed gene expression and metabolite accumulation profiles of cyanobacteria and green microalgae during nutrient limitation-stimulated α-polyglucan accumulation. We also focus on the enhancement in cyanobacterial glycogen production via the genetic engineering of glycolysis, CO2 concentration mechanism, and photosynthetic light-harvesting protein based on the polyglucan accumulation mechanism. The combined strategies of cultivation design and genetic engineering should be considered for further enhancement of polyglucan productivity for bioethanol production. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

What makes a reach movement effortful? Physical effort discounting supports common minimization principles in decision making and motor control.

Directory of Open Access Journals (Sweden)

Pierre Morel

2017-06-01

Full Text Available When deciding between alternative options, a rational agent chooses on the basis of the desirability of each outcome, including associated costs. As different options typically result in different actions, the effort associated with each action is an essential cost parameter. How do humans discount physical effort when deciding between movements? We used an action-selection task to characterize how subjective effort depends on the parameters of arm transport movements and controlled for potential confounding factors such as delay discounting and performance. First, by repeatedly asking subjects to choose between 2 arm movements of different amplitudes or durations, performed against different levels of force, we identified parameter combinations that subjects experienced as identical in effort (isoeffort curves. Movements with a long duration were judged more effortful than short-duration movements against the same force, while movement amplitudes did not influence effort. Biomechanics of the movements also affected effort, as movements towards the body midline were preferred to movements away from it. Second, by introducing movement repetitions, we further determined that the cost function for choosing between effortful movements had a quadratic relationship with force, while choices were made on the basis of the logarithm of these costs. Our results show that effort-based action selection during reaching cannot easily be explained by metabolic costs. Instead, force-loaded reaches, a widely occurring natural behavior, imposed an effort cost for decision making similar to cost functions in motor control. Our results thereby support the idea that motor control and economic choice are governed by partly overlapping optimization principles.

A review of parameters and heuristics for guiding metabolic pathfinding.

Science.gov (United States)

Kim, Sarah M; Peña, Matthew I; Moll, Mark; Bennett, George N; Kavraki, Lydia E

2017-09-15

Recent developments in metabolic engineering have led to the successful biosynthesis of valuable products, such as the precursor of the antimalarial compound, artemisinin, and opioid precursor, thebaine. Synthesizing these traditionally plant-derived compounds in genetically modified yeast cells introduces the possibility of significantly reducing the total time and resources required for their production, and in turn, allows these valuable compounds to become cheaper and more readily available. Most biosynthesis pathways used in metabolic engineering applications have been discovered manually, requiring a tedious search of existing literature and metabolic databases. However, the recent rapid development of available metabolic information has enabled the development of automated approaches for identifying novel pathways. Computer-assisted pathfinding has the potential to save biochemists time in the initial discovery steps of metabolic engineering. In this paper, we review the parameters and heuristics used to guide the search in recent pathfinding algorithms. These parameters and heuristics capture information on the metabolic network structure, compound structures, reaction features, and organism-specificity of pathways. No one metabolic pathfinding algorithm or search parameter stands out as the best to use broadly for solving the pathfinding problem, as each method and parameter has its own strengths and shortcomings. As assisted pathfinding approaches continue to become more sophisticated, the development of better methods for visualizing pathway results and integrating these results into existing metabolic engineering practices is also important for encouraging wider use of these pathfinding methods.

A Consensus Genome-scale Reconstruction of Chinese Hamster Ovary Cell Metabolism

DEFF Research Database (Denmark)

Hefzi, Hooman; Ang, Kok Siong; Hanscho, Michael

2016-01-01

Chinese hamster ovary (CHO) cells dominate biotherapeutic protein production and are widely used in mammalian cell line engineering research. To elucidate metabolic bottlenecks in protein production and to guide cell engineering and bioprocess optimization, we reconstructed the metabolic pathways...

Glycosylation Engineering

DEFF Research Database (Denmark)

Clausen, Henrik; Wandall, Hans H.; Steentoft, Catharina

2017-01-01

Knowledge of the cellular pathways of glycosylation across phylogeny provides opportunities for designing glycans via genetic engineering in a wide variety of cell types including bacteria, fungi, plant cells, and mammalian cells. The commercial demand for glycosylation engineering is broad......, including production of biological therapeutics with defined glycosylation (Chapter 57). This chapter describes how knowledge of glycan structures and their metabolism (Parts I–III of this book) has led to the current state of glycosylation engineering in different cell types. Perspectives for rapid...

Solvent production by engineered Ralstonia eutropha: channeling carbon to biofuel.

Science.gov (United States)

Chakravarty, Jayashree; Brigham, Christopher J

2018-06-01

Microbial production of solvents like acetone and butanol was a couple of the first industrial fermentation processes to gain global importance. These solvents are important feedstocks for the chemical and biofuel industry. Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H 2 and CO 2 under aerobic conditions. This bacterium is a natural producer of polyhydroxyalkanoate biopolymers. Recently, with the advances in the development of genetic engineering tools, the range of metabolites R. eutropha can produce has enlarged. Its ability to utilize various carbon sources renders it an interesting candidate host for synthesis of renewable biofuel and solvent production. This review focuses on progress in metabolic engineering of R. eutropha for the production of alcohols, terpenes, methyl ketones, and alka(e)nes using various resources. Biological synthesis of solvents still presents the challenge of high production costs and competition from chemical synthesis. Better understanding of R. eutropha biology will support efforts to engineer and develop superior microbial strains for solvent production. Continued research on multiple fronts is required to engineer R. eutropha for truly sustainable and economical solvent production.

Stoichiometric network constraints on xylose metabolism by recombinant Saccharomyces cerevisiae

Science.gov (United States)

Yong-Su Jin; Thomas W. Jeffries

2004-01-01

Metabolic pathway engineering is constrained by the thermodynamic and stoichiometric feasibility of enzymatic activities of introduced genes. Engineering of xylose metabolism in Saccharomyces cerevisiae has focused on introducing genes for the initial xylose assimilation steps from Pichia stipitis, a xylose-fermenting yeast, into S. cerevisiae, a yeast raditionally...

Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism.

Science.gov (United States)

Xu, Li-Qin; Liu, Yong-Jun; Yao, Kang; Liu, Hao-Hao; Tao, Xin-Yi; Wang, Feng-Qing; Wei, Dong-Zhi

2016-02-22

The catabolism of sterols in mycobacteria is highly important due to its close relevance in the pathogenesis of pathogenic strains and the biotechnological applications of nonpathogenic strains for steroid synthesis. However, some key metabolic steps remain unknown. In this study, the hsd4A gene from Mycobacterium neoaurum ATCC 25795 was investigated. The encoded protein, Hsd4A, was characterized as a dual-function enzyme, with both 17β-hydroxysteroid dehydrogenase and β-hydroxyacyl-CoA dehydrogenase activities in vitro. Using a kshAs-null strain of M. neoaurum ATCC 25795 (NwIB-XII) as a model, Hsd4A was further confirmed to exert dual-function in sterol catabolism in vivo. The deletion of hsd4A in NwIB-XII resulted in the production of 23,24-bisnorcholenic steroids (HBCs), indicating that hsd4A plays a key role in sterol side-chain degradation. Therefore, two competing pathways, the AD and HBC pathways, were proposed for the side-chain degradation. The proposed HBC pathway has great value in illustrating the production mechanism of HBCs in sterol catabolism and in developing HBCs producing strains for industrial application via metabolic engineering. Through the combined modification of hsd4A and other genes, three HBCs producing strains were constructed that resulted in promising productivities of 0.127, 0.109 and 0.074 g/l/h, respectively.

Development of a High-Efficiency Transformation Method and Implementation of Rational Metabolic Engineering for the Industrial Butanol Hyperproducer Clostridium saccharoperbutylacetonicum Strain N1-4.

Science.gov (United States)

Herman, Nicolaus A; Li, Jeffrey; Bedi, Ripika; Turchi, Barbara; Liu, Xiaoji; Miller, Michael J; Zhang, Wenjun

2017-01-15

While a majority of academic studies concerning acetone, butanol, and ethanol (ABE) production by Clostridium have focused on Clostridium acetobutylicum, other members of this genus have proven to be effective industrial workhorses despite the inability to perform genetic manipulations on many of these strains. To further improve the industrial performance of these strains in areas such as substrate usage, solvent production, and end product versatility, transformation methods and genetic tools are needed to overcome the genetic intractability displayed by these species. In this study, we present the development of a high-efficiency transformation method for the industrial butanol hyperproducer Clostridium saccharoperbutylacetonicum strain N1-4 (HMT) ATCC 27021. Following initial failures, we found that the key to creating a successful transformation method was the identification of three distinct colony morphologies (types S, R, and I), which displayed significant differences in transformability. Working with the readily transformable type I cells (transformation efficiency, 1.1 × 10 6 CFU/μg DNA), we performed targeted gene deletions in C. saccharoperbutylacetonicum N1-4 using a homologous recombination-mediated allelic exchange method. Using plasmid-based gene overexpression and targeted knockouts of key genes in the native acetone-butanol-ethanol (ABE) metabolic pathway, we successfully implemented rational metabolic engineering strategies, yielding in the best case an engineered strain (Clostridium saccharoperbutylacetonicum strain N1-4/pWIS13) displaying an 18% increase in butanol titers and 30% increase in total ABE titer (0.35 g ABE/g sucrose) in batch fermentations. Additionally, two engineered strains overexpressing aldehyde/alcohol dehydrogenases (encoded by adh11 and adh5) displayed 8.5- and 11.8-fold increases (respectively) in batch ethanol production. This paper presents the first steps toward advanced genetic engineering of the industrial butanol

Model-based design of bistable cell factories for metabolic engineering.

Science.gov (United States)

Srinivasan, Shyam; Cluett, William R; Mahadevan, Radhakrishnan

2018-04-15

Metabolism can exhibit dynamic phenomena like bistability due to the presence of regulatory motifs like the positive feedback loop. As cell factories, microorganisms with bistable metabolism can have a high and a low product flux at the two stable steady states, respectively. The exclusion of metabolic regulation and network dynamics limits the ability of pseudo-steady state stoichiometric models to detect the presence of bistability, and reliably assess the outcomes of design perturbations to metabolic networks. Using kinetic models of metabolism, we assess the change in the bistable characteristics of the network, and suggest designs based on perturbations to the positive feedback loop to enable the network to produce at its theoretical maximum rate. We show that the most optimal production design in parameter space, for a small bistable metabolic network, may exist at the boundary of the bistable region separating it from the monostable region of low product fluxes. The results of our analysis can be broadly applied to other bistable metabolic networks with similar positive feedback network topologies. This can complement existing model-based design strategies by providing a smaller number of feasible designs that need to be tested in vivo. http://lmse.biozone.utoronto.ca/downloads/. krishna.mahadevan@utoronto.ca. Supplementary data are available at Bioinformatics online.

Overview of NASA/OAST efforts related to manufacturing technology

Science.gov (United States)

Saunders, N. T.

1976-01-01

An overview of some of NASA's current efforts related to manufacturing technology and some possible directions for the future are presented. The topics discussed are: computer-aided design, composite structures, and turbine engine components.

Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Hou, Jin; Lages, Nuno; Oldiges, M.

2009-01-01

to induce widespread changes in metabolism. We present a detailed analysis of the impact of perturbations in redox cofactors in the cytosol or mitochondria on glucose and energy metabolism in Saccharomyces cerevisiae to aid metabolic engineering decisions that involve cofactor engineering. We enhanced NADH...... oxidation by introducing NADH oxidase or alternative oxidase, its ATP-mediated conversion to NADPH using NADH kinase as well as the interconversion of NADH and NADPH independent of ATP by the soluble, non-proton-translocating bacterial transhydrogenase. Decreasing cytosolic NADH level lowered glycerol...

Policy efforts used to develop awareness aimed at increased students' scientific literacy and career choices in mathematics, science and engineering

Science.gov (United States)

Whitfield, Frank Albert

The lack of an adequate supply of human resources in science and engineering has been well documented. Efforts from a number of agencies, such as the National Science Foundation, have been implemented to alleviate this national problem. However, it is unclear what concerted efforts state agencies are taking to increase the number of African American students' scientific literacy, and career choices in science and engineering. The purpose of this study was to select a talent pool of African American students who are academically able to pursue a career in a math-based major. The selection of this talent pool lead to the recommendation of an encouragement process model to be used by the Tennessee Board of Regents (TBR) system to encourage the selectees of this talent pool to enter math-based programs at TBR universities. An integrated literature review was conducted. This review includes perspectives on national, state, and local educational policy decisions which affect educational purposes, institutional governance and secondary-postsecondary linkages. Existing TBR system data were analyzed and tabulated. This tabulated data along with the recommended model will be offered to the TBR system for possible adoption. The results of these data support the methodological notion that there are an appreciable number of potential TBR system African American students academically able to enter math related majors who, however, may be reluctant to choose a career direction in a math-based career field. Implications of this study and suggestions for further research are discussed. On an applied level, the study might suggest to other states ways in which to deal with similar problems.

Genetic and metabolic engineering for microbial production of poly-γ-glutamic acid.

Science.gov (United States)

Cao, Mingfeng; Feng, Jun; Sirisansaneeyakul, Sarote; Song, Cunjiang; Chisti, Yusuf

2018-05-28

Poly-γ-glutamic acid (γ-PGA) is a natural biopolymer of glutamic acid. The repeating units of γ-PGA may be derived exclusively from d-glutamic acid, or l-glutamic acid, or both. The monomer units are linked by amide bonds between the α-amino group and the γ-carboxylic acid group. γ-PGA is biodegradable, edible and water-soluble. It has numerous existing and emerging applications in processing of foods, medicines and cosmetics. This review focuses on microbial production of γ-PGA via genetically and metabolically engineered recombinant bacteria. Strategies for improving production of γ-PGA include modification of its biosynthesis pathway, enhancing the production of its precursor (glutamic acid), and preventing loss of the precursor to competing byproducts. These and other strategies are discussed. Heterologous synthesis of γ-PGA in industrial bacterial hosts that do not naturally produce γ-PGA is discussed. Emerging trends and the challenges affecting the production of γ-PGA are reviewed. Copyright © 2018. Published by Elsevier Inc.

Preliminary engineering cost trends for highway projects.

Science.gov (United States)

2011-10-21

Preliminary engineering (PE) for a highway project encompasses two efforts: planning to minimize the physical, social, and human environmental impacts of projects and engineering design to deliver the best alternative. PE efforts begin years in advan...

Engineering Microbial Metabolite Dynamics and Heterogeneity.

Science.gov (United States)

Schmitz, Alexander C; Hartline, Christopher J; Zhang, Fuzhong

2017-10-01

As yields for biological chemical production in microorganisms approach their theoretical maximum, metabolic engineering requires new tools, and approaches for improvements beyond what traditional strategies can achieve. Engineering metabolite dynamics and metabolite heterogeneity is necessary to achieve further improvements in product titers, productivities, and yields. Metabolite dynamics, the ensemble change in metabolite concentration over time, arise from the need for microbes to adapt their metabolism in response to the extracellular environment and are important for controlling growth and productivity in industrial fermentations. Metabolite heterogeneity, the cell-to-cell variation in a metabolite concentration in an isoclonal population, has a significant impact on ensemble productivity. Recent advances in single cell analysis enable a more complete understanding of the processes driving metabolite heterogeneity and reveal metabolic engineering targets. The authors present an overview of the mechanistic origins of metabolite dynamics and heterogeneity, why they are important, their potential effects in chemical production processes, and tools and strategies for engineering metabolite dynamics and heterogeneity. The authors emphasize that the ability to control metabolite dynamics and heterogeneity will bring new avenues of engineering to increase productivity of microbial strains. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of Saccharomyces cerevisiae

Directory of Open Access Journals (Sweden)

Ishii Jun

2011-01-01

Full Text Available Abstract Background The development of novel yeast strains with increased tolerance toward inhibitors in lignocellulosic hydrolysates is highly desirable for the production of bio-ethanol. Weak organic acids such as acetic and formic acids are necessarily released during the pretreatment (i.e. solubilization and hydrolysis of lignocelluloses, which negatively affect microbial growth and ethanol production. However, since the mode of toxicity is complicated, genetic engineering strategies addressing yeast tolerance to weak organic acids have been rare. Thus, enhanced basic research is expected to identify target genes for improved weak acid tolerance. Results In this study, the effect of acetic acid on xylose fermentation was analyzed by examining metabolite profiles in a recombinant xylose-fermenting strain of Saccharomyces cerevisiae. Metabolome analysis revealed that metabolites involved in the non-oxidative pentose phosphate pathway (PPP [e.g. sedoheptulose-7-phosphate, ribulose-5-phosphate, ribose-5-phosphate and erythrose-4-phosphate] were significantly accumulated by the addition of acetate, indicating the possibility that acetic acid slows down the flux of the pathway. Accordingly, a gene encoding a PPP-related enzyme, transaldolase or transketolase, was overexpressed in the xylose-fermenting yeast, which successfully conferred increased ethanol productivity in the presence of acetic and formic acid. Conclusions Our metabolomic approach revealed one of the molecular events underlying the response to acetic acid and focuses attention on the non-oxidative PPP as a target for metabolic engineering. An important challenge for metabolic engineering is identification of gene targets that have material importance. This study has demonstrated that metabolomics is a powerful tool to develop rational strategies to confer tolerance to stress through genetic engineering.

Metabolic engineering of Chinese hamster ovary cells: towards a bioengineered heparin.

Science.gov (United States)

Baik, Jong Youn; Gasimli, Leyla; Yang, Bo; Datta, Payel; Zhang, Fuming; Glass, Charles A; Esko, Jeffrey D; Linhardt, Robert J; Sharfstein, Susan T

2012-03-01

Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS/heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS/heparin biosynthesis might be necessary. Copyright © 2012 Elsevier Inc. All rights reserved.

Engineering chemical interactions in microbial communities.

Science.gov (United States)

Kenny, Douglas J; Balskus, Emily P

2018-03-05

Microbes living within host-associated microbial communities (microbiotas) rely on chemical communication to interact with surrounding organisms. These interactions serve many purposes, from supplying the multicellular host with nutrients to antagonizing invading pathogens, and breakdown of chemical signaling has potentially negative consequences for both the host and microbiota. Efforts to engineer microbes to take part in chemical interactions represent a promising strategy for modulating chemical signaling within these complex communities. In this review, we discuss prominent examples of chemical interactions found within host-associated microbial communities, with an emphasis on the plant-root microbiota and the intestinal microbiota of animals. We then highlight how an understanding of such interactions has guided efforts to engineer microbes to participate in chemical signaling in these habitats. We discuss engineering efforts in the context of chemical interactions that enable host colonization, promote host health, and exclude pathogens. Finally, we describe prominent challenges facing this field and propose new directions for future engineering efforts.

Toward solar biodiesel production from CO2 using engineered cyanobacteria.

Science.gov (United States)

Woo, Han Min; Lee, Hyun Jeong

2017-05-01

Metabolic engineering of cyanobacteria has received attention as a sustainable strategy to convert carbon dioxide to various biochemicals including fatty acid-derived biodiesel. Recently, Synechococcus elongatus PCC 7942, a model cyanobacterium, has been engineered to convert CO2 to fatty acid ethyl esters (FAEEs) as biodiesel. Modular pathway has been constructed for FAEE production. Several metabolic engineering strategies were discussed to improve the production levels of FAEEs, including host engineering by improving CO2 fixation rate and photosynthetic efficiency. In addition, protein engineering of key enzyme in S. elongatus PCC 7942 was implemented to address issues on FAEE secretions toward sustainable FAEE production from CO2. Finally, advanced metabolic engineering will promote developing biosolar cell factories to convert CO2 to feasible amount of FAEEs toward solar biodiesel. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Metabolic modeling of synthesis gas fermentation in bubble column reactors.

Science.gov (United States)

Chen, Jin; Gomez, Jose A; Höffner, Kai; Barton, Paul I; Henson, Michael A

2015-01-01

A promising route to renewable liquid fuels and chemicals is the fermentation of synthesis gas (syngas) streams to synthesize desired products such as ethanol and 2,3-butanediol. While commercial development of syngas fermentation technology is underway, an unmet need is the development of integrated metabolic and transport models for industrially relevant syngas bubble column reactors. We developed and evaluated a spatiotemporal metabolic model for bubble column reactors with the syngas fermenting bacterium Clostridium ljungdahlii as the microbial catalyst. Our modeling approach involved combining a genome-scale reconstruction of C. ljungdahlii metabolism with multiphase transport equations that govern convective and dispersive processes within the spatially varying column. The reactor model was spatially discretized to yield a large set of ordinary differential equations (ODEs) in time with embedded linear programs (LPs) and solved using the MATLAB based code DFBAlab. Simulations were performed to analyze the effects of important process and cellular parameters on key measures of reactor performance including ethanol titer, ethanol-to-acetate ratio, and CO and H2 conversions. Our computational study demonstrated that mathematical modeling provides a complementary tool to experimentation for understanding, predicting, and optimizing syngas fermentation reactors. These model predictions could guide future cellular and process engineering efforts aimed at alleviating bottlenecks to biochemical production in syngas bubble column reactors.

A modular metabolic engineering approach for the production of 1,2-propanediol from glycerol by Saccharomyces cerevisiae.

Science.gov (United States)

Islam, Zia-Ul; Klein, Mathias; Aßkamp, Maximilian R; Ødum, Anders S R; Nevoigt, Elke

2017-11-01

Compared to sugars, a major advantage of using glycerol as a feedstock for industrial bioprocesses is the fact that this molecule is more reduced than sugars. A compound whose biotechnological production might greatly profit from the substrate's higher reducing power is 1,2-propanediol (1,2-PDO). Here we present a novel metabolic engineering approach to produce 1,2-PDO from glycerol in S. cerevisiae. Apart from implementing the heterologous methylglyoxal (MG) pathway for 1,2-PDO formation from dihydroxyacetone phosphate (DHAP) and expressing a heterologous glycerol facilitator, the employed genetic modifications included the replacement of the native FAD-dependent glycerol catabolic pathway by the 'DHA pathway' for delivery of cytosolic NADH and the reduction of triosephosphate isomerase (TPI) activity for increased precursor (DHAP) supply. The choice of the medium had a crucial impact on both the strength of the metabolic switch towards fermentation in general (as indicated by the production of ethanol and 1,2-PDO) and on the ratio at which these two fermentation products were formed. For example, virtually no 1,2-PDO but only ethanol was formed in synthetic glycerol medium with urea as the nitrogen source. When nutrient-limited complex YG medium was used, significant amounts of 1,2-PDO were formed and it became obvious that the concerted supply of NADH and DHAP are essential for boosting 1,2-PDO production. Additionally, optimizing the flux into the MG pathway improved 1,2-PDO formation at the expense of ethanol. Cultivation of the best-performing strain in YG medium and a controlled bioreactor set-up resulted in a maximum titer of > 4gL -1 1,2-PDO which, to the best of our knowledge, has been the highest titer of 1,2-PDO obtained in yeast so far. Surprisingly, significant 1,2-PDO production was also obtained in synthetic glycerol medium after changing the nitrogen source towards ammonium sulfate and adding a buffer. Copyright © 2017 International Metabolic

Mathematical modelling of metabolism

DEFF Research Database (Denmark)

Gombert, Andreas Karoly; Nielsen, Jens

2000-01-01

Mathematical models of the cellular metabolism have a special interest within biotechnology. Many different kinds of commercially important products are derived from the cell factory, and metabolic engineering can be applied to improve existing production processes, as well as to make new processes...... availability of genomic information and powerful analytical techniques, mathematical models also serve as a tool for understanding the cellular metabolism and physiology....... available. Both stoichiometric and kinetic models have been used to investigate the metabolism, which has resulted in defining the optimal fermentation conditions, as well as in directing the genetic changes to be introduced in order to obtain a good producer strain or cell line. With the increasing...

Production of cinnamic and p-hydroxycinnamic acids in engineered microbes

Directory of Open Access Journals (Sweden)

Alejandra eVargas-Tah

2015-08-01

Full Text Available The aromatic compounds cinnamic and p-hydroxycinnamic acids are phenylpropanoids having applications as precursors for the synthesis of thermoplastics, flavoring, cosmetic and health products. These two aromatic acids can be obtained by chemical synthesis or extraction from plant tissues. However, both manufacturing processes have shortcomings such as the generation of toxic subproducts or a low concentration in plant material. Alternative production methods are being developed to enable the biotechnological production of cinnamic and p-hydroxycinnamic acids by genetically engineering various microbial hosts, including Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida and Streptomyces lividans. The natural capacity to synthesize these aromatic acids is not existent in these microbial species. Therefore, genetic modification have been performed that include the heterologous expression of genes encoding phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities, which catalyze the conversion of L-phenylalanine and L-tyrosine to cinnamic acid and p-hydroxycinnamic acid, respectively. Additional host modifications include the metabolic engineering to increase carbon flow from central metabolism to the L-phenylalanine or L-tyrosine biosynthetic pathways. These strategies include the expression of feedback insensitive mutant versions of enzymes from the aromatic pathways, as well as genetic modifications to central carbon metabolism to increase biosynthetic availability of precursors phosphoenolpyruvate and erythrose-4-phosphate. These efforts have been complemented with strain optimization for the utilization of raw material, including various simple carbon sources, as well as sugar polymers and sugar mixtures derived from plant biomass. A systems biology approach to production strains characterization has been limited so far and should yield important data for future strain improvement.

Dissecting the genetic and metabolic mechanisms of adaptation to the knockout of a major metabolic enzyme in Escherichia coli

DEFF Research Database (Denmark)

Long, Christopher P.; Gonzalez, Jacqueline E.; Feist, Adam M.

2018-01-01

Unraveling the mechanisms of microbial adaptive evolution following genetic or environmental challenges is of fundamental interest in biological science and engineering. When the challenge is the loss of a metabolic enzyme, adaptive responses can also shed significant insight into metabolic...

Building Inclusive Engineering Identities: Implications for Changing Engineering Culture

Science.gov (United States)

Atadero, Rebecca A.; Paguyo, Christina H.; Rambo-Hernandez, Karen E.; Henderson, Heather L.

2018-01-01

Ongoing efforts to broaden the participation of women and people of colour in engineering degree programmes and careers have had limited success. This paper describes a different approach to broadening participation that seeks to work with all students and develop inclusive engineering identities. Researchers worked with the instructors of two…

Altered Levels of Aroma and Volatiles by Metabolic Engineering of Shikimate Pathway Genes in Tomato Fruits

Directory of Open Access Journals (Sweden)

Vered Tzin

2015-06-01

Full Text Available The tomato (Solanum lycopersicum fruit is an excellent source of antioxidants, dietary fibers, minerals and vitamins and therefore has been referred to as a “functional food”. Ripe tomato fruits produce a large number of specialized metabolites including volatile organic compounds. These volatiles serve as key components of the tomato fruit flavor, participate in plant pathogen and herbivore defense, and are used to attract seed dispersers. A major class of specialized metabolites is derived from the shikimate pathway followed by aromatic amino acid biosynthesis of phenylalanine, tyrosine and tryptophan. We attempted to modify tomato fruit flavor by overexpressing key regulatory genes in the shikimate pathway. Bacterial genes encoding feedback-insensitive variants of 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthase (DAHPS; AroG209-9 and bi-functional Chorismate Mutase/Prephenate Dehydratase (CM/PDT; PheA12 were expressed under the control of a fruit-specific promoter. We crossed these transgenes to generate tomato plants expressing both the AroG209 and PheA12 genes. Overexpression of the AroG209-9 gene had a dramatic effect on the overall metabolic profile of the fruit, including enhanced levels of multiple volatile and non-volatile metabolites. In contrast, the PheA12 overexpression line exhibited minor metabolic effects compared to the wild type fruit. Co-expression of both the AroG209-9 and PheA12 genes in tomato resulted overall in a similar metabolic effect to that of expressing only the AroG209-9 gene. However, the aroma ranking attributes of the tomato fruits from PheA12//AroG209-9 were unique and different from those of the lines expressing a single gene, suggesting a contribution of the PheA12 gene to the overall metabolic profile. We suggest that expression of bacterial genes encoding feedback-insensitive enzymes of the shikimate pathway in tomato fruits provides a useful metabolic engineering tool for the modification of

Molecular Cloning Designer Simulator (MCDS): All-in-one molecular cloning and genetic engineering design, simulation and management software for complex synthetic biology and metabolic engineering projects.

Science.gov (United States)

Shi, Zhenyu; Vickers, Claudia E

2016-12-01

Molecular Cloning Designer Simulator (MCDS) is a powerful new all-in-one cloning and genetic engineering design, simulation and management software platform developed for complex synthetic biology and metabolic engineering projects. In addition to standard functions, it has a number of features that are either unique, or are not found in combination in any one software package: (1) it has a novel interactive flow-chart user interface for complex multi-step processes, allowing an integrated overview of the whole project; (2) it can perform a user-defined workflow of cloning steps in a single execution of the software; (3) it can handle multiple types of genetic recombineering, a technique that is rapidly replacing classical cloning for many applications; (4) it includes experimental information to conveniently guide wet lab work; and (5) it can store results and comments to allow the tracking and management of the whole project in one platform. MCDS is freely available from https://mcds.codeplex.com.

VRML metabolic network visualizer.

Science.gov (United States)

Rojdestvenski, Igor

2003-03-01

A successful date collection visualization should satisfy a set of many requirements: unification of diverse data formats, support for serendipity research, support of hierarchical structures, algorithmizability, vast information density, Internet-readiness, and other. Recently, virtual reality has made significant progress in engineering, architectural design, entertainment and communication. We experiment with the possibility of using the immersive abstract three-dimensional visualizations of the metabolic networks. We present the trial Metabolic Network Visualizer software, which produces graphical representation of a metabolic network as a VRML world from a formal description written in a simple SGML-type scripting language.

Nuclear propulsion systems engineering

International Nuclear Information System (INIS)

Madsen, W.W.; Neuman, J.E.: Van Haaften, D.H.

1992-01-01

The Nuclear Energy for Rocket Vehicle Application (NERVA) program of the 1960's and early 1970's was dramatically successful, with no major failures during the entire testing program. This success was due in large part to the successful development of a systems engineering process. Systems engineering, properly implemented, involves all aspects of the system design and operation, and leads to optimization of theentire system: cost, schedule, performance, safety, reliability, function, requirements, etc. The process must be incorporated from the very first and continued to project completion. This paper will discuss major aspects of the NERVA systems engineering effort, and consider the implications for current nuclear propulsion efforts

Development of Next Generation Synthetic Biology Tools for Use in Streptomyces venezuelae

DEFF Research Database (Denmark)

Phelan, Ryan M.; Sachs, Daniel; Petkiewicz, Shayne J.

2017-01-01

precludes rapid and predictable metabolic engineering that is possible in hosts such as Escherichia coli or Saccharomyces cerevisiae. In an effort to improve genetic tools for Streptomyces venezuelae, we developed a suite of standardized, orthogonal integration vectors and an improved method to monitor...... expression system. These tools advance S. venezuelae to be a practical host for future metabolic engineering efforts....

Engine Torque Control of Spark Ignition Engine using Fuzzy Gain Scheduling

OpenAIRE

Aris Triwiyatno

2012-01-01

In the spark ignition engine system, driver convenience is very dependent on satisfying engine torque appropriate with the throttle position given by the driver. Unfortunately, sometimes the fulfillment of engine torque is not in line with fuel saving efforts. This requires the development of high performance and robust power train controllers. One way to potentially meet these performance requirements is to introduce a method of controlling engine torque using fuzzy gain scheduling. By using...

Analysis and design of a genetic circuit for dynamic metabolic engineering.

Science.gov (United States)

Anesiadis, Nikolaos; Kobayashi, Hideki; Cluett, William R; Mahadevan, Radhakrishnan

2013-08-16

Recent advances in synthetic biology have equipped us with new tools for bioprocess optimization at the genetic level. Previously, we have presented an integrated in silico design for the dynamic control of gene expression based on a density-sensing unit and a genetic toggle switch. In the present paper, analysis of a serine-producing Escherichia coli mutant shows that an instantaneous ON-OFF switch leads to a maximum theoretical productivity improvement of 29.6% compared to the mutant. To further the design, global sensitivity analysis is applied here to a mathematical model of serine production in E. coli coupled with a genetic circuit. The model of the quorum sensing and the toggle switch involves 13 parameters of which 3 are identified as having a significant effect on serine concentration. Simulations conducted in this reduced parameter space further identified the optimal ranges for these 3 key parameters to achieve productivity values close to the maximum theoretical values. This analysis can now be used to guide the experimental implementation of a dynamic metabolic engineering strategy and reduce the time required to design the genetic circuit components.

African American Adolescent Female Identification with Engineering and Participation in Engineering Education

Science.gov (United States)

Cornick, Shayla L.

2012-01-01

Experiences that females have during middle and high school have been found to influence the perceptions that they have of their ability to be successful as an engineer and the value that they place on participating in engineering education. Engineering education continues to suffer from a lack of female participation. Several efforts have been…

SOFTWARE EFFORT ESTIMATION FRAMEWORK TO IMPROVE ORGANIZATION PRODUCTIVITY USING EMOTION RECOGNITION OF SOFTWARE ENGINEERS IN SPONTANEOUS SPEECH

Directory of Open Access Journals (Sweden)

B.V.A.N.S.S. Prabhakar Rao

2015-10-01

Full Text Available Productivity is a very important part of any organisation in general and software industry in particular. Now a day’s Software Effort estimation is a challenging task. Both Effort and Productivity are inter-related to each other. This can be achieved from the employee’s of the organization. Every organisation requires emotionally stable employees in their firm for seamless and progressive working. Of course, in other industries this may be achieved without man power. But, software project development is labour intensive activity. Each line of code should be delivered from software engineer. Tools and techniques may helpful and act as aid or supplementary. Whatever be the reason software industry has been suffering with success rate. Software industry is facing lot of problems in delivering the project on time and within the estimated budget limit. If we want to estimate the required effort of the project it is significant to know the emotional state of the team member. The responsibility of ensuring emotional contentment falls on the human resource department and the department can deploy a series of systems to carry out its survey. This analysis can be done using a variety of tools, one such, is through study of emotion recognition. The data needed for this is readily available and collectable and can be an excellent source for the feedback systems. The challenge of recognition of emotion in speech is convoluted primarily due to the noisy recording condition, the variations in sentiment in sample space and exhibition of multiple emotions in a single sentence. The ambiguity in the labels of training set also increases the complexity of problem addressed. The existing models using probabilistic models have dominated the study but present a flaw in scalability due to statistical inefficiency. The problem of sentiment prediction in spontaneous speech can thus be addressed using a hybrid system comprising of a Convolution Neural Network and

Effects of physical training with different intensities of effort on lipid metabolism in rats submitted to the neonatal application of alloxan

Directory of Open Access Journals (Sweden)

Ribeiro Carla

2012-10-01

revealed that intermittent training increased serum levels in alloxan animals Conclusion Thus, it was concluded that physical training at different intensities of effort is of great importance in attenuation and control of changes in the lipid metabolism in alloxan animals.

Engineering performance metrics

Science.gov (United States)

Delozier, R.; Snyder, N.

1993-03-01

Implementation of a Total Quality Management (TQM) approach to engineering work required the development of a system of metrics which would serve as a meaningful management tool for evaluating effectiveness in accomplishing project objectives and in achieving improved customer satisfaction. A team effort was chartered with the goal of developing a system of engineering performance metrics which would measure customer satisfaction, quality, cost effectiveness, and timeliness. The approach to developing this system involved normal systems design phases including, conceptual design, detailed design, implementation, and integration. The lessons teamed from this effort will be explored in this paper. These lessons learned may provide a starting point for other large engineering organizations seeking to institute a performance measurement system accomplishing project objectives and in achieving improved customer satisfaction. To facilitate this effort, a team was chartered to assist in the development of the metrics system. This team, consisting of customers and Engineering staff members, was utilized to ensure that the needs and views of the customers were considered in the development of performance measurements. The development of a system of metrics is no different than the development of any type of system. It includes the steps of defining performance measurement requirements, measurement process conceptual design, performance measurement and reporting system detailed design, and system implementation and integration.

Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

Science.gov (United States)

Witthoff, Sabrina; Schmitz, Katja; Niedenführ, Sebastian; Nöh, Katharina; Noack, Stephan

2015-01-01

Methanol is already an important carbon feedstock in the chemical industry, but it has found only limited application in biotechnological production processes. This can be mostly attributed to the inability of most microbial platform organisms to utilize methanol as a carbon and energy source. With the aim to turn methanol into a suitable feedstock for microbial production processes, we engineered the industrially important but nonmethylotrophic bacterium Corynebacterium glutamicum toward the utilization of methanol as an auxiliary carbon source in a sugar-based medium. Initial oxidation of methanol to formaldehyde was achieved by heterologous expression of a methanol dehydrogenase from Bacillus methanolicus, whereas assimilation of formaldehyde was realized by implementing the two key enzymes of the ribulose monophosphate pathway of Bacillus subtilis: 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The recombinant C. glutamicum strain showed an average methanol consumption rate of 1.7 ± 0.3 mM/h (mean ± standard deviation) in a glucose-methanol medium, and the culture grew to a higher cell density than in medium without methanol. In addition, [13C]methanol-labeling experiments revealed labeling fractions of 3 to 10% in the m + 1 mass isotopomers of various intracellular metabolites. In the background of a C. glutamicum Δald ΔadhE mutant being strongly impaired in its ability to oxidize formaldehyde to CO2, the m + 1 labeling of these intermediates was increased (8 to 25%), pointing toward higher formaldehyde assimilation capabilities of this strain. The engineered C. glutamicum strains represent a promising starting point for the development of sugar-based biotechnological production processes using methanol as an auxiliary substrate. PMID:25595770

Monitoring Bone Tissue Engineered (BTE) Constructs Based on the Shifting Metabolism of Differentiating Stem Cells.

Science.gov (United States)

Simmons, Aaron D; Sikavitsas, Vassilios I

2018-01-01

Ever-increasing demand for bone grafts necessitates the realization of clinical implementation of bone tissue engineered constructs. The predominant hurdle to implementation remains to be securing FDA approval, based on the lack of viable methods for the rigorous monitoring of said constructs. The study presented herein details a method for such monitoring based on the shifting metabolism of mesenchymal stem cells (MSCs) as they differentiate into osteoblasts. To that end, rat MSCs seeded on 85% porous spunbonded poly(L-lactic acid) scaffolds were cultured in flow perfusion bioreactors with baseline or osteoinductive media, and levels of key physio-metabolic markers (oxygen, glucose, osteoprotegerin, and osteocalcin) were monitored throughout culture. Comparison of these non-destructively obtained values and current standard destructive analyses demonstrated key trends useful for the concurrent real-time monitoring of construct cellularity and maturation. Principle among these is the elucidation of the ratio of the rates of oxygen uptake to glucose consumption as a powerful quality marker. This ratio, supported on a physiological basis, has been shown herein to be reliable in the determination of both construct maturation (defined as osteoblastic differentiation and accompanying mineralization) and construct cellularity. Supplementary monitoring of OPG and OCN are shown to provide further validation of such metrics.

Yeast metabolic engineering for hemicellulosic ethanol production

Science.gov (United States)

Jennifer Van Vleet; Thomas W. Jeffries

2009-01-01

Efficient fermentation of hemicellulosic sugars is critical for the bioconversion of lignocellulosics to ethanol. Efficient sugar uptake through the heterologous expression of yeast and fungal xylose/glucose transporters can improve fermentation if other metabolic steps are not rate limiting. Rectification of cofactor imbalances through heterologous expression of...

Intracellular cellobiose metabolism and its applications in lignocellulose-based biorefineries.

Science.gov (United States)

Parisutham, Vinuselvi; Chandran, Sathesh-Prabu; Mukhopadhyay, Aindrila; Lee, Sung Kuk; Keasling, Jay D

2017-09-01

Complete hydrolysis of cellulose has been a key characteristic of biomass technology because of the limitation of industrial production hosts to use cellodextrin, the partial hydrolysis product of cellulose. Cellobiose, a β-1,4-linked glucose dimer, is a major cellodextrin of the enzymatic hydrolysis (via endoglucanase and exoglucanase) of cellulose. Conversion of cellobiose to glucose is executed by β-glucosidase. The complete extracellular hydrolysis of celluloses has several critical barriers in biomass technology. An alternative bioengineering strategy to make the bioprocessing less challenging is to engineer microbes with the abilities to hydrolyze and assimilate the cellulosic-hydrolysate cellodextrin. Microorganisms engineered to metabolize cellobiose rather than the monomeric glucose can provide several advantages for lignocellulose-based biorefineries. This review describes the recent advances and challenges in engineering efficient intracellular cellobiose metabolism in industrial hosts. This review also describes the limitations of and future prospectives in engineering intracellular cellobiose metabolism. Copyright © 2017 Elsevier Ltd. All rights reserved.

Metabolic Engineering of Yeast to Produce Fatty Acid-derived Biofuels: Bottlenecks and Solutions

Directory of Open Access Journals (Sweden)

Jiayuan eSheng

2015-06-01

Full Text Available Fatty acid-derived biofuels can be a better solution than bioethanol to replace petroleum fuel, since they have similar energy content and combustion properties as current transportation fuels. The environmentally friendly microbial fermentation process has been used to synthesize advanced biofuels from renewable feedstock. Due to their robustness as well as the high tolerance to fermentation inhibitors and phage contamination, yeast strains such as Saccharomyces cerevisiae and Yarrowia lipolytica have attracted tremendous attention in recent studies regarding the production of fatty acid-derived biofuels, including fatty acids, fatty acid ethyl esters, fatty alcohols, and fatty alkanes. However, the native yeast strains cannot produce fatty acids and fatty acid-derived biofuels in large quantities. To this end, we have summarized recent publications in this review on metabolic engineering of yeast strains to improve the production of fatty acid-derived biofuels, identified the bottlenecks that limit the productivity of biofuels, and categorized the appropriate approaches to overcome these obstacles.

Metabolic engineering of plant monoterpenes, sesquiterpenes and diterpenes--current status and future opportunities.

Science.gov (United States)

Lange, B Markus; Ahkami, Amirhossein

2013-02-01

Terpenoids (a.k.a. isoprenoids) represent the most diverse class of natural products found in plants, with tens of thousands of reported structures. Plant-derived terpenoids have a multitude of pharmaceutical and industrial applications, but the natural resources for their extraction are often limited and, in many cases, synthetic routes are not commercially viable. Some of the most valuable terpenoids are not accumulated in model plants or crops, and genetic resources for breeding of terpenoid natural product traits are thus poorly developed. At present, metabolic engineering, either in the native producer or a heterologous host, is the only realistic alternative to improve yield and accessibility. In this review article, we will evaluate the state of the art of modulating the biosynthetic pathways for the production of mono-, sesqui- and diterpenes in plants. © 2012 The Authors Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

BioMet Toolbox: genome-wide analysis of metabolism

DEFF Research Database (Denmark)

Cvijovic, M.; Olivares Hernandez, Roberto; Agren, R.

2010-01-01

The rapid progress of molecular biology tools for directed genetic modifications, accurate quantitative experimental approaches, high-throughput measurements, together with development of genome sequencing has made the foundation for a new area of metabolic engineering that is driven by metabolic...

Aum Shinrikyo’s Nuclear and Chemical Weapons Development Efforts

Directory of Open Access Journals (Sweden)

Andrea A. Nehorayoff

2016-03-01

Full Text Available This article details the terrorist activities of the Japanese cult, Aum Shinrikyo, from the perspective of its complex engineering efforts aimed at producing nuclear and chemical weapons. The experience of this millenarian organization illustrates that even violent non-state actors with considerable wealth and resources at their disposal face numerous obstacles to realizing their destructive aspirations. Specifically, Aum’s attempts at complex engineering were stymied by a combination of unchecked fantastical thinking, self-imposed ideological constraints, and a capricious leadership. The chapter highlights each of these mechanisms, as well as the specific ways in which they constrained the decision-making process and the implementation of the complex engineering tasks associated with their unconventional weapons development.

Protein design for pathway engineering.

Science.gov (United States)

Eriksen, Dawn T; Lian, Jiazhang; Zhao, Huimin

2014-02-01

Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. Copyright © 2013 Elsevier Inc. All rights reserved.

Metabolic engineering of the chloroplast genome reveals that the yeast ArDH gene confers enhanced tolerance to salinity and drought in plants

Directory of Open Access Journals (Sweden)

Muhammad Sarwar Khan

2015-09-01

Full Text Available Osmoprotectants stabilize proteins and membranes against the denaturing effect of high concentrations of salts and other harmful solutes. In yeast, arabitol dehydrogenase (ArDH reduces D-ribulose to D-arabitol where D-ribulose is derived by dephosphorylating D-ribulose-5-PO4 in the oxidized pentose pathway. Osmotolerance in plants could be developed through metabolic engineering of chloroplast genome by introducing genes encoding polyols. Here, we report that ArDH expression in chloroplasts confers tolerance to NaCl (up to 400 mM. Transgenic plants compared to wild type survived for four to five weeks on 400 mM NaCl. Nevertheless, plants remained green and grew normal on concentrations up to 350 mM NaCl. Further, a-week-old seedlings were also challenged with poly ethylene glycol (PEG, up to 6% in the liquid medium, considering that membranes and proteins are protected under stress conditions due to accumulation of arabitol in chloroplasts. Seedlings were tolerant to 6% PEG, suggesting that ARDH enzyme maintains integrity of membranes in chloroplasts under drought conditions via metabolic engineering. Hence, the gene could be expressed in agronomic plants to withstand abiotic stresses.

Effort to increase an engine performance using electrical ignition system for motor vehicle

Directory of Open Access Journals (Sweden)

I Wayan Bandem Adnyana

2012-11-01

Full Text Available Increasing engine performances using electrical ignition system on motor vehicle. In accordance with the development oftechnology, improvisation of automotive is created in order to increase the performance of engine. The method to increase thisperformance has been done by modify the ignition system, where the conventional method of ignition system which uses contactbreaker substituted by using capacitor. The improvisation of ignition system has been tested by increasing the speed and load onstationary condition. Results show that the improvisation of ignition system by using capacitor increases the effective power andreduce the specific fuel consumption of engine and reduce the gas emission of CO.

Building inclusive engineering identities: implications for changing engineering culture

Science.gov (United States)

Atadero, Rebecca A.; Paguyo, Christina H.; Rambo-Hernandez, Karen E.; Henderson, Heather L.

2018-05-01

Ongoing efforts to broaden the participation of women and people of colour in engineering degree programmes and careers have had limited success. This paper describes a different approach to broadening participation that seeks to work with all students and develop inclusive engineering identities. Researchers worked with the instructors of two first-year engineering courses to integrate curriculum activities designed to promote the formation of engineering identities and build an appreciation for how diversity and inclusion strengthen engineering practice. Multilevel modelling results indicated positive effects of the intervention on appreciation for diversity but no effects on engineering identity, and qualitative results indicated students learned the most about diversity not through one of the intervention activities, but through team projects in the courses. We also describe lessons learned in how to teach engineering students about diversity in ways that are relevant to engineering.

Effects of introducing heterologous pathways on microbial metabolism with respect to metabolic optimality

DEFF Research Database (Denmark)

Kim, Hyun Uk; Kim, Byoungjin; Seung, Do Young

2014-01-01

reactions are more frequently introduced into various microbial hosts. The genome-scale metabolic simulations of Escherichia coli strains engineered to produce 1,4-butanediol, 1,3-propanediol, and amorphadiene suggest that microbial metabolism shows much different responses to the introduced heterologous...... reactions in a strain-specific manner than typical gene knockouts in terms of the energetic status (e.g., ATP and biomass generation) and chemical production capacity. The 1,4-butanediol and 1,3-propanediol producers showed greater metabolic optimality than the wild-type strains and gene knockout mutants...... for the energetic status, while the amorphadiene producer was metabolically less optimal. For the optimal chemical production capacity, additional gene knockouts were most effective for the strain producing 1,3-propanediol, but not for the one producing 1,4-butanediol. These observations suggest that strains having...

Biochemistry engineering

International Nuclear Information System (INIS)

Jang, Ho Nam

1993-01-01

This deals with biochemistry engineering with nine chapters. It explains bionics on development and prospect, basics of life science on classification and structure, enzyme and metabolism, fundamentals of chemical engineering on viscosity, shear rate, PFR, CSTR, mixing, dispersion, measurement and response, Enzyme kinetics, competitive inhibition, pH profile, temperature profile, stoichiometry and fermentation kinetics, bio-reactor on Enzyme-reactor and microorganism-reactor, measurement and processing on data acquisition and data processing, separation and purification, waste water treatment and economics of bionics process.

Synthetic Biology and Metabolic Engineering Approaches and Its Impact on Non-Conventional Yeast and Biofuel Production

Energy Technology Data Exchange (ETDEWEB)

Madhavan, Aravind [Biotechnology Division, National Institute for Interdisciplinary Science and Technology, Council of Scientific and Industrial Research, Trivandrum (India); Rajiv Gandhi Centre for Biotechnology, Trivandrum (India); Jose, Anju Alphonsa; Binod, Parameswaran; Sindhu, Raveendran, E-mail: sindhurgcb@gmail.com; Sukumaran, Rajeev K. [Biotechnology Division, National Institute for Interdisciplinary Science and Technology, Council of Scientific and Industrial Research, Trivandrum (India); Pandey, Ashok [Biotechnology Division, National Institute for Interdisciplinary Science and Technology, Council of Scientific and Industrial Research, Trivandrum (India); Center for Innovative and Applied Bioprocessing, Mohali, Punjab (India); Castro, Galliano Eulogio [Dpt. Ingeniería Química, Ambiental y de los Materiales Edificio, Universidad de Jaén, Jaén (Spain)

2017-04-25

The increasing fossil fuel scarcity has led to an urgent need to develop alternative fuels. Currently microorganisms have been extensively used for the production of first-generation biofuels from lignocellulosic biomass. Yeast is the efficient producer of bioethanol among all existing biofuels option. Tools of synthetic biology have revolutionized the field of microbial cell factories especially in the case of ethanol and fatty acid production. Most of the synthetic biology tools have been developed for the industrial workhorse Saccharomyces cerevisiae. The non-conventional yeast systems have several beneficial traits like ethanol tolerance, thermotolerance, inhibitor tolerance, genetic diversity, etc., and synthetic biology have the power to expand these traits. Currently, synthetic biology is slowly widening to the non-conventional yeasts like Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, and Yarrowia lipolytica. Herein, we review the basic synthetic biology tools that can apply to non-conventional yeasts. Furthermore, we discuss the recent advances employed to develop efficient biofuel-producing non-conventional yeast strains by metabolic engineering and synthetic biology with recent examples. Looking forward, future synthetic engineering tools’ development and application should focus on unexplored non-conventional yeast species.

Synthetic Biology and Metabolic Engineering Approaches and Its Impact on Non-Conventional Yeast and Biofuel Production

Directory of Open Access Journals (Sweden)

Raveendran Sindhu

2017-04-01

Full Text Available The increasing fossil fuel scarcity has led to an urgent need to develop alternative fuels. Currently microorganisms have been extensively used for the production of first-generation biofuels from lignocellulosic biomass. Yeast is the efficient producer of bioethanol among all existing biofuels option. Tools of synthetic biology have revolutionized the field of microbial cell factories especially in the case of ethanol and fatty acid production. Most of the synthetic biology tools have been developed for the industrial workhorse Saccharomyces cerevisiae. The non-conventional yeast systems have several beneficial traits like ethanol tolerance, thermotolerance, inhibitor tolerance, genetic diversity, etc., and synthetic biology have the power to expand these traits. Currently, synthetic biology is slowly widening to the non-conventional yeasts like Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, and Yarrowia lipolytica. Herein, we review the basic synthetic biology tools that can apply to non-conventional yeasts. Furthermore, we discuss the recent advances employed to develop efficient biofuel-producing non-conventional yeast strains by metabolic engineering and synthetic biology with recent examples. Looking forward, future synthetic engineering tools’ development and application should focus on unexplored non-conventional yeast species.

Synthetic Biology and Metabolic Engineering Approaches and Its Impact on Non-Conventional Yeast and Biofuel Production

International Nuclear Information System (INIS)

Madhavan, Aravind; Jose, Anju Alphonsa; Binod, Parameswaran; Sindhu, Raveendran; Sukumaran, Rajeev K.; Pandey, Ashok; Castro, Galliano Eulogio

2017-01-01

The increasing fossil fuel scarcity has led to an urgent need to develop alternative fuels. Currently microorganisms have been extensively used for the production of first-generation biofuels from lignocellulosic biomass. Yeast is the efficient producer of bioethanol among all existing biofuels option. Tools of synthetic biology have revolutionized the field of microbial cell factories especially in the case of ethanol and fatty acid production. Most of the synthetic biology tools have been developed for the industrial workhorse Saccharomyces cerevisiae. The non-conventional yeast systems have several beneficial traits like ethanol tolerance, thermotolerance, inhibitor tolerance, genetic diversity, etc., and synthetic biology have the power to expand these traits. Currently, synthetic biology is slowly widening to the non-conventional yeasts like Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, and Yarrowia lipolytica. Herein, we review the basic synthetic biology tools that can apply to non-conventional yeasts. Furthermore, we discuss the recent advances employed to develop efficient biofuel-producing non-conventional yeast strains by metabolic engineering and synthetic biology with recent examples. Looking forward, future synthetic engineering tools’ development and application should focus on unexplored non-conventional yeast species.

Programmed evolution for optimization of orthogonal metabolic output in bacteria.

Directory of Open Access Journals (Sweden)

Todd T Eckdahl

Full Text Available Current use of microbes for metabolic engineering suffers from loss of metabolic output due to natural selection. Rather than combat the evolution of bacterial populations, we chose to embrace what makes biological engineering unique among engineering fields - evolving materials. We harnessed bacteria to compute solutions to the biological problem of metabolic pathway optimization. Our approach is called Programmed Evolution to capture two concepts. First, a population of cells is programmed with DNA code to enable it to compute solutions to a chosen optimization problem. As analog computers, bacteria process known and unknown inputs and direct the output of their biochemical hardware. Second, the system employs the evolution of bacteria toward an optimal metabolic solution by imposing fitness defined by metabolic output. The current study is a proof-of-concept for Programmed Evolution applied to the optimization of a metabolic pathway for the conversion of caffeine to theophylline in E. coli. Introduced genotype variations included strength of the promoter and ribosome binding site, plasmid copy number, and chaperone proteins. We constructed 24 strains using all combinations of the genetic variables. We used a theophylline riboswitch and a tetracycline resistance gene to link theophylline production to fitness. After subjecting the mixed population to selection, we measured a change in the distribution of genotypes in the population and an increased conversion of caffeine to theophylline among the most fit strains, demonstrating Programmed Evolution. Programmed Evolution inverts the standard paradigm in metabolic engineering by harnessing evolution instead of fighting it. Our modular system enables researchers to program bacteria and use evolution to determine the combination of genetic control elements that optimizes catabolic or anabolic output and to maintain it in a population of cells. Programmed Evolution could be used for applications in

Programmed Evolution for Optimization of Orthogonal Metabolic Output in Bacteria

Science.gov (United States)

Eckdahl, Todd T.; Campbell, A. Malcolm; Heyer, Laurie J.; Poet, Jeffrey L.; Blauch, David N.; Snyder, Nicole L.; Atchley, Dustin T.; Baker, Erich J.; Brown, Micah; Brunner, Elizabeth C.; Callen, Sean A.; Campbell, Jesse S.; Carr, Caleb J.; Carr, David R.; Chadinha, Spencer A.; Chester, Grace I.; Chester, Josh; Clarkson, Ben R.; Cochran, Kelly E.; Doherty, Shannon E.; Doyle, Catherine; Dwyer, Sarah; Edlin, Linnea M.; Evans, Rebecca A.; Fluharty, Taylor; Frederick, Janna; Galeota-Sprung, Jonah; Gammon, Betsy L.; Grieshaber, Brandon; Gronniger, Jessica; Gutteridge, Katelyn; Henningsen, Joel; Isom, Bradley; Itell, Hannah L.; Keffeler, Erica C.; Lantz, Andrew J.; Lim, Jonathan N.; McGuire, Erin P.; Moore, Alexander K.; Morton, Jerrad; Nakano, Meredith; Pearson, Sara A.; Perkins, Virginia; Parrish, Phoebe; Pierson, Claire E.; Polpityaarachchige, Sachith; Quaney, Michael J.; Slattery, Abagael; Smith, Kathryn E.; Spell, Jackson; Spencer, Morgan; Taye, Telavive; Trueblood, Kamay; Vrana, Caroline J.; Whitesides, E. Tucker

2015-01-01

Current use of microbes for metabolic engineering suffers from loss of metabolic output due to natural selection. Rather than combat the evolution of bacterial populations, we chose to embrace what makes biological engineering unique among engineering fields – evolving materials. We harnessed bacteria to compute solutions to the biological problem of metabolic pathway optimization. Our approach is called Programmed Evolution to capture two concepts. First, a population of cells is programmed with DNA code to enable it to compute solutions to a chosen optimization problem. As analog computers, bacteria process known and unknown inputs and direct the output of their biochemical hardware. Second, the system employs the evolution of bacteria toward an optimal metabolic solution by imposing fitness defined by metabolic output. The current study is a proof-of-concept for Programmed Evolution applied to the optimization of a metabolic pathway for the conversion of caffeine to theophylline in E. coli. Introduced genotype variations included strength of the promoter and ribosome binding site, plasmid copy number, and chaperone proteins. We constructed 24 strains using all combinations of the genetic variables. We used a theophylline riboswitch and a tetracycline resistance gene to link theophylline production to fitness. After subjecting the mixed population to selection, we measured a change in the distribution of genotypes in the population and an increased conversion of caffeine to theophylline among the most fit strains, demonstrating Programmed Evolution. Programmed Evolution inverts the standard paradigm in metabolic engineering by harnessing evolution instead of fighting it. Our modular system enables researchers to program bacteria and use evolution to determine the combination of genetic control elements that optimizes catabolic or anabolic output and to maintain it in a population of cells. Programmed Evolution could be used for applications in energy

Initial Efforts in Characterizing Radiation and Plasma Effects on Space Assets: Bridging the Space Environment, Engineering and User Community

Science.gov (United States)

Zheng, Y.; Ganushkina, N. Y.; Guild, T. B.; Jiggens, P.; Jun, I.; Mazur, J. E.; Meier, M. M.; Minow, J. I.; Pitchford, D. A.; O'Brien, T. P., III; Shprits, Y.; Tobiska, W. K.; Xapsos, M.; Rastaetter, L.; Jordanova, V. K.; Kellerman, A. C.; Fok, M. C. H.

2017-12-01

The Community Coordinated Modeling Center (CCMC) has been leading the community-wide model validation projects for many years. Such effort has been broadened and extended via the newly-launched International Forum for Space Weather Modeling Capabilities Assessment (https://ccmc.gsfc.nasa.gov/assessment/), Its objective is to track space weather models' progress and performance over time, which is critically needed in space weather operations. The Radiation and Plasma Effects Working Team is working on one of the many focused evaluation topics and deals with five different subtopics: Surface Charging from 10s eV to 40 keV electrons, Internal Charging due to energetic electrons from hundreds keV to several MeVs. Single Event Effects from solar energetic particles (SEPs) and galactic cosmic rays (GCRs) (several MeV to TeVs), Total Dose due to accumulation of doses from electrons (>100 KeV) and protons (> 1 MeV) in a broad energy range, and Radiation Effects from SEPs and GCRs at aviation altitudes. A unique aspect of the Radiation and Plasma Effects focus area is that it bridges the space environments, engineering and user community. This presentation will summarize the working team's progress in metrics discussion/definition and the CCMC web interface/tools to facilitate the validation efforts. As an example, tools in the areas of surface charging/internal charging will be demoed.

Advanced biotechnology: metabolically engineered cells for the bio-based production of chemicals and fuels, materials, and health-care products.

Science.gov (United States)

Becker, Judith; Wittmann, Christoph

2015-03-09

Corynebacterium glutamicum, Escherichia coli, and Saccharomyces cerevisiae in particular, have become established as important industrial workhorses in biotechnology. Recent years have seen tremendous progress in their advance into tailor-made producers, driven by the upcoming demand for sustainable processes and renewable raw materials. Here, the diversity and complexity of nature is simultaneously a challenge and a benefit. Harnessing biodiversity in the right manner through synergistic progress in systems metabolic engineering and chemical synthesis promises a future innovative bio-economy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

System-level perturbations of cell metabolism using CRISPR/Cas9

Energy Technology Data Exchange (ETDEWEB)

Jakočiūnas, Tadas [Technical Univ. of Denmark, Lyngby (Denmark); Jensen, Michael K. [Technical Univ. of Denmark, Lyngby (Denmark); Keasling, Jay D. [Technical Univ. of Denmark, Lyngby (Denmark); Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)

2017-03-30

CRISPR/Cas9 (clustered regularly interspaced palindromic repeats and the associated protein Cas9) techniques have made genome engineering and transcriptional reprogramming studies much more advanced and cost-effective. For metabolic engineering purposes, the CRISPR-based tools have been applied to single and multiplex pathway modifications and transcriptional regulations. The effectiveness of these tools allows researchers to implement genome-wide perturbations, test model-guided genome editing strategies, and perform transcriptional reprogramming perturbations in a more advanced manner than previously possible. In this mini-review we highlight recent studies adopting CRISPR/Cas9 for systems-level perturbations and model-guided metabolic engineering.

Student Interest in Engineering Design-Based Science

Science.gov (United States)

Selcen Guzey, S.; Moore, Tamara J.; Morse, Gillian

2016-01-01

Current reform efforts in science education around the world call on teachers to use integrated approaches to teach science. As a part of such reform efforts in the United States, engineering practices and engineering design have been identified in K-12 science education standards. However, there is relatively little is known about effective ways…

Recent Stirling engine loss - understanding results

International Nuclear Information System (INIS)

Tew, R.C.; Thieme, L.G.; Dudenhoefer, J.E.

1994-01-01

For several years, the National Aeronautics and Space Administration and other US Government agencies have been funding experimental and analytical efforts to improve the understanding of Stirling thermodynamic losses. NASA's objective is to improve Stirling engine design capability to support the development of new engines for space power. An overview of these efforts was last given at the 1988 IECEC. Recent results of this research are reviewed

Metabolic engineering of monoterpene biosynthesis in tomato fruits via introduction of the non-canonical substrate neryl diphosphate.

Science.gov (United States)

Gutensohn, Michael; Nguyen, Thuong T H; McMahon, Richard D; Kaplan, Ian; Pichersky, Eran; Dudareva, Natalia

2014-07-01

Recently it was shown that monoterpenes in tomato trichomes (Solanum lycopersicum) are synthesized by phellandrene synthase 1 (PHS1) from the non-canonical substrate neryl diphosphate (NPP), the cis-isomer of geranyl diphosphate (GPP). As PHS1 accepts both NPP and GPP substrates forming different monoterpenes, it was overexpressed in tomato fruits to test if NPP is also available in a tissue highly active in carotenoid production. However, transgenic fruits overexpressing PHS1 produced only small amounts of GPP-derived PHS1 monoterpene products, indicating the absence of endogenous NPP. Therefore, NPP formation was achieved by diverting the metabolic flux from carotenoids via expression of tomato neryl diphosphate synthase 1 (NDPS1). NDPS1 transgenic fruits produced NPP-derived monoterpenes, including nerol, neral and geranial, while displaying reduced lycopene content. NDPS1 co-expression with PHS1 resulted in a monoterpene blend, including β-phellandrene, similar to that produced from NPP by PHS1 in vitro and in trichomes. Unexpectedly, PHS1×NDPS1 fruits showed recovery of lycopene levels compared to NDPS1 fruits, suggesting that redirection of metabolic flux is only partially responsible for the reduction in carotenoids. In vitro assays demonstrated that NPP serves as an inhibitor of geranylgeranyl diphosphate synthase, thus its consumption by PHS1 leads to recovery of lycopene levels. Monoterpenes produced in PHS1×NDPS1 fruits contributed to direct plant defense negatively affecting feeding behavior of the herbivore Helicoverpa zea and displaying antifungal activity against Botrytis cinerea. These results show that NPP-derived terpenoids can be produced in plant tissues; however, NPP has to be consumed to avoid negative impacts on plant metabolism. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Tissue Engineering of the Penis

Directory of Open Access Journals (Sweden)

Manish N. Patel

2011-01-01

Full Text Available Congenital disorders, cancer, trauma, or other conditions of the genitourinary tract can lead to significant organ damage or loss of function, necessitating eventual reconstruction or replacement of the damaged structures. However, current reconstructive techniques are limited by issues of tissue availability and compatibility. Physicians and scientists have begun to explore tissue engineering and regenerative medicine strategies for repair and reconstruction of the genitourinary tract. Tissue engineering allows the development of biological substitutes which could potentially restore normal function. Tissue engineering efforts designed to treat or replace most organs are currently being undertaken. Most of these efforts have occurred within the past decade. However, before these engineering techniques can be applied to humans, further studies are needed to ensure the safety and efficacy of these new materials. Recent progress suggests that engineered urologic tissues and cell therapy may soon have clinical applicability.

Development of Computational Tools for Metabolic Model Curation, Flux Elucidation and Strain Design

Energy Technology Data Exchange (ETDEWEB)

Maranas, Costas D

2012-05-21

An overarching goal of the Department of Energy mission is the efficient deployment and engineering of microbial and plant systems to enable biomass conversion in pursuit of high energy density liquid biofuels. This has spurred the pace at which new organisms are sequenced and annotated. This torrent of genomic information has opened the door to understanding metabolism in not just skeletal pathways and a handful of microorganisms but for truly genome-scale reconstructions derived for hundreds of microbes and plants. Understanding and redirecting metabolism is crucial because metabolic fluxes are unique descriptors of cellular physiology that directly assess the current cellular state and quantify the effect of genetic engineering interventions. At the same time, however, trying to keep pace with the rate of genomic data generation has ushered in a number of modeling and computational challenges related to (i) the automated assembly, testing and correction of genome-scale metabolic models, (ii) metabolic flux elucidation using labeled isotopes, and (iii) comprehensive identification of engineering interventions leading to the desired metabolism redirection.

Student employment and study effort for engineering students

DEFF Research Database (Denmark)

Clemmensen, Line Katrine Harder; Harder, D. E.

2015-01-01

more than those in studies from e.g. UK and US [3, 4, 5]. A similar trend was seen in a study from Norway [6]. Government financial support seems to limit the amount of hours spent on paid work but not the percentage of students who take on paid work. Thus, full-time studies with benefits of increased...... capabilities and experience gained through employment could be aided by proper policies. Additionally, one of the highest impacts on study activity was the perceived study environment. As the engineering students have four hours per week of interaction with an instructor for each five ECTS...... to answer if the full-time student is under demise in these settings as opposed to settings without financial support [1, 2]. The research consisted of a web-based survey amongst all students at the Technical University of Denmark (DTU). The students in this survey had fewer employment hours and studied...

Enzyme and metabolic engineering for the production of novel biopolymers: crossover of biological and chemical processes.

Science.gov (United States)

Matsumoto, Ken'ichiro; Taguchi, Seiichi

2013-12-01

The development of synthetic biology has transformed microbes into useful factories for producing valuable polymers and/or their precursors from renewable biomass. Recent progress at the interface of chemistry and biology has enabled the production of a variety of new biopolymers with properties that substantially differ from their petroleum-derived counterparts. This review touches on recent trials and achievements in the field of biopolymer synthesis, including chemo-enzymatically synthesized aliphatic polyesters, wholly biosynthesized lactate-based polyesters, polyhydroxyalkanoates and other unusual bacterially synthesized polyesters. The expanding diversities in structure and the material properties of biopolymers are key for exploring practical applications. The enzyme and metabolic engineering approaches toward this goal are discussed by shedding light on the successful case studies. Copyright © 2013 Elsevier Ltd. All rights reserved.

Catabolism of biomass-derived sugars in fungi and metabolic engineering as a tool for organic acid production

Energy Technology Data Exchange (ETDEWEB)

Koivistoinen, O.

2013-11-01

The use of metabolic engineering as a tool for production of biochemicals and biofuels requires profound understanding of cell metabolism. The pathways for the most abundant and most important hexoses have already been studied quite extensively but it is also important to get a more complete picture of sugar catabolism. In this thesis, catabolic pathways of L-rhamnose and D-galactose were studied in fungi. Both of these hexoses are present in plant biomass, such as in hemicellulose and pectin. Galactoglucomannan, a type of hemicellulose that is especially rich in softwood, is an abundant source of D-galactose. As biotechnology is moving from the usage of edible and easily metabolisable carbon sources towards the increased use of lignocellulosic biomass, it is important to understand how the different sugars can be efficiently turned into valuable biobased products. Identification of the first fungal L-rhamnose 1-dehydrogenase gene, which codes for the first enzyme of the fungal catabolic L-rhamnose pathway, showed that the protein belongs to a protein family of short-chain alcohol dehydrogenases. Sugar dehydrogenases oxidising a sugar to a sugar acid are not very common in fungi and thus the identification of the L-rhamnose dehydrogenase gene provides more understanding of oxidative sugar catabolism in eukaryotic microbes. Further studies characterising the L-rhamnose cluster in the yeast Scheffersomyces stipitis including the expression of the L-rhamnonate dehydratase in Saccharomyces cerevisiae finalised the biochemical characterisation of the enzymes acting on the pathway. In addition, more understanding of the regulation and evolution of the pathway was gained. D-Galactose catabolism was studied in the filamentous fungus Aspergillus niger. Two genes coding for the enzymes of the oxido-reductive pathway were identified. Galactitol dehydrogenase is the second enzyme of the pathway converting galactitol to L-xylo-3-hexulose. The galactitol dehydrogenase encoding

Systems Engineering Force XXI: Experimental Analysis Integration and Systems Engineering Support to the Force XXI Design Effort

National Research Council Canada - National Science Library

Brock, H

1996-01-01

.... Effective needs are critical because the definition of a successful design effort is meeting or exceeding the effective needs of the client or stakeholder group in a cost-effective, high-quality way...

Metabolic and Transcriptional Response to Cofactor Perturbations in Escherichia coli

DEFF Research Database (Denmark)

Holm, Anders Koefoed; Blank, L.M.; Oldiges, M.

2010-01-01

Metabolic cofactors such as NADH and ATP play important roles in a large number of cellular reactions, and it is of great interest to dissect the role of these cofactors in different aspects of metabolism. Toward this goal, we overexpressed NADH oxidase and the soluble F1-ATPase in Escherichia coli...... of redox and energy metabolism and should help in developing metabolic engineering strategies in E. coli....

Advances in metabolic pathway and strain engineering paving the way for sustainable production of chemical building blocks.

Science.gov (United States)

Chen, Yun; Nielsen, Jens

2013-12-01

Bio-based production of chemical building blocks from renewable resources is an attractive alternative to petroleum-based platform chemicals. Metabolic pathway and strain engineering is the key element in constructing robust microbial chemical factories within the constraints of cost effective production. Here we discuss how the development of computational algorithms, novel modules and methods, omics-based techniques combined with modeling refinement are enabling reduction in development time and thus advance the field of industrial biotechnology. We further discuss how recent technological developments contribute to the development of novel cell factories for the production of the building block chemicals: adipic acid, succinic acid and 3-hydroxypropionic acid. Copyright © 2013 Elsevier Ltd. All rights reserved.

Metabolic engineering of ethanol production in Thermoanaerobacter mathranii

Energy Technology Data Exchange (ETDEWEB)

Shou Yao

2010-11-15

Strain BG1 is a xylanolytic, thermophilic, anaerobic, Gram-positive bacterium originally isolated from an Icelandic hot spring. The strain belongs to the species Thermoanaerobacter mathranii. The strain ferments glucose, xylose, arabinose, galactose and mannose simultaneously and produces ethanol, acetate, lactate, CO{sub 2}, and H2 as fermentation end-products. As a potential ethanol producer from lignocellulosic biomass, tailor-made BG1 strain with the metabolism redirected to produce ethanol is needed. Metabolic engineering of T. mathranii BG1 is therefore necessary to improve ethanol production. Strain BG1 contains four alcohol dehydrogenase (ADH) encoding genes. They are adhA, adhB, bdhA and adhE encoding primary alcohol dehydrogenase, secondary alcohol dehydrogenase, butanol dehydrogenase and bifunctional alcohol/acetaldehyde dehydrogenase, respectively. The presence in an organism of multiple alcohol dehydrogenases with overlapping specificities makes the determination of the specific role of each ADH difficult. Deletion of each individual adh gene in the strain revealed that the adhE deficient mutant strain fails to produce ethanol as the fermentation product. The bifunctional alcohol/acetaldehyde dehydrogenase, AdhE, is therefore proposed responsible for ethanol production in T. mathranii BG1, by catalyzing sequential NADH-dependent reductions of acetyl-CoA to acetaldehyde and then to ethanol under fermentative conditions. Moreover, AdhE was conditionally expressed from a xylose-induced promoter in a recombinant strain (BG1E1) with a concomitant deletion of a lactate dehydrogenase. Over-expression of AdhE in strain BG1E1 with xylose as a substrate facilitates the production of ethanol at an increased yield. With a cofactor-dependent ethanol production pathway in T. mathranii BG1, it may become crucial to regenerate cofactor to increase the ethanol yield. Feeding the cells with a more reduced carbon source, such as mannitol, was shown to increase ethanol

MicrobesFlux: a web platform for drafting metabolic models from the KEGG database

Directory of Open Access Journals (Sweden)

Feng Xueyang

2012-08-01

Full Text Available Abstract Background Concurrent with the efforts currently underway in mapping microbial genomes using high-throughput sequencing methods, systems biologists are building metabolic models to characterize and predict cell metabolisms. One of the key steps in building a metabolic model is using multiple databases to collect and assemble essential information about genome-annotations and the architecture of the metabolic network for a specific organism. To speed up metabolic model development for a large number of microorganisms, we need a user-friendly platform to construct metabolic networks and to perform constraint-based flux balance analysis based on genome databases and experimental results. Results We have developed a semi-automatic, web-based platform (MicrobesFlux for generating and reconstructing metabolic models for annotated microorganisms. MicrobesFlux is able to automatically download the metabolic network (including enzymatic reactions and metabolites of ~1,200 species from the KEGG database (Kyoto Encyclopedia of Genes and Genomes and then convert it to a metabolic model draft. The platform also provides diverse customized tools, such as gene knockouts and the introduction of heterologous pathways, for users to reconstruct the model network. The reconstructed metabolic network can be formulated to a constraint-based flux model to predict and analyze the carbon fluxes in microbial metabolisms. The simulation results can be exported in the SBML format (The Systems Biology Markup Language. Furthermore, we also demonstrated the platform functionalities by developing an FBA model (including 229 reactions for a recent annotated bioethanol producer, Thermoanaerobacter sp. strain X514, to predict its biomass growth and ethanol production. Conclusion MicrobesFlux is an installation-free and open-source platform that enables biologists without prior programming knowledge to develop metabolic models for annotated microorganisms in the KEGG

An overview of the NASA rotary engine research program

Science.gov (United States)

Meng, P. R.; Hady, W. F.

1984-01-01

A brief overview and technical highlights of the research efforts and studies on rotary engines over the last several years at the NASA Lewis Research Center are presented. The test results obtained from turbocharged rotary engines and preliminary results from a high performance single rotor engine were discussed. Combustion modeling studies of the rotary engine and the use of a Laser Doppler Velocimeter to confirm the studies were examined. An in-house program in which a turbocharged rotary engine was installed in a Cessna Skymaster for ground test studies was reviewed. Details are presented on single rotor stratified charge rotary engine research efforts, both in-house and on contract.

Metabolic Engineering of the Actinomycete Amycolatopsis sp. Strain ATCC 39116 towards Enhanced Production of Natural Vanillin.

Science.gov (United States)

Fleige, Christian; Meyer, Florian; Steinbüchel, Alexander

2016-06-01

The Gram-positive bacterium Amycolatopsis sp. ATCC 39116 is used for the fermentative production of natural vanillin from ferulic acid on an industrial scale. The strain is known for its outstanding tolerance to this toxic product. In order to improve the productivity of the fermentation process, the strain's metabolism was engineered for higher final concentrations and molar yields. Degradation of vanillin could be decreased by more than 90% through deletion of the vdh gene, which codes for the central vanillin catabolism enzyme, vanillin dehydrogenase. This mutation resulted in improvement of the final concentration of vanillin by more than 2.2 g/liter, with a molar yield of 80.9%. Further improvement was achieved with constitutive expression of the vanillin anabolism genes ech and fcs, coding for the enzymes feruloyl-coenzyme A (CoA) synthetase (fcs) and enoyl-CoA hydratase/aldolase (ech). The transcription of both genes was shown to be induced by ferulic acid, which explains the unwanted adaptation phase in the fermentation process before vanillin was efficiently produced by the wild-type cells. Through the constitutive and enhanced expression of the two genes, the adaptation phase was eliminated and a final vanillin concentration of 19.3 g/liter, with a molar yield of 94.9%, was obtained. Moreover, an even higher final vanillin concentration of 22.3 g/liter was achieved, at the expense of a lower molar yield, by using an improved feeding strategy. This is the highest reported vanillin concentration reached in microbial fermentation processes without extraction of the product. Furthermore, the vanillin was produced almost without by-products, with a molar yield that nearly approached the theoretical maximum. Much effort has been put into optimization of the biotechnological production of natural vanillin. The demand for this compound is growing due to increased consumer concerns regarding chemically produced food additives. Since this compound is toxic to most

Gender-Specific Covariations between Competencies, Interest and Effort during Science Learning in Virtual Environments.

Science.gov (United States)

Christophel, Eva; Schnotz, Wolfgang

2017-01-01

Women are still underrepresented in engineering courses although some German universities offer separate women's engineering courses which include virtual STEM learning environments. To outline information about fundamental aspects relevant for virtual STEM learning, one has to reveal which similarities both genders in virtual learning show. Moreover, the question arises as to whether there are in fact differences in the virtual science learning of female and male learners. Working with virtual STEM learning environments requires strategic and arithmetic-operative competences. Even if we assume that female and male learners have similar competences levels, their correlational pattern of competences, motivational variables, and invested effort during virtual STEM learning might differ. If such gender differences in the correlations between cognitive and motivational variables and learning behavior were revealed, it would be possible to finetune study conditions for female students in a separate engineering course and shape virtual STEM learning in a more gender-appropriate manner. That might support an increase in the number of women in engineering courses. To reveal the differences and similarities between female and male learners, a field study was conducted with 56 students (female = 27, male = 29) as part of the Open MINT Labs project (the German term for Open STEM Labs, OML). The participants had to complete a virtual STEM learning environment during their regular science lessons. The data were collected with questionnaires. The results revealed that the strategic competences of both genders were positively correlated with situational interest in the virtual learning environment. This result shows the big impact strategic competences have for both genders regarding their situational interest. In contrast, the correlations between mental effort and competences differed between female and male participants. Especially female learners' mental effort decreased if

Manage changes in the requirements definition through a collaborative effort

CSIR Research Space (South Africa)

Joseph-Malherbe, S

2009-08-01

Full Text Available to the engineering effort. A history of changes made to the data repository should be kept throughout the SE process. The software development community refers to such a history of changes as version control. This will enable the systems engineer to generate a list... of changes at any point during the development process, showing the time of the change and by whom the change was introduced. By sharing the change history with stakeholders, they can see how the model evolved and what the rationale was for each change...

Aerospace engineering curriculum for the 21st century

Science.gov (United States)

Simitses, George J.

1995-01-01

The second year of the study was devoted to completing the information-gathering phase of this redesign effort, using the conclusions from that activity to prepare the initial structure for the new curriculum, publicizing activities to a wider engineering forum, and preparing the department faculty (Aerospace Engineering and Engineering Mechanics at University of Cincinnati) for the roles they will play in the curriculum redesign and implementation. These activities are summarized briefly in this progress report. Attached is a paper resulting from the data acquisition of this effort, 'Educating Aerospace Engineers for the Twenty-First Century: Results of a Survey.'

Control of fluxes towards antibiotics and the role of primary metabolism in production of antibiotics

DEFF Research Database (Denmark)

Gunnarsson, Nina; Eliasson Lantz, Anna; Nielsen, Jacob

2004-01-01

Yield improvements in antibiotic-producing strains have classically been obtained through random mutagenesis and screening. An attractive alternative to this strategy is the rational design of producer strains via metabolic engineering, an approach that offers the possibility to increase yields...... in the metabolic network. Here we describe and discuss available methods for identification of these steps, both in antibiotic biosynthesis pathways and in the primary metabolism, which serves as the supplier of precursors and cofactors for the secondary metabolism. Finally, the importance of precursor...... and cofactor supply from primary metabolism in the biosynthesis of different types of antibiotics is discussed and recent developments in metabolic engineering towards increased product yields in antibiotic producing strains are reviewed....

Dysregulated metabolism contributes to oncogenesis

Science.gov (United States)

Hirschey, Matthew D.; DeBerardinis, Ralph J.; Diehl, Anna Mae E.; Drew, Janice E.; Frezza, Christian; Green, Michelle F.; Jones, Lee W.; Ko, Young H.; Le, Anne; Lea, Michael A.; Locasale, Jason W.; Longo, Valter D.; Lyssiotis, Costas A.; McDonnell, Eoin; Mehrmohamadi, Mahya; Michelotti, Gregory; Muralidhar, Vinayak; Murphy, Michael P.; Pedersen, Peter L.; Poore, Brad; Raffaghello, Lizzia; Rathmell, Jeffrey C.; Sivanand, Sharanya; Vander Heiden, Matthew G.; Wellen, Kathryn E.

2015-01-01

Cancer is a disease characterized by unrestrained cellular proliferation. In order to sustain growth, cancer cells undergo a complex metabolic rearrangement characterized by changes in metabolic pathways involved in energy production and biosynthetic processes. The relevance of the metabolic transformation of cancer cells has been recently included in the updated version of the review “Hallmarks of Cancer”, where the dysregulation of cellular metabolism was included as an emerging hallmark. While several lines of evidence suggest that metabolic rewiring is orchestrated by the concerted action of oncogenes and tumor suppressor genes, in some circumstances altered metabolism can play a primary role in oncogenesis. Recently, mutations of cytosolic and mitochondrial enzymes involved in key metabolic pathways have been associated with hereditary and sporadic forms of cancer. Together, these results suggest that aberrant metabolism, once seen just as an epiphenomenon of oncogenic reprogramming, plays a key role in oncogenesis with the power to control both genetic and epigenetic events in cells. In this review, we discuss the relationship between metabolism and cancer, as part of a larger effort to identify a broad-spectrum of therapeutic approaches. We focus on major alterations in nutrient metabolism and the emerging link between metabolism and epigenetics. Finally, we discuss potential strategies to manipulate metabolism in cancer and tradeoffs that should be considered. More research on the suite of metabolic alterations in cancer holds the potential to discover novel approaches to treat it. PMID:26454069

Metabolic engineering of a diazotrophic bacterium improves ammonium release and biofertilization of plants and microalgae.

Science.gov (United States)

Ambrosio, Rafael; Ortiz-Marquez, Juan Cesar Federico; Curatti, Leonardo

2017-03-01

The biological nitrogen fixation carried out by some Bacteria and Archaea is one of the most attractive alternatives to synthetic nitrogen fertilizers. However, with the exception of the symbiotic rhizobia-legumes system, progress towards a more extensive realization of this goal has been slow. In this study we manipulated the endogenous regulation of both nitrogen fixation and assimilation in the aerobic bacterium Azotobacter vinelandii. Substituting an exogenously inducible promoter for the native promoter of glutamine synthetase produced conditional lethal mutant strains unable to grow diazotrophically in the absence of the inducer. This mutant phenotype could be reverted in a double mutant strain bearing a deletion in the nifL gene that resulted in constitutive expression of nif genes and increased production of ammonium. Under GS non-inducing conditions both the single and the double mutant strains consistently released very high levels of ammonium (>20mM) into the growth medium. The double mutant strain grew and excreted high levels of ammonium under a wider range of concentrations of the inducer than the single mutant strain. Induced mutant cells could be loaded with glutamine synthetase at different levels, which resulted in different patterns of extracellular ammonium accumulation afterwards. Inoculation of the engineered bacteria into a microalgal culture in the absence of sources of C and N other than N 2 and CO 2 from the air, resulted in a strong proliferation of microalgae that was suppressed upon addition of the inducer. Both single and double mutant strains also promoted growth of cucumber plants in the absence of added N-fertilizer, while this property was only marginal in the parental strain. This study provides a simple synthetic genetic circuit that might inspire engineering of optimized inoculants that efficiently channel N 2 from the air into crops. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All

Multi-office engineering

International Nuclear Information System (INIS)

Cowle, E.S.; Hall, L.D.; Koss, P.; Saheb, E.; Setrakian, V.

1995-01-01

This paper addresses the viability of multi-office project engineering as has been made possible in a large part by the computer age. Brief discussions are provided on two past projects describing the authors' initial efforts at multi-office engineering, and an in-depth discussion is provided on a current Bechtel project that demonstrates their multi-office engineering capabilities. Efficiencies and cost savings associated with executing an engineering project from multiple office locations was identified as a viable and cost-effective execution approach. The paper also discusses how the need for multi-office engineering came about, what is required to succeed, and where they are going from here. Furthermore, it summarizes the benefits to their clients and to Bechtel

Metabolism of chlorofluorocarbons and polybrominated compounds by Pseudomonas putida G786(pHG-2) via an engineered metabolic pathway.

Science.gov (United States)

Hur, H G; Sadowsky, M J; Wackett, L P

1994-11-01

The recombinant bacterium Pseudomonas putida G786(pHG-2) metabolizes pentachloroethane to glyoxylate and carbon dioxide, using cytochrome P-450CAM and toluene dioxygenase to catalyze consecutive reductive and oxidative dehalogenation reactions (L.P. Wackett, M.J. Sadowsky, L.N. Newman, H.-G. Hur, and S. Li, Nature [London] 368:627-629, 1994). The present study investigated metabolism of brominated and chlorofluorocarbon compounds by the recombinant strain. Under anaerobic conditions, P. putida G786(pHG-2) reduced 1,1,2,2-tetrabromoethane, 1,2-dibromo-1,2-dichloroethane, and 1,1,1,2-tetrachloro-2,2-difluoroethane to products bearing fewer halogen substituents. Under aerobic conditions, P. putida G786(pHG-2) oxidized cis- and trans-1,2-dibromoethenes, 1,1-dichloro-2,2-difluoroethene, and 1,2-dichloro-1-fluoroethene. Several compounds were metabolized by sequential reductive and oxidative reactions via the constructed metabolic pathway. For example, 1,1,2,2-tetrabromoethane was reduced by cytochrome P-450CAM to 1,2-dibromoethenes, which were subsequently oxidized by toluene dioxygenase. The same pathway metabolized 1,1,1,2-tetrachloro-2,2-difluoroethane to oxalic acid as one of the final products. The results obtained in this study indicate that P. putida G786(pHG-2) metabolizes polyfluorinated, chlorinated, and brominated compounds and further demonstrates the value of using a knowledge of catabolic enzymes and recombinant DNA technology to construct useful metabolic pathways.

Non-invasive Assessments of Adipose Tissue Metabolism In Vitro.

Science.gov (United States)

Abbott, Rosalyn D; Borowsky, Francis E; Quinn, Kyle P; Bernstein, David L; Georgakoudi, Irene; Kaplan, David L

2016-03-01

Adipose tissue engineering is a diverse area of research where the developed tissues can be used to study normal adipose tissue functions, create disease models in vitro, and replace soft tissue defects in vivo. Increasing attention has been focused on the highly specialized metabolic pathways that regulate energy storage and release in adipose tissues which affect local and systemic outcomes. Non-invasive, dynamic measurement systems are useful to track these metabolic pathways in the same tissue model over time to evaluate long term cell growth, differentiation, and development within tissue engineering constructs. This approach reduces costs and time in comparison to more traditional destructive methods such as biochemical and immunochemistry assays and proteomics assessments. Towards this goal, this review will focus on important metabolic functions of adipose tissues and strategies to evaluate them with non-invasive in vitro methods. Current non-invasive methods, such as measuring key metabolic markers and endogenous contrast imaging will be explored.

Contract engineers cut costs, and flexibility

International Nuclear Information System (INIS)

Bhargava, V.M.

1991-01-01

Today's rapid expansion of technology is an important reason for considering hiring contract engineering labor for your power plant because technical specialization is an important aspect of the contract engineering industry. This paper reports that another reason is addressing peak work loads. Any amount of technical assistance is available to assist in meeting schedules. Although a typical human resources department of a utility or architect/engineer may contain a few hundred active resumes, a contract engineering firm may have upwards of 40,000 resumes on file from all parts of the country. Because contract workers know that contract agencies keep abreast of client needs, they are more apt to forward updated resumes to such agencies. Used properly, a contract engineering company's services will augment the efforts of a human resources group and, in the case of some smaller companies, actually take the place of all internal recruiting efforts

Metabolite secretion in microorganisms: the theory of metabolic overflow put to the test

DEFF Research Database (Denmark)

Pinu, Farhana R.; Granucci, Ninna; Daniell, James

2018-01-01

Introduction Microbial cells secrete many metabolites during growth, including important intermediates of the central carbon metabolism. This has not been taken into account by researchers when modeling microbial metabolism for metabolic engineering and systems biology studies. Materials and Meth...

Supercomputer and cluster performance modeling and analysis efforts:2004-2006.

Energy Technology Data Exchange (ETDEWEB)

Sturtevant, Judith E.; Ganti, Anand; Meyer, Harold (Hal) Edward; Stevenson, Joel O.; Benner, Robert E., Jr. (.,; .); Goudy, Susan Phelps; Doerfler, Douglas W.; Domino, Stefan Paul; Taylor, Mark A.; Malins, Robert Joseph; Scott, Ryan T.; Barnette, Daniel Wayne; Rajan, Mahesh; Ang, James Alfred; Black, Amalia Rebecca; Laub, Thomas William; Vaughan, Courtenay Thomas; Franke, Brian Claude

2007-02-01

This report describes efforts by the Performance Modeling and Analysis Team to investigate performance characteristics of Sandia's engineering and scientific applications on the ASC capability and advanced architecture supercomputers, and Sandia's capacity Linux clusters. Efforts to model various aspects of these computers are also discussed. The goals of these efforts are to quantify and compare Sandia's supercomputer and cluster performance characteristics; to reveal strengths and weaknesses in such systems; and to predict performance characteristics of, and provide guidelines for, future acquisitions and follow-on systems. Described herein are the results obtained from running benchmarks and applications to extract performance characteristics and comparisons, as well as modeling efforts, obtained during the time period 2004-2006. The format of the report, with hypertext links to numerous additional documents, purposefully minimizes the document size needed to disseminate the extensive results from our research.

Building Open in Engineering

OpenAIRE

Berg, Devin

2018-01-01

A description of ongoing efforts to build a culture for open in engineering. I will overview some of the tools that have been developed to promote open practices in engineering and discuss some of the barriers to adoption within the field.Presented 27 March 2018 at the "E"ffordabilitiy Summit, Menomonie, WI.

Acetone production with metabolically engineered strains of Acetobacterium woodii.

Science.gov (United States)

Hoffmeister, Sabrina; Gerdom, Marzena; Bengelsdorf, Frank R; Linder, Sonja; Flüchter, Sebastian; Öztürk, Hatice; Blümke, Wilfried; May, Antje; Fischer, Ralf-Jörg; Bahl, Hubert; Dürre, Peter

2016-07-01

Expected depletion of oil and fossil resources urges the development of new alternative routes for the production of bulk chemicals and fuels beyond petroleum resources. In this study, the clostridial acetone pathway was used for the formation of acetone in the acetogenic bacterium Acetobacterium woodii. The acetone production operon (APO) containing the genes thlA (encoding thiolase A), ctfA/ctfB (encoding CoA transferase), and adc (encoding acetoacetate decarboxylase) from Clostridium acetobutylicum were cloned under the control of the thlA promoter into four vectors having different replicons for Gram-positives (pIP404, pBP1, pCB102, and pCD6). Stable replication was observed for all constructs. A. woodii [pJIR_actthlA] achieved the maximal acetone concentration under autotrophic conditions (15.2±3.4mM). Promoter sequences of the genes ackA from A. woodii and pta-ack from C. ljungdahlii were determined by primer extension (PEX) and cloned upstream of the APO. The highest acetone production in recombinant A. woodii cells was achieved using the promoters PthlA and Ppta-ack. Batch fermentations using A. woodii [pMTL84151_actthlA] in a bioreactor revealed that acetate concentration had an effect on the acetone production, due to the high Km value of the CoA transferase. In order to establish consistent acetate concentration within the bioreactor and to increase biomass, a continuous fermentation process for A. woodii was developed. Thus, acetone productivity of the strain A. woodii [pMTL84151_actthlA] was increased from 1.2mgL(-1)h(-1) in bottle fermentation to 26.4mgL(-1)h(-1) in continuous gas fermentation. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

IEEE [Institute of Electrical and Electronics Engineers] standards and nuclear software quality engineering

International Nuclear Information System (INIS)

Daughtrey, T.

1988-01-01