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      Engineering an electroactive Escherichia coli for the microbial electrosynthesis of succinate from glucose and CO 2

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          Abstract

          Background

          Electrochemical energy is a key factor of biosynthesis, and is necessary for the reduction or assimilation of substrates such as CO 2. Previous microbial electrosynthesis (MES) research mainly utilized naturally electroactive microbes to generate non-specific products.

          Results

          In this research, an electroactive succinate-producing cell factory was engineered in E. coli T110(pMtrABC, pFccA-CymA) by expressing mtrABC, fccA and cymA from Shewanella oneidensis MR-1, which can utilize electricity to reduce fumarate. The electroactive T110 strain was further improved by incorporating a carbon concentration mechanism (CCM). This strain was fermented in an MES system with neutral red as the electron carrier and supplemented with HCO 3 +, which produced a succinate yield of 1.10 mol/mol glucose—a 1.6-fold improvement over the parent strain T110.

          Conclusions

          The strain T110(pMtrABC, pFccA-CymA, pBTCA) is to our best knowledge the first electroactive microbial cell factory engineered to directly utilize electricity for the production of a specific product. Due to the versatility of the E. coli platform, this pioneering research opens the possibility of engineering various other cell factories to utilize electricity for bioproduction.

          Electronic supplementary material

          The online version of this article (10.1186/s12934-019-1067-3) contains supplementary material, which is available to authorized users.

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          Most cited references44

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          Conversion of wastes into bioelectricity and chemicals by using microbial electrochemical technologies.

          Waste biomass is a cheap and relatively abundant source of electrons for microbes capable of producing electrical current outside the cell. Rapidly developing microbial electrochemical technologies, such as microbial fuel cells, are part of a diverse platform of future sustainable energy and chemical production technologies. We review the key advances that will enable the use of exoelectrogenic microorganisms to generate biofuels, hydrogen gas, methane, and other valuable inorganic and organic chemicals. Moreover, we examine the key challenges for implementing these systems and compare them to similar renewable energy technologies. Although commercial development is already underway in several different applications, ranging from wastewater treatment to industrial chemical production, further research is needed regarding efficiency, scalability, system lifetimes, and reliability.
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            j5 DNA assembly design automation software.

            Recent advances in Synthetic Biology have yielded standardized and automatable DNA assembly protocols that enable a broad range of biotechnological research and development. Unfortunately, the experimental design required for modern scar-less multipart DNA assembly methods is frequently laborious, time-consuming, and error-prone. Here, we report the development and deployment of a web-based software tool, j5, which automates the design of scar-less multipart DNA assembly protocols including SLIC, Gibson, CPEC, and Golden Gate. The key innovations of the j5 design process include cost optimization, leveraging DNA synthesis when cost-effective to do so, the enforcement of design specification rules, hierarchical assembly strategies to mitigate likely assembly errors, and the instruction of manual or automated construction of scar-less combinatorial DNA libraries. Using a GFP expression testbed, we demonstrate that j5 designs can be executed with the SLIC, Gibson, or CPEC assembly methods, used to build combinatorial libraries with the Golden Gate assembly method, and applied to the preparation of linear gene deletion cassettes for E. coli. The DNA assembly design algorithms reported here are generally applicable to broad classes of DNA construction methodologies and could be implemented to supplement other DNA assembly design tools. Taken together, these innovations save researchers time and effort, reduce the frequency of user design errors and off-target assembly products, decrease research costs, and enable scar-less multipart and combinatorial DNA construction at scales unfeasible without computer-aided design.
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              Towards Electrosynthesis in Shewanella: Energetics of Reversing the Mtr Pathway for Reductive Metabolism

              Bioelectrochemical systems rely on microorganisms to link complex oxidation/reduction reactions to electrodes. For example, in Shewanella oneidensis strain MR-1, an electron transfer conduit consisting of cytochromes and structural proteins, known as the Mtr respiratory pathway, catalyzes electron flow from cytoplasmic oxidative reactions to electrodes. Reversing this electron flow to drive microbial reductive metabolism offers a possible route for electrosynthesis of high value fuels and chemicals. We examined electron flow from electrodes into Shewanella to determine the feasibility of this process, the molecular components of reductive electron flow, and what driving forces were required. Addition of fumarate to a film of S. oneidensis adhering to a graphite electrode poised at −0.36 V versus standard hydrogen electrode (SHE) immediately led to electron uptake, while a mutant lacking the periplasmic fumarate reductase FccA was unable to utilize electrodes for fumarate reduction. Deletion of the gene encoding the outer membrane cytochrome-anchoring protein MtrB eliminated 88% of fumarate reduction. A mutant lacking the periplasmic cytochrome MtrA demonstrated more severe defects. Surprisingly, disruption of menC, which prevents menaquinone biosynthesis, eliminated 85% of electron flux. Deletion of the gene encoding the quinone-linked cytochrome CymA had a similar negative effect, which showed that electrons primarily flowed from outer membrane cytochromes into the quinone pool, and back to periplasmic FccA. Soluble redox mediators only partially restored electron transfer in mutants, suggesting that soluble shuttles could not replace periplasmic protein-protein interactions. This work demonstrates that the Mtr pathway can power reductive reactions, shows this conduit is functionally reversible, and provides new evidence for distinct CymA:MtrA and CymA:FccA respiratory units.
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                Author and article information

                Contributors
                wuzaiqiang99@163.com
                wang.junsong@gmail.com
                liu_jun@tib.cas.cn
                wyps2103@163.com
                bi_ch@tib.cas.cn
                zhang_xl@tib.cas.cn
                Journal
                Microb Cell Fact
                Microb. Cell Fact
                Microbial Cell Factories
                BioMed Central (London )
                1475-2859
                28 January 2019
                28 January 2019
                2019
                : 18
                : 15
                Affiliations
                [1 ]ISNI 0000 0000 9116 9901, GRID grid.410579.e, Center for Molecular Metabolism, School of Environmental and Biological Engineering, , Nanjing University of Science and Technology, ; Nanjing, 210094 China
                [2 ]ISNI 0000000119573309, GRID grid.9227.e, Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, , Chinese Academy of Sciences, ; 32 West 7th Ave, Tianjin Airport Economic Park, Tianjin, 300308 China
                Author information
                http://orcid.org/0000-0002-1940-8511
                Article
                1067
                10.1186/s12934-019-1067-3
                6348651
                30691454
                f5ab1535-edbf-42bc-9e60-90c78a33d736
                © The Author(s) 2019

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 23 May 2018
                : 20 January 2019
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 31522002
                Funded by: Natural Science Foundation of Tianjin Municipal Science and Technology Commission (CN)
                Award ID: 15JCYBJC49400
                Funded by: Tianjin Key Technology R&D program of Tianjin Municipal Science and Technology Commission
                Award ID: 11ZCZDSY08600
                Categories
                Research
                Custom metadata
                © The Author(s) 2019

                Biotechnology
                microbial electrosynthesis,bioelectrochemical systems,succinate,co2 fixation
                Biotechnology
                microbial electrosynthesis, bioelectrochemical systems, succinate, co2 fixation

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