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      Reorganization of a synthetic microbial consortium for one-step vitamin C fermentation

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          Abstract

          Background

          In the industry, the conventional two-step fermentation method was used to produce 2-keto- l-gulonic acid (2-KGA), the precursor of vitamin C, by three strains, namely, Gluconobacter oxydans, Bacillus spp. and Ketogulonicigenium vulgare. Despite its high production efficiency, the long incubation period and an additional second sterilization process inhibit the further development. Therefore, we aimed to reorganize a synthetic consortium of G. oxydans and K. vulgare for one-step fermentation of 2-KGA and enhance the symbiotic interaction between microorganisms to perform better.

          Results

          During the fermentation, competition for sorbose of G. oxydans arose when co-cultured with K. vulgare. In this study, the competition between the two microbes was alleviated and their mutualism was enhanced by deleting genes involved in sorbose metabolism of G. oxydans. In the engineered synthetic consortium (H 6 + Kv), the yield of 2-KGA (mol/mol) against d-sorbitol reached 89.7 % within 36 h, increased by 29.6 %. Furthermore, metabolomic analysis was used to verify the enhancement of the symbiotic relationship and to provide us potential strategies for improving the synthetic consortium. Additionally, a significant redistribution of metabolism occurred by co-culturing the K. vulgare with the engineered G. oxydans, mainly reflected in the increased TCA cycle, purine, and fatty acid metabolism.

          Conclusions

          We reorganized and optimized a synthetic consortium of G. oxydans and K. vulgare to produce 2-KGA directly from d-sorbitol. The yield of 2-KGA was comparable to that of the conventional two-step fermentation. The metabolic interaction between the strains was further investigated by metabolomics, which verified the enhancement of the mutualism between the microbes and gave us a better understanding of the synthetic consortium.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12934-016-0418-6) contains supplementary material, which is available to authorized users.

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          Most cited references21

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          Natural strategies for the spatial optimization of metabolism in synthetic biology.

          Metabolism is a highly interconnected web of chemical reactions that power life. Though the stoichiometry of metabolism is well understood, the multidimensional aspects of metabolic regulation in time and space remain difficult to define, model and engineer. Complex metabolic conversions can be performed by multiple species working cooperatively and exchanging metabolites via structured networks of organisms and resources. Within cells, metabolism is spatially regulated via sequestration in subcellular compartments and through the assembly of multienzyme complexes. Metabolic engineering and synthetic biology have had success in engineering metabolism in the first and second dimensions, designing linear metabolic pathways and channeling metabolic flux. More recently, engineering of the third dimension has improved output of engineered pathways through isolation and organization of multicell and multienzyme complexes. This review highlights natural and synthetic examples of three-dimensional metabolism both inter- and intracellularly, offering tools and perspectives for biological design.
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            Microbial metabolic exchange--the chemotype-to-phenotype link.

            The function of microbial interactions is to enable microorganisms to survive by establishing a homeostasis between microbial neighbors and local environments. A microorganism can respond to environmental stimuli using metabolic exchange-the transfer of molecular factors, including small molecules and proteins. Microbial interactions not only influence the survival of the microbes but also have roles in morphological and developmental processes of the organisms themselves and their neighbors. This, in turn, shapes the entire habitat of these organisms. Here we highlight our current understanding of metabolic exchange as well as the emergence of new technologies that are allowing us to eavesdrop on microbial conversations comprising dozens to hundreds of secreted metabolites that control the behavior, survival and differentiation of members of the community. The goal of the rapidly advancing field studying multifactorial metabolic exchange is to devise a microbial 'Rosetta stone' in order to understand the language by which microbial interactions are negotiated and, ultimately, to control the outcome of these conversations.
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              Ethanol tolerance in the yeast Saccharomyces cerevisiae is dependent on cellular oleic acid content.

              In this investigation, we examined the effects of different unsaturated fatty acid compositions of Saccharomyces cerevisiae on the growth-inhibiting effects of ethanol. The unsaturated fatty acid (UFA) composition of S. cerevisiae is relatively simple, consisting almost exclusively of the mono-UFAs palmitoleic acid (Delta(9)Z-C(16:1)) and oleic acid (Delta(9)Z-C(18:1)), with the former predominating. Both UFAs are formed in S. cerevisiae by the oxygen- and NADH-dependent desaturation of palmitic acid (C(16:0)) and stearic acid (C(18:0)), respectively, catalyzed by a single integral membrane desaturase encoded by the OLE1 gene. We systematically altered the UFA composition of yeast cells in a uniform genetic background (i) by genetic complementation of a desaturase-deficient ole1 knockout strain with cDNA expression constructs encoding insect desaturases with distinct regioselectivities (i.e., Delta(9) and Delta(11)) and substrate chain-length preferences (i.e., C(16:0) and C(18:0)); and, (ii) by supplementation of the same strain with synthetic mono-UFAs. Both experimental approaches demonstrated that oleic acid is the most efficacious UFA in overcoming the toxic effects of ethanol in growing yeast cells. Furthermore, the only other UFA tested that conferred a nominal degree of ethanol tolerance is cis-vaccenic acid (Delta(11)Z-C(18:1)), whereas neither Delta(11)Z-C(16:1) nor palmitoleic acid (Delta(9)Z-C(16:1)) conferred any ethanol tolerance. We also showed that the most ethanol-tolerant transformant, which expresses the insect desaturase TniNPVE, produces twice as much oleic acid as palmitoleic acid in the absence of ethanol and undergoes a fourfold increase in the ratio of oleic acid to palmitoleic acid in response to exposure to 5% ethanol. These findings are consistent with the hypothesis that ethanol tolerance in yeast results from incorporation of oleic acid into lipid membranes, effecting a compensatory decrease in membrane fluidity that counteracts the fluidizing effects of ethanol.
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                Author and article information

                Contributors
                enxuwang@tju.edu.cn
                mzding@tju.edu.cn
                qianma1987@tju.edu.cn
                dongxiutao@126.com
                yjyuan@MIT.EDU
                Journal
                Microb Cell Fact
                Microb. Cell Fact
                Microbial Cell Factories
                BioMed Central (London )
                1475-2859
                25 January 2016
                25 January 2016
                2016
                : 15
                : 21
                Affiliations
                [ ]Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072 People’s Republic of China
                [ ]SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300072 People’s Republic of China
                Article
                418
                10.1186/s12934-016-0418-6
                4727326
                26809519
                8ed2254a-4123-4e8e-b008-6286691acc04
                © Wang et al. 2016

                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
                : 19 November 2015
                : 11 January 2016
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100002855, Ministry of Science and Technology of the People's Republic of China (CN);
                Award ID: “973” Program: 2014CB745100
                Award ID: “863” Program: 2012AA02A701
                Award Recipient :
                Funded by: The National Natural Science Foundation of China
                Award ID: Major Program: 21390203
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2016

                Biotechnology
                synthetic microbial consortium,reorganization,one-step fermentation,interaction,metabolomics

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