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      Complete Biosynthesis of Anthocyanins Using E. coli Polycultures

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          ABSTRACT

          Fermentation-based chemical production strategies provide a feasible route for the rapid, safe, and sustainable production of a wide variety of important chemical products, ranging from fuels to pharmaceuticals. These strategies have yet to find wide industrial utilization due to their inability to economically compete with traditional extraction and chemical production methods. Here, we engineer for the first time the complex microbial biosynthesis of an anthocyanin plant natural product, starting from sugar. This was accomplished through the development of a synthetic, 4-strain Escherichia coli polyculture collectively expressing 15 exogenous or modified pathway enzymes from diverse plants and other microbes. This synthetic consortium-based approach enables the functional expression and connection of lengthy pathways while effectively managing the accompanying metabolic burden. The de novo production of specific anthocyanin molecules, such as calistephin, has been an elusive metabolic engineering target for over a decade. The utilization of our polyculture strategy affords milligram-per-liter production titers. This study also lays the groundwork for significant advances in strain and process design toward the development of cost-competitive biochemical production hosts through nontraditional methodologies.

          IMPORTANCE

          To efficiently express active extensive recombinant pathways with high flux in microbial hosts requires careful balance and allocation of metabolic resources such as ATP, reducing equivalents, and malonyl coenzyme A (malonyl-CoA), as well as various other pathway-dependent cofactors and precursors. To address this issue, we report the design, characterization, and implementation of the first synthetic 4-strain polyculture. Division of the overexpression of 15 enzymes and transcription factors over 4 independent strain modules allowed for the division of metabolic burden and for independent strain optimization for module-specific metabolite needs. This study represents the most complex synthetic consortia constructed to date for metabolic engineering applications and provides a new paradigm in metabolic engineering for the reconstitution of extensive metabolic pathways in nonnative hosts.

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          Most cited references 32

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          Genome-scale metabolic network modeling results in minimal interventions that cooperatively force carbon flux towards malonyl-CoA.

          Malonyl-coenzyme A is an important precursor metabolite for the biosynthesis of polyketides, flavonoids and biofuels. However, malonyl-CoA naturally synthesized in microorganisms is consumed for the production of fatty acids and phospholipids leaving only a small amount available for the production of other metabolic targets in recombinant biosynthesis. Here we present an integrated computational and experimental approach aimed at improving the intracellular availability of malonyl-CoA in Escherichia coli. We used a customized version of the recently developed OptForce methodology to predict a minimal set of genetic interventions that guarantee a prespecified yield of malonyl-CoA in E. coli strain BL21 Star™. In order to validate the model predictions, we have successfully constructed an E. coli recombinant strain that exhibits a 4-fold increase in the levels of intracellular malonyl-CoA compared to the wild type strain. Furthermore, we demonstrate the potential of this E. coli strain for the production of plant-specific secondary metabolites naringenin (474mg/L) with the highest yield ever achieved in a lab-scale fermentation process. Combined effect of the genetic interventions was found to be synergistic based on a developed analysis method that correlates genetic modification to cell phenotype, specifically the identified knockout targets (ΔfumC and ΔsucC) and overexpression targets (ACC, PGK, GAPD and PDH) can cooperatively force carbon flux towards malonyl-CoA. The presented strategy can also be readily expanded for the production of other malonyl-CoA-derived compounds like polyketides and biofuels. Copyright © 2011 Elsevier Inc. All rights reserved.
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            Optimization of a heterologous pathway for the production of flavonoids from glucose.

            The development of efficient microbial processes for the production of flavonoids has been a metabolic engineering goal for the past several years, primarily due to the purported health-promoting effects of these compounds. Although significant strides have been made recently in improving strain titers and yields, current fermentation strategies suffer from two major drawbacks-(1) the requirement for expensive phenylpropanoic precursors supplemented into the media and (2) the need for two separate media formulations for biomass/protein generation and flavonoid production. In this study, we detail the construction of a series of strains capable of bypassing both of these problems. A four-step heterologous pathway consisting of the enzymes tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI) was assembled within two engineered l-tyrosine Escherichia coli overproducers in order to enable the production of the main flavonoid precursor naringenin directly from glucose. During the course of this investigation, we discovered that extensive optimization of both enzyme sources and relative gene expression levels was required to achieve high quantities of both p-coumaric acid and naringenin accumulation. Once this metabolic balance was achieved, however, such strains were found to be capable of producing 29 mg/l naringenin from glucose and up to 84 mg/l naringenin with the addition of the fatty acid enzyme inhibitor, cerulenin. These results were obtained through cultivation of E. coli in a single minimal medium formulation without additional precursor supplementation, thus paving the way for the development of a simple and economical process for the microbial production of flavonoids directly from glucose. Copyright © 2011 Elsevier Inc. All rights reserved.
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              Engineering microbial consortia for controllable outputs

              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 robust 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 frameworks, systems-level knowledge can markedly improve our ability to predict the fate and functioning of consortia. Here 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.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                mBio
                MBio
                mbio
                mbio
                mBio
                mBio
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                2150-7511
                6 June 2017
                May-Jun 2017
                : 8
                : 3
                Affiliations
                [a ]Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, USA
                [b ]Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York, USA
                [c ]Department of Chemistry, Rensselaer Polytechnic Institute, Troy, New York, USA
                [d ]Department of Chemistry, Hamilton College, Clinton, New York, USA
                [e ]State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, China
                [f ]Beijing Key Laboratory of Bioactive Substances and Functional Food, Beijing Union University, Beijing, China
                Korea Advanced Institute of Science and Technology
                Author notes
                Address correspondence to Mattheos A. G. Koffas, koffam@ 123456rpi.edu .
                Article
                mBio00621-17
                10.1128/mBio.00621-17
                5461408
                Copyright © 2017 Jones et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

                Page count
                supplementary-material: 4, Figures: 5, Tables: 1, Equations: 0, References: 40, Pages: 9, Words: 5277
                Product
                Funding
                Funded by: National Science Foundation (NSF) https://doi.org/10.13039/100000001
                Award ID: 1549767
                Award Recipient : John Andrew Jones Award Recipient : Victoria Vernacchio Award Recipient : Shannon Collins Award Recipient : Yu Xiu Award Recipient : Jacob Englaender Award Recipient : Brady Fletcher Cress Award Recipient : Mattheos A. Koffas
                Categories
                Research Article
                Custom metadata
                May/June 2017

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