Simultaneous utilization of glucose, xylose and arabinose in the presence of acetate by a consortium of Escherichia coli strains

Xia, Tian; Eiteman, Mark A.; Altman, Elliot

doi:10.1186/1475-2859-11-77

Cited by 73 publications

(57 citation statements)

References 35 publications

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“…However, previous research on microbial consortia was primarily concerned with the study of mixed population stability and dynamic interactions (7)(8)(9)(10)(11), although a few recent studies have reported the engineering of microbial consortia for utilization of simple sugars to make small molecules of central carbon metabolism, such as ethanol and lactate (12,13). Progress was made recently, when a full n-butanol pathway was expressed in two separate E. coli cells to achieve higher production (14) and when a bacterium-yeast coculture was used to address the difficulties of functional reconstitution of a pathway involving prokaryotic and eukaryotic enzymes in a consortium, and thus improved production of complex pharmaceutical molecules (15).…”

Section: -Hydroxybenzoic Acidmentioning

confidence: 99%

Engineering Escherichia coli coculture systems for the production of biochemical products

Zhang

Pereira

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

263

199

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Engineering microbial consortia to express complex biosynthetic pathways efficiently for the production of valuable compounds is a promising approach for metabolic engineering and synthetic biology. Here, we report the design, optimization, and scale-up of an Escherichia coli-E. coli coculture that successfully overcomes fundamental microbial production limitations, such as high-level intermediate secretion and low-efficiency sugar mixture utilization. For the production of the important chemical cis,cis-muconic acid, we show that the coculture approach achieves a production yield of 0.35 g/g from a glucose/xylose mixture, which is significantly higher than reported in previous reports. By efficiently producing another compound, 4-hydroxybenzoic acid, we also demonstrate that the approach is generally applicable for biosynthesis of other important industrial products.metabolic engineering | microbial coculture | muconic acid | 4-hydroxybenzoic acidM etabolic engineering and synthetic biology have made great strides in constructing and optimizing metabolic pathways for biochemical product synthesis in a pure culture (1, 2). There are, however, situations where this approach may be limited, as in the cases where (i) a single host cell cannot provide an optimal environment for the functioning of all pathway enzymes, (ii) biosynthetic efficiency is reduced due to overwhelming metabolic stress from the overexpression of long and complex pathways (3, 4), or (iii) pathway intermediates are secreted yielding undesired byproducts and reducing substrate utilization (5, 6). The above limitations can potentially be overcome through the use of rationally designed microbial coculture systems. Different from previous modular engineering approaches, such coculturebased systems completely modularize and segregate a biosynthetic pathway into two separate cells, each of which carries a portion of the pathway, and thus can be engineered independently to achieve optimal functioning of the combined pathway.The concept of microbial cocultures is not new. However, previous research on microbial consortia was primarily concerned with the study of mixed population stability and dynamic interactions (7-11), although a few recent studies have reported the engineering of microbial consortia for utilization of simple sugars to make small molecules of central carbon metabolism, such as ethanol and lactate (12, 13). Progress was made recently, when a full n-butanol pathway was expressed in two separate E. coli cells to achieve higher production (14) and when a bacterium-yeast coculture was used to address the difficulties of functional reconstitution of a pathway involving prokaryotic and eukaryotic enzymes in a consortium, and thus improved production of complex pharmaceutical molecules (15). Here, we expand the generality of the coculture engineering by demonstrating that microbial cocultures can also be engineered to overcome more universal challenges in metabolic engineering, including high-level intermediate secretion and low-efficiency s...

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Section: -Hydroxybenzoic Acidmentioning

confidence: 99%

Engineering Escherichia coli coculture systems for the production of biochemical products

Zhang

Pereira

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

263

199

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“…We have previously studied D-xylose and L-arabinose metabolism in E. coli that lacks the ability to metabolize D-glucose due to knockouts in the ptsG, manZ, and glk genes (1)(2)(3). Recently, small but consistent amounts (about 50 mg/liter) of D-glucose were observed as the accumulated end product when E. coli ptsG manZ glk was grown on 5 g/liter of either pentose in a defined medium (unpublished data).…”

mentioning

confidence: 99%

Accumulation of d -Glucose from Pentoses by Metabolically Engineered Escherichia coli

Xia

Han

Costanzo

et al. 2015

Appl Environ Microbiol

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T he pentose phosphate (PP) pathway interconverts phosphosugars having 3 to 7 carbon atoms principally by the action of the reversible enzymes transketolase and transaldolase. During the consumption of hexoses such as D-glucose or D-fructose, entry of carbon into this pathway provides many microorganisms, including Escherichia coli, the means to generate the reduced cofactor NADPH and to synthesize specific building-block compounds derived from intermediates of this pathway (e.g., phenylalanine, histidine, and ribose). For microorganisms having the requisite kinases and sugar transport mechanisms, the PP pathway also provides convenient entry points for the catabolism of many other sugars, including D-xylose and L-arabinose (49).We have previously studied D-xylose and L-arabinose metabolism in E. coli that lacks the ability to metabolize D-glucose due to knockouts in the ptsG, manZ, and glk genes (1-3). Recently, small but consistent amounts (about 50 mg/liter) of D-glucose were observed as the accumulated end product when E. coli ptsG manZ glk was grown on 5 g/liter of either pentose in a defined medium (unpublished data). How might D-glucose be derived from these pentoses?Both D-xylose and L-arabinose are converted to the common intermediate D-xylulose-5-phosphate (D-xylulose-5P) (Fig. 1), which via the PP pathway partitions to 67% D-fructose-6P and 33% D-glyceraldehyde-3P without the involvement of ATP:(1) During growth of cells having a complete glycolytic pathway, the 2 mol of D-fructose-6P formed via equation 1 readily generates 4 mol of D-glyceraldehyde-3P. For D-glucose to accumulate from pentoses in cells prevented from metabolizing D-glucose, we reasoned that some D-fructose-6P generated from these pentoses (i.e., by equation 1) is converted "back" to D-glucose and that once formed, the D-glucose is unable to reenter metabolism in the triple knockout strain. We furthermore hypothesized that even more D-glucose would accumulate from pentoses in cells that were further constrained from metabolizing D-fructose-6P or D-glucose-6P.Because D-fructose-6P conversion to D-glyceraldehyde-3P is ubiquitous in wild-type organisms, D-glucose is not typically considered a product of D-xylose or L-arabinose metabolism, and the conversion of these pentoses to readily available D-glucose would in itself not seem to be an economically viable process. However, if the yields and rates were sufficiently large, the accumulation of hexoses directly from pentoses might advance the use of lignocellulosic hydrolysates with organisms, such as Saccharomyces cerevisiae, which metabolize D-glucose readily but are natively unable to consume pentoses. Moreover, conversion of 5-carbon saccharides into 6-carbon saccharides derived from D-fructose-6P offers a unique platform both to build carbon length and potentially to generate compounds in industrially relevant organisms such as E. coli that might not be possible under typical conditions in which products of D-fructose-6P do not accumulate.The objectives of this study were to examine ...

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“…The propensity for specialist subpopulation development can be deduced from the modeling-quantified substrate distances, and ALE studies can be designed accordingly, depending on the desired outcome. If a single dominant genotype were desired, such as when optimizing a genetically engineered strain (39), an environment favoring generalist development could be selected; if overall culture performance were instead the important factor, then an environment favoring specialists would not need to be avoided, allowing naturally evolved specialists to substitute for artificially engineered microbial consortia that would have the same collective phenotype (40,41).…”

Section: Discussionmentioning

confidence: 99%

Laboratory Evolution to Alternating Substrate Environments Yields Distinct Phenotypic and Genetic Adaptive Strategies

Sandberg

Lloyd

Palsson

et al. 2017

Appl Environ Microbiol

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Adaptive laboratory evolution (ALE) experiments are often designed to maintain a static culturing environment to minimize confounding variables that could influence the adaptive process, but dynamic nutrient conditions occur frequently in natural and bioprocessing settings. To study the nature of carbon substrate fitness tradeoffs, we evolved batch cultures of Escherichia coli via serial propagation into tubes alternating between glucose and either xylose, glycerol, or acetate. Genome sequencing of evolved cultures revealed several genetic changes preferentially selected for under dynamic conditions and different adaptation strategies depending on the substrates being switched between; in some environments, a persistent "generalist" strain developed, while in another, two "specialist" subpopulations arose that alternated dominance. Diauxic lag phenotype varied across the generalists and specialists, in one case being completely abolished, while gene expression data distinguished the transcriptional strategies implemented by strains in pursuit of growth optimality. Genome-scale metabolic modeling techniques were then used to help explain the inherent substrate differences giving rise to the observed distinct adaptive strategies. This study gives insight into the population dynamics of adaptation in an alternating environment and into the underlying metabolic and genetic mechanisms. Furthermore, ALE-generated optimized strains have phenotypes with potential industrial bioprocessing applications.IMPORTANCE Evolution and natural selection inexorably lead to an organism's improved fitness in a given environment, whether in a laboratory or natural setting. However, despite the frequent natural occurrence of complex and dynamic growth environments, laboratory evolution experiments typically maintain simple, static culturing environments so as to reduce selection pressure complexity. In this study, we investigated the adaptive strategies underlying evolution to fluctuating environments by evolving Escherichia coli to conditions of frequently switching growth substrate. Characterization of evolved strains via a number of different data types revealed the various genetic and phenotypic changes implemented in pursuit of growth optimality and how these differed across the different growth substrates and switching protocols. This work not only helps to establish general principles of adaptation to complex environments but also suggests strategies for experimental design to achieve desired evolutionary outcomes.KEYWORDS adaptive laboratory evolution, Escherichia coli, adaptive mutations, phenotypic variation I n heterotrophs such as Escherichia coli, catabolism of carbon substrates is the driving force behind the energy generation and chemical synthesis necessary for homeostasis and anabolism (1). Although glucose is the most readily metabolized carbohydrate

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Simultaneous utilization of glucose, xylose and arabinose in the presence of acetate by a consortium of Escherichia coli strains

Cited by 73 publications

References 35 publications

Engineering Escherichia coli coculture systems for the production of biochemical products

Engineering Escherichia coli coculture systems for the production of biochemical products

Accumulation of d -Glucose from Pentoses by Metabolically Engineered Escherichia coli

Laboratory Evolution to Alternating Substrate Environments Yields Distinct Phenotypic and Genetic Adaptive Strategies

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