Metabolic flux analysis ofEscherichia coli expressing theBacillus subtilis acetolactate synthase in batch and continuous cultures

Aristidou, Aristos; San, Ka‐Yiu; Bennett, George N.

doi:10.1002/(sici)1097-0290(19990620)63:6<737::aid-bit12>3.0.co;2-9

Cited by 32 publications

(21 citation statements)

References 43 publications

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“…Metabolic flux analysis based on stoichiometric constraints constitutes a straightforward way to visualize the operativity of the entire metabolic network in anaerobic E. coli cells (4,56,67). Under these growth conditions, the carbon source is mostly converted into fermentation products and, to a lesser extent, into biomass (13,38).…”

Section: Resultsmentioning

confidence: 99%

Manipulation of the Anoxic Metabolism in Escherichia coli by ArcB Deletion Variants in the ArcBA Two-Component System

Bidart

Ruiz

Almeida

et al. 2012

Appl Environ Microbiol

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Bioprocesses conducted under conditions with restricted O 2 supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative anaerobe Escherichia coli has elaborate sensing and signal transduction mechanisms for redox control in response to the availability of O 2 and other electron acceptors. The ArcBA two-component system consists of ArcB, a membrane-associated sensor kinase, and ArcA, the cognate response regulator. The tripartite hybrid kinase ArcB possesses a transmembrane, a PAS, a primary transmitter (H1), a receiver (D1), and a phosphotransfer (H2) domain. Metabolic fluxes were compared under anoxic conditions in a wild-type E. coli strain, its ⌬arcB derivative, and two partial arcB deletion mutants in which ArcB lacked either the H1 domain or the PAS-H1-D1 domains. These analyses revealed that elimination of different segments in ArcB determines a distinctive distribution of D-glucose catabolic fluxes, different from that observed in the ⌬arcB background. Metabolite profiles, enzyme activity levels, and gene expression patterns were also investigated in these strains. Relevant alterations were observed at the P-enol-pyruvate/pyruvate and acetyl coenzyme A metabolic nodes, and the formation of reduced fermentation metabolites, such as succinate, D-lactate, and ethanol, was favored in the mutant strains to different extents compared to the wild-type strain. These phenotypic traits were associated with altered levels of the enzymatic activities operating at these nodes, as well as with elevated NADH/NAD ؉ ratios. Thus, targeted modification of global regulators to obtain different metabolic flux distributions under anoxic conditions is emerging as an attractive tool for metabolic engineering purposes.A noxic fermentation of different carbon sources by Escherichia coli is increasingly gaining momentum in biotechnological setups designed to obtain reduced biochemicals. Relevant examples in this sense include (but are certainly not limited to) the production of ethanol (31, 58, 69), succinate (64), D-lactate (45), and polyhydroxyalkanoates (24, 40), often by using redox and/or regulatory E. coli mutants as the biocatalyst. These metabolic engineering approaches underscore the need for a complete understanding of the cell physiology and metabolic network operativity under anoxic growth conditions. In fact, the relative lack of knowledge on the cellular wiring of these regulatory networks under conditions relevant to both laboratory and industrial applications represents a hurdle that has to be overcome for the efficient design of industrial processes. Metabolic fluxes through the central carbon pathways constitute the backbone of cell metabolism and represent the in vivo reaction rates of cognate enzymatic steps (62). The observed fluxome is the phenotypic consequence of both gene transcription and translation, as well as of the enzymatic activity and the regulation exerted at the metabolite level (48). Fluxome analysis is thus a useful approach to...

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Section: Resultsmentioning

confidence: 99%

Manipulation of the Anoxic Metabolism in Escherichia coli by ArcB Deletion Variants in the ArcBA Two-Component System

Bidart

Ruiz

Almeida

et al. 2012

Appl Environ Microbiol

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“…This is because the production of acetate is coupled to ATP production through the action of acetate kinase and provides a major portion of the ATP produced during anaerobic growth (41). E. coli expressing ␣-ALS from B. subtilis in batch cultures synthesized less acetate, and, as a result, had a lower ATP yield, than the corresponding wild-type strain (42).…”

Section: Discussionmentioning

confidence: 99%

Acetoin Synthesis Acquisition Favors Escherichia coli Growth at Low pH

Vivijs

Moons

Aertsen

et al. 2014

Appl Environ Microbiol

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Some members of the family Enterobacteriaceae ferment sugars via the mixed-acid fermentation pathway. This yields large amounts of acids, causing strong and sometimes even lethal acidification of the environment. Other family members employ the 2,3-butanediol fermentation pathway, which generates comparatively less acidic and more neutral end products, such as acetoin and 2,3-butanediol. In this work, we equipped Escherichia coli MG1655 with the budAB operon, encoding the acetoin pathway, from Serratia plymuthica RVH1 and investigated how this affected the ability of E. coli to cope with acid stress during growth. Acetoin fermentation prevented lethal medium acidification by E. coli in lysogeny broth (LB) supplemented with glucose. It also supported growth and higher stationary-phase cell densities in acidified LB broth with glucose (pH 4.10 to 4.50) and in tomato juice (pH 4.40 to 5.00) and reduced the minimal pH at which growth could be initiated. On the other hand, the acetoin-producing strain was outcompeted by the nonproducer in a mixed-culture experiment at low pH, suggesting a fitness cost associated with acetoin production. Finally, we showed that acetoin production profoundly changes the appearance of E. coli on several diagnostic culture media. Natural E. coli strains that have laterally acquired budAB genes may therefore have escaped detection thus far. This study demonstrates the potential importance of acetoin fermentation in the ecology of E. coli in the food chain and contributes to a better understanding of the microbiological stability and safety of acidic foods.

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“…By-products of E. coli anaerobic fermentation, e.g. acetate, succinate, formate, lactate and ethanol (Aristidou et at., 1999) influence culture environments and lead to an undesired decrease of pH, which negatively affects both biomass concentration and cell morphology. Accurate pH control at specific set points does not directly address the issue of fermentative metabolism, but is necessary for exploring optimum culture conditions with higher yield for biomass or products.…”

Section: Discussionmentioning

confidence: 99%

Microbioreactors for Bioprocess Development

Zhang

Perozziello

Boccazzi

et al. 2007

JALA: Journal of the Association for Laboratory Automation

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As a step toward high-throughput bioprocess development, we present design, fabrication, and characterization of polymer based microbioreactors integrated with automated sensors and actuators. The devices are realized, in increasing levels of complexity, in poly(dimethylsiloxane) and poly(methyl methacrylate) by micromachining and multilayer thermal compression bonding procedures. Online optical measurements for optical density, pH, and dissolved oxygen are integrated. Active mixing is made possible by a miniature magnetic stir bar. Plug-in-and-flow microfluidic connectors and fabricated polymer micro-optical lenses/connectors are integrated in the microbioreactors for fast set up and easy operation. Application examples demonstrate the feasibility of culturing microbial cells, specifically Escherichia coli, in 150 μL-volume bioreactors in batch, continuous, and fed-batch operations. (JALA 2007;12:143–51)

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Metabolic flux analysis ofEscherichia coli expressing theBacillus subtilis acetolactate synthase in batch and continuous cultures

Cited by 32 publications

References 43 publications

Manipulation of the Anoxic Metabolism in Escherichia coli by ArcB Deletion Variants in the ArcBA Two-Component System

Manipulation of the Anoxic Metabolism in Escherichia coli by ArcB Deletion Variants in the ArcBA Two-Component System

Acetoin Synthesis Acquisition Favors Escherichia coli Growth at Low pH

Microbioreactors for Bioprocess Development

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