The Effects of Feed and Intracellular Pyruvate Levels on the Redistribution of Metabolic Fluxes in Escherichia coli

Yang, Yea-Tyng; Bennett, George N.; San, Ka‐Yiu

doi:10.1006/mben.2000.0166

Cited by 66 publications

(42 citation statements)

References 39 publications

(32 reference statements)

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“…The k cat and K m for acetyl-CoA of CimA3.7 improved about threefold over the wild-type levels. However, the K m for pyruvate of CimA3.7 increased over wild-type CimA, although this may not be crucial for the activity in vivo as the cellular concentration of pyruvate (7.5 mM) (20) is much higher than the K m for pyruvate (0.34 mM) of CimA3.7.…”

Section: Resultsmentioning

confidence: 87%

Directed Evolution of Methanococcus jannaschii Citramalate Synthase for Biosynthesis of 1-Propanol and 1-Butanol by Escherichia coli

Atsumi

Liao

2008

Appl Environ Microbiol

228

159

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Biofuels synthesized from renewable resources are of increasing interest because of global energy and environmental problems. We have previously demonstrated production of higher alcohols from Escherichia coli using a 2-keto acid-based pathway. Here, we took advantage of the growth phenotype associated with 2-keto acid deficiency to construct a hyperproducer of 1-propanol and 1-butanol by evolving citramalate synthase (CimA) from Methanococcus jannaschii. This new pathway, which directly converts pyruvate to 2-ketobutyrate, bypasses threonine biosynthesis and represents the shortest keto acid-mediated pathway for producing 1-propanol and 1-butanol from glucose. Directed evolution of CimA enhanced the specific activity over a wide temperature range (30 to 70°C). The best CimA variant was found to be insensitive to feedback inhibition by isoleucine in addition to the improved activity. This CimA variant enabled 9-and 22-fold higher production levels of 1-propanol and 1-butanol, respectively, compared to the strain expressing the wild-type CimA. This work demonstrates (i) the first production of 1-propanol and 1-butanol using the citramalate pathway and (ii) the benefit of the 2-keto acid pathway that enables a growth-based evolutionary strategy to improve the production of non-growth-related products.

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Section: Resultsmentioning

confidence: 87%

Directed Evolution of Methanococcus jannaschii Citramalate Synthase for Biosynthesis of 1-Propanol and 1-Butanol by Escherichia coli

Atsumi

Liao

2008

Appl Environ Microbiol

228

159

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“…However, the capacity of FocA to export the central metabolic intermediate pyruvate seems contradictory, in particular because the uptake of pyruvate is an active process (31,32). Under conditions of mixed-acid fermentation, extracellular pyruvate was detected when glucose was supplied (22), suggesting that intracellular levels of pyruvate may rise to a point where FocA again can serve as an overflow.…”

Section: Discussionmentioning

confidence: 99%

The formate channel FocA exports the products of mixed-acid fermentation

Lü

Schwarzer

et al. 2012

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

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Formate is a major metabolite in the anaerobic fermentation of glucose by many enterobacteria. It is translocated across cellular membranes by the pentameric ion channel/transporter FocA that, together with the nitrite channel NirC, forms the formate/nitrite transporter (FNT) family of membrane transport proteins. Here we have carried out an electrophysiological analysis of FocA from Salmonella typhimurium to characterize the channel properties and assess its specificity toward formate and other possible permeating ions. Single-channel currents for formate, hypophosphite and nitrite revealed two mechanistically distinct modes of gating that reflect different types of structural rearrangements in the transport channel of each FocA protomer. Moreover, FocA did not conduct cations or divalent anions, but the chloride anion was identified as further transported species, along with acetate, lactate and pyruvate. Formate, acetate and lactate are major end products of anaerobic mixed-acid fermentation, the pathway where FocA is predominantly required, so that this channel is ideally adapted to act as a multifunctional export protein to prevent their intracellular accumulation. Because of the high degree of conservation in the residues forming the transport channel among FNT family members, the flexibility in conducting multiple molecules is most likely a general feature of these proteins.electrophysiology | planar lipid bilayer | formate channel

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“…Each NADH molecule must be oxidized by reducing an electron acceptor, such as acetaldehyde, or by biosynthetic reactions. Addition of pyruvate to media has been shown to increase the intracellular pyruvate pool in E. coli (Yang et al 2001), which in turn increases the ratio of potential electron acceptors for oxidation of NADH from glycolysis. Acetyl-CoA, pyruvate and upstream metabolites in the Embden-Meyerhof-Parnas pathway are used for the biosynthesis of approx.…”

Section: Discussionmentioning

confidence: 99%

Ethanol synthesis from glycerol by Escherichia coli redox mutants expressing adhE from Leuconostoc mesenteroides

Nikel

Ramírez

Pettinari

et al. 2010

Journal of Applied Microbiology

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Aims: Analysis of the physiology and metabolism of Escherichia coli arcA and creC mutants expressing a bifunctional alcohol‐acetaldehyde dehydrogenase from Leuconostoc mesenteroides growing on glycerol under oxygen‐restricted conditions. The effect of an ldhA mutation and different growth medium modifications was also assessed. Methods and Results: Expression of adhE in E. coli CT1061 [arcA creC(Con)] resulted in a 1·4‐fold enhancement in ethanol synthesis. Significant amounts of lactate were produced during micro‐oxic cultures and strain CT1061LE, in which fermentative lactate dehydrogenase was deleted, produced up to 6·5 ± 0·3 g l−1 ethanol in 48 h. Escherichia coli CT1061LE derivatives resistant to >25 g l−1 ethanol were obtained by metabolic evolution. Pyruvate and acetaldehyde addition significantly increased both biomass and ethanol concentrations, probably by overcoming acetyl‐coenzyme A (CoA) shortage. Yeast extract also promoted growth and ethanol synthesis, and this positive effect was mainly attributable to its vitamin content. Two‐stage bioreactor cultures were conducted in a minimal medium containing 100 μg l−1 calcium d‐pantothenate to evaluate oxic acetyl‐CoA synthesis followed by a switch into fermentative conditions. Ethanol reached 15·4 ± 0·9 g l−1 with a volumetric productivity of 0·34 ± 0·02 g l−1 h−1. Conclusions: Escherichia coli responded to adhE over‐expression by funnelling carbon and reducing equivalents into a highly reduced metabolite, ethanol. Acetyl‐CoA played a key role in micro‐oxic ethanol synthesis and growth. Significance and Impact of the Study: Insight into the micro‐oxic metabolism of E. coli growing on glycerol is essential for the development of efficient industrial processes for reduced biochemicals production from this substrate, with special relevance to biofuels synthesis.

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The Effects of Feed and Intracellular Pyruvate Levels on the Redistribution of Metabolic Fluxes in Escherichia coli

Cited by 66 publications

References 39 publications

Directed Evolution of Methanococcus jannaschii Citramalate Synthase for Biosynthesis of 1-Propanol and 1-Butanol by Escherichia coli

Directed Evolution of Methanococcus jannaschii Citramalate Synthase for Biosynthesis of 1-Propanol and 1-Butanol by Escherichia coli

The formate channel FocA exports the products of mixed-acid fermentation

Ethanol synthesis from glycerol by Escherichia coli redox mutants expressing adhE from Leuconostoc mesenteroides

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