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

• Published in: Journal of applied microbiology Vol. 109; no. 2; pp. 492 - 504
• Main Authors: Nikel, P.I, Ramirez, M.C, Pettinari, M.J, Méndez, B.S, Galvagno, M.A
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01-08-2010; Blackwell Publishing Ltd; Blackwell; Oxford University Press
• Subjects: Acetaldehyde; Acetaldehyde dehydrogenase; Acetyl Coenzyme A - metabolism; acetyl-CoA availability; Alcohol Dehydrogenase - genetics; Alcohol Dehydrogenase - metabolism; alcohol-acetaldehyde dehydrogenase; Aldehyde Oxidoreductases - genetics; Aldehyde Oxidoreductases - metabolism; Bacteria; Biofuels; Biological and medical sciences; Bioreactors; Calcium; Coenzyme A; Dehydrogenase; Dehydrogenases; E coli; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Ethanol; Ethanol - metabolism; Fundamental and applied biological sciences. Psychology; Glycerol; Glycerol - metabolism; heterologous gene expression; L-Lactate dehydrogenase; Lactate dehydrogenase; Lactic acid; Leuconostoc - enzymology; Leuconostoc mesenteroides; Metabolism; Metabolites; microaerobiosis; microbial physiology; Microbiology; Mutants; Mutation; Oxidation-Reduction; Pyruvic acid; Substrates; Synthesis; Yeasts; Lactic acid bacteria; microaerobiosis; Applied microbiology; Escherichia coli; heterologous gene expression; Alcohol dehydrogenase; Acetaldehyde; microbial physiology; Bacteria; Micrococcales; Physiology; acetyl-CoA availability; Enterobacteriaceae; Availability; Leuconostoc mesenteroides; Ethanol; Enzyme; Glycerol; Gene expression; Metabolism; alcohol-acetaldehyde dehydrogenase; Streptococcaceae; Oxidoreductases; Microorganism; Mutation; glycerol metabolism
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/j.1365-2672.2010.04668.x

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. 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. 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. 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.