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


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 microg 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.


CONCLUSIONS
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.
 Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, Buenos Aires, Argentina.| Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina.
P I
PI
Nikel
 Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, Buenos Aires, Argentina.
M C
MC
Ramirez
 Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina.
M J
MJ
Pettinari
 Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina.
B S
BS
Méndez
 Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, Buenos Aires, Argentina.| Departamento de Ingeniería Química, Facultad de Ingeniería, Universidad de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina.
M A
MA
Galvagno
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism*; D007934:Leuconostoc / Q000201:enzymology*; D009154:Mutation; D010084:Oxidation-Reduction
D000431:Ethanol; D000105:Acetyl Coenzyme A; D000426:Alcohol Dehydrogenase; D000445:Aldehyde Oxidoreductases; C018699:aldehyde dehydrogenase (NAD(P)+); D005990:Glycerol