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Appl. Environ. Microbiol. doi:10.1128/AEM.00625-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Metabolic engineering of Bacillus subtilis for ethanol production: Lactate dehydrogenase plays a key role in the fermentative metabolism

Departamento de Ingeniería Celular y Biocatálisis, Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México. Cuernavaca, Mor., México

^* To whom correspondence should be addressed. Email: alfredo{at}ibt.unam.mx.

	Abstract

Wild type Bacillus subtilis ferments 20 g/L glucose in 48 h, producing lactate and butanediol, but not ethanol or acetate. To construct an ethanologenic B. subtilis strain, homologous recombination was used to disrupt the native lactate dehydrogenase gene (ldh) by chromosomal insertion of the Zymomonas mobilis pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) genes under the control of the ldh native promoter. Values of intracellular PDC and ADHII enzymatic activities of engineered B. subtilis BS35 strain were similar to those found in an ethanologenic Escherichia coli strain. BS35 produced ethanol and butanediol; however, cell growth and glucose consumption were reduced by 70 and 65%, respectively, in comparison to the progenitor strain. To eliminate butanediol production, the acetolactate synthase gene (alsS) was inactivated. In the BS36 (BS35 alsS ^-) strain, ethanol production was enhanced with high yield (89% of the theoretical); however, cell growth and glucose consumption remained low. Interestingly, kinetic characterization of LDH from B. subtilis showed that it is able to oxidize NADH and NADPH. Expression of the UDHA transhydrogenase from E. coli allowed a partial recovery of cell growth, and an early onset of ethanol production. Beyond pyruvate-to-lactate conversion and NADH oxidation, an additional key physiological role of LDH for glucose consumption under fermentative conditions is suggested. Long-term cultivation showed that 8.9 g/L of ethanol can be obtained using BS37 (BS35 alsS ^-udhA ⁺) strain. As far as we know, this is the highest ethanol titer and yield reported with a B. subtilis strain.