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Applied and Environmental Microbiology, August 2007, p. 5190-5198, Vol. 73, No. 16
0099-2240/07/$08.00+0     doi:10.1128/AEM.00625-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Metabolic Engineering of Bacillus subtilis for Ethanol Production: Lactate Dehydrogenase Plays a Key Role in Fermentative Metabolism

Susana Romero,¹ Enrique Merino,² Francisco Bolívar,¹ Guillermo Gosset,¹ and Alfredo Martinez^1*

Departamento de Ingeniería Celular y Biocatálisis,¹ Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México²

Received 19 March 2007/ Accepted 1 June 2007

Wild-type Bacillus subtilis ferments 20 g/liter 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 (LDH) gene (ldh) by chromosomal insertion of the Zymomonas mobilis pyruvate decarboxylase gene (pdc) and alcohol dehydrogenase II gene (adhB) under the control of the ldh native promoter. The values of the intracellular PDC and ADHII enzymatic activities of the engineered B. subtilis BS35 strain were similar to those found in an ethanologenic Escherichia coli strain. BS35 produced ethanol and butanediol; however, the cell growth and glucose consumption rates were reduced by 70 and 65%, respectively, in comparison to those in the progenitor strain. To eliminate butanediol production, the acetolactate synthase gene (alsS) was inactivated. In the BS36 strain (BS35 alsS), ethanol production was enhanced, with a high yield (89% of the theoretical); however, the cell growth and glucose consumption rates remained low. Interestingly, kinetic characterization of LDH from B. subtilis showed that it is able to oxidize NADH and NADPH. The expression of the transhydrogenase encoded by udhA from E. coli allowed a partial recovery of the cell growth rate 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/liter of ethanol can be obtained using strain BS37 (BS35 alsS udhA⁺). As far as we know, this is the highest ethanol titer and yield reported with a B. subtilis strain.

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* Corresponding author. Mailing address: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, A.P. 510-3 Cuernavaca, Mor. 62250, México. Phone: (52-777) 3291601. Fax: (52-777) 3172388. E-mail: alfredo{at}ibt.unam.mx

Published ahead of print on 22 June 2007.

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Applied and Environmental Microbiology, August 2007, p. 5190-5198, Vol. 73, No. 16
0099-2240/07/$08.00+0     doi:10.1128/AEM.00625-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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