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

Abstract

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 delta 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 delta alsS udhA+). As far as we know, this is the highest ethanol titer and yield reported with a B. subtilis strain.

FIG. 1.

Bacillus subtilis fermentation pathways. The heterologous pathway used in this study is highlighted with a dashed-line ellipse. PDC and ADHII are from Z. mobilis. ALDC, acetolactate decarboxylase; AR, acetoin reductase; PDH, pyruvate dehydrogenase; CoA, coenzyme A; ALDH, acetaldehyde dehydrogenase; PTA, phosphotransacetylase; ACK, acetate kinase. (Modified from references and with permission.)

FIG. 2.

Vectors constructed to inactivate (A) the B. subtilis ldh gene (ldh_Bs) by chromosomal integration of the Z. mobilis pdc gene (pdc_Zm) and adhB_Zm, (B) alsS_Bs by chromosomal integration of the Spt cassette, and (C) alsS_Bs by chromosomal integration of E. coli udhA and the Spt cassette.

FIG. 3.

Characterization of B. subtilis strains under fermentative conditions in LB medium containing glucose (20 g/liter). Cell mass formation (A), glucose consumption (B), lactate (C), butanediol (D), and ethanol (E) production of B. subtilis strains CH1 (□), BS35 (▵), BS36 (○), and BS37 (⋄). B. subtilis CH1 (▪) was included as a control, using LB medium without glucose. The error bars represent the variations of duplicated experiments.

FIG. 4.

Characterization of B. subtilis BS37 in LB supplemented with glucose (Glc; 20 g/liter) and pyruvate (Pyr; 2 g/liter) under fermentative conditions. Cell mass (□), consumed glucose (○), consumed pyruvate (▿), and ethanol (EtOH) production (⋄). The error bars represent the variations of duplicated experiments.

FIG. 5.

Long-term fermentation of B. subtilis BS37 in LB supplemented with glucose (20 g/liter). Cell mass (□), glucose consumption (○), and ethanol production (⋄). The error bars represent the variations of duplicated experiments.