Effectiveness of the Bacillus subtilis genome-reduced strain as an ethanol production host.

We investigated the performance of a genome-reduced strain of Bacillus subtilis MGB874, whose 0.87 Mbp of genomic DNA was cumulatively deleted, as an ethanol production host. A recombinant strain A267_EtOH was constructed by introducing the pdc and adhB genes from Zymomonas mobilis, both of which were expressed from an isopropyl-β-d-1-thiogalactopyranoside-inducible spac promoter, into the A267 strain, a tryptophan prototrophic derivative of the MGB874 with disruption of metabolic pathways for producing lactic acid, acetic acid, and acetoin. Focusing on the stationary phase in fed-batch fermentation, 1.6 g L-1 ethanol was produced by the A267_EtOH strain after 144 h. Moreover, its ethanol production further increased by approximately 3.7-fold (5.9 g L-1) at 80 h through replacing the spac promoter for expressing pdc and adhB genes with the lytR promoter and the yield was about 112%. These results indicate that the MGB874 is an effective host for ethanol production during the stationary phase.