Surface display of Acetobacter pasteurianus AdhA on Bacillus subtilis spores to enhance ethanol tolerance for liquor industrial potential

Bacillus subtilis have always been regarded as one of the most important functional bacteria for flavor formation in liquor industry. During the flavor formation process, B. subtilis facilitate the conversion of ethanol into aroma compounds by esterification and Maillard reactions mainly due to their vigorous enzymatic secretion and metabolic systems. However, high concentrations of ethanol could be a significant stressor to inhibit Bacillus spp. propagation and thus impair their effective flavor-forming ability. Therefore, in order to obtain improved B. subtilis strains with enhanced ethanol tolerance for liquor industrial potential, we adopted spore surface display strategy in the present study. Acetobacter pasteurianus alcohol dehydrogenase A (adhA) gene was employed as foreign gene, and recombinant integrative plasmid pJS700-adhA was constructed by using B. subtilis spore coat protein CotC as a fusion partner. A combination of amylase activity assay, site-directed PCR identification, Western blot analysis, and immunofluorescence microscopy sequentially demonstrated that AdhA was successfully expressed and displayed on recombinant B. subtilis mutant spore surface. In addition, ethanol tolerance assay showed that the recombinant mutant exhibited enhanced ethanol resistance than its wild-type form when confronted with high concentrations of ethanol, indicating both of its validity in methodology and application potential for liquor industry.