Development of Pdc-Adh bifunctional enzyme fusion for ethanol production in Bacillus subtilis

Bioethanol is one of the most important biofuels according to its combustion characteristics. Bacillus subtilis, Gram-positive bacterium with potent enzyme secretion system and widespread application in production of industrial chemicals, seems to be a good candidate for bioethanol production. A new approach was taken in this research to construct an ethanologenic B. subtilis strain by heterologous expression of a recombinant fusion gene of Zymomonas mobilis pyruvate decarboxylase (pdc) and Saccharomyces cerevisiae alcohol dehydrogenase1 (adh1) in a strain of B. subtlis WB600 in which the lactate dehydrogenase coding gene was genetically knocked-out. The resulting strain was able to produce bioethanol in 2YTG medium supplemented with 6% waste potato flour. During a 5-day incubation at 30°C, 120 RPM, maximum ethanol concentration of about 7.19 g/L was obtained after 48 h and a final concentration of 5.25 g/L could be measured at the end of the incubation period. B. subtilis WB600 Δldh harboring empty pHY300PLK vector was used as control with no detectable ethanol production. The results show that the Pdc-Adh1 fusion as a bifunctional enzyme can afford microbial ethanol production. Current study is the first report on ethanol production in B. subtilis by development of Pdc-Adh bifunctional enzyme fusion.