Construction Of Butanol-producing Pathway From Clostridium Saccharobutylicum In Escherichia Coli

With worldwide increasing concern on energy crisis and environmental issues such as global warming, biofuels derived from renewable non-food substrates attracts more and more attention. Compared with ethanol as a traditional biofuel, butanol exhibits better properties and becomes a research hotspot in recent years due to its higher energy content, less corrosive and less water soluble.Several key genes(thl A, bcs-operon/crt-bcd1-etf B2-fix B2-hbd and adh E) related to butanol pathway from Clostridium saccharobutylicum DSM13864 were cloned into the coexpression plasmids p ETDuet-1 and p RSFDuet-1 to construct the recombinant plasmids p ETDuet-bcs and p RSFDuet-thl A-adh E, respectively. Then the recombinant plasmids were transformed into Escherichia coli JM109(DE3), and a butanol-producing recombinant E. coli ECJ0 strain was successfully constructedThe recombinant E. coli ECJ0 was used to produce butanol under the semi-anaerobic condition with M9 medium, and 25.4 mg·L-1 butanol was obtained after 60 h fermentation. After the medium optimization, butanol concentration can reached to 34.1 mg·L-1 with TB as the fermentation medium. We investigated the effect of oxygen on butanol production under obligate anaerobic and semi-anaerobic fermentation, respectively. The result shows that almost similar butanol concentration was obtained under the two different conditions. However, no butanol production was detected under aerobic condition. After the optimization of IPTG inducing concentration, the highest butanol concentration of 54.1 mg·L-1 was obtained at IPTG concentration of 0.05 mmol·L-1.Several metabolic byproducts were detected in the fermentation broth such as ethanol, acetate and butyrate. Therefore, the deletion of key genes related to the byproduct pathway was performed to reduce the byproducts concentration and increase butanol production. The key gene ldh A encoding lactate dehydrogenase in lactate synthesis pathway was knocked out, then the co-expression plasmids above were transformed into E. coli JM109(DE3)Δldh A to construct the recombinant E. coli ECJ1. E. coli ECJ1 can produce butanol concentration of 97.5 mg·L-1, which was nearly 2-fold of that produced by E. coli ECJ0. After optimization of carbon source, the highest butanol concentration of 142.1 mg·L-1 was obtained with glucose as the unique carbon source. The initial inducing cell density was optimized to be OD600 0.8, under which the highest butanol concentration of 143.2 mg·L-1 was achieved.