Cloning of butanol synthetic pathway genes from Clostridium acetobutylicum in heterologous host

Abstract

newline Despite having tremendous potential as a fuel, butanol s toxicity to the microbes is a major bottleneck. An alternative microbial host Staphylococcus sciuri for butanol production possessing higher level of tolerance has been isolated which was able to tolerate up to 2.25% v/v butanol with 70% growth as compared to control. Four possible gene products as Universal stress protein UspA, 2 general stress proteins (GSP) and one other stress protein (GlsB/YeaQ/YmgE family) have been predicted to play a possible role in butanol tolerance. Another microbe Bacillus subtilis was isolated from a soil column which showed approximately 70% growth as compared to control at 1.75% butanol. Its TEM studies indicated the thickening of extracellular capsule up to many folds in the presence of butanol as compared to control. Butanol synthesis pathway genes thl, hbd, crt, bcd, etfA, etfB were cloned in pTZ57R/T and TOPO pET101/D plasmids. The resulting plasmids were cloned into E.coli BL21(DE3) and E.coli Dh5and#945;. The recombinant strains showed very high level of expression of genes thl, hbd and bcd-etfA-etfB than few earlier studies. High tolerance to butanol in case of alternative hosts and high expression of genes in case of Escherichia coli both pave a path for exploring each of these as butanol production hosts. newline