Cloning and expression of key genes of butanol synthetic pathway in Escherichia coli

Abstract:

Abstract:［Objective］We constructed a recombinant Escherichia coli strain for butanol production by cloning the cDNA sequence of the key butanol synthetic pathway genes from Clostridium acetobutylicum ATCC824．［Methods］ We amplified the genes of thil，adhE2 and BCS operon by PCR with C.acetobutylicum ATCC824 genome as a template. We constructed the recombinant strain E.coli pBAT (BCS operon-adhE2-thil/pTrc99a/MG1655)．We used 0.1 mmol/l Isopropyl beta-D-thiogalactopyranoside (IPTG) to induce the recombinant E.coli pBAT for 5 h for recombinant protein expression. We measured acetyl-CoA acetyltransferase (THL)，β-hydroxybutyryl-CoA dehydrogenase (HBD)，3-hydroxybutyryl-CoA dehydratase (CRT)， butyryl-CoA dehydrogenase (BCD) and butyraldehyde dehydrogenase (BYDH)/butanol dehydrogenase (BDH) activities in E.coli MG1655 and E.coli pBAT． The fermentation of E.coli pBAT was done in flask in aerobic，micro-aerobic and anaerobic mode separately．［Results］ In the recombinant E． coli pBAT，THL activity was 0.160 U/mg protein，about 30 times higher than that of E.coli MG1655． HBD activity was 5 times higher than that of E.coli MG1655.CRT activity was 1.53 U/mg protein whereas not detectable in E.coli MG1655. BCD activity was about 32 times higher than that of E.coli MG1655.In addition，the results show that nbutanol could be produced under anaerobic and micro-aerobic conditions． The maximum n-buntanol concentration of 84 mg/l was detected in cultivation broth.［Conclusion］ The key genes of butanol synthetic pathway were expressed in E.coli and the recombinant strains would offer an alternative strategy for butanol biosynthesis.