Cloning and expression of key genes of butanol synthetic pathway in Escherichia coli
丁醇合成途径关键酶基因在大肠杆菌中的克隆和表达

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 n-butanol 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.