丁醇合成途径关键酶基因在大肠杆菌中的克隆和表达

摘要

[目的]克隆丙酮丁醇梭状芽胞杆菌(Clostridium acetobutylicum)ATCC824丁醇合成途径关键酶基因,构建产丁醇的工程大肠杆菌.[方法]以C.acetobutylicum ATCC824基因组为模板,分别扩增丁醇合成途径关键酶基因thil adhE2和BCS operon(crt-bcd-etfB-etfA-hbd)基因序列,构建BCS operon-adhE2-thil/pTrc99a/MG1655 (pBAT).重组菌E.coli pBAT采用0.1 mmol异丙基-β-硫代半乳糖苷(IPTG)诱导5h,测定乙酰基转移酶(THL)、3-羟基丁酰辅酶A脱氢酶(HBD)、3-羟基丁酰辅酶A脱水酶(CRT)、丁酰辅酶A脱氢酶(BCD)、醛醇脱氢酶(BYDH/BDH)的酶活.并以该基因工程菌作为发酵菌种,采用好氧、厌氧和微好氧三种培养方式,检测丁醇产量.[结果]酶活测定结果显示:THL酶活达到0.160 U/mg protein,酶活力提高了近30倍；HBD酶活力提高了近5倍；CRT酶活达到1.53 U/mg protein,野生菌株无此酶活；BCD酶活力提高了32倍；BYDH/BDH酶活力无显著提高.3种发酵培养结果显示在微好氧和厌氧条件下,均有丁醇产生,且丁醇的最大产量约为84 mg/L.[结论]本实验通过构建产丁醇基因工程大肠杆菌,实现了丁醇关键酶基因在大肠杆菌中的活性表达以及发酵产丁醇,为发酵法生产丁醇开辟了一条新的途径.%[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.