丁醇合成途径关键酶基因在大肠杆菌中的克隆和表达

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

【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5 h,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.