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

摘要:

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1.53U/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。

关键词:关键词: 丙酮丁醇梭状芽胞杆菌，大肠杆菌，正丁醇，酶活，厌氧发酵

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.

Key words:Keywords: Clostridium acetobutylicum，Escherichia coli，n-butanol，enzyme activities，anaerobic-fermentation