糖丁基梭菌产丁醇途径在大肠杆菌中的构建及发酵

Construction of butanol-producing pathway from Clostridium saccharobutylicum in Escherichia coli JM109( DE3) and its fermentation

【目的】通过克隆来源于糖丁基梭菌(Clostridium saccharobutylicum DSM13864)丁醇合成途径的关键酶基因(thl A,bcs-operon和adh E),构建产丁醇大肠杆菌。【方法】以Clostridium saccharobutylicum DSM13864的基因组为模板,分别扩增丁醇途径关键酶基因thl A,bcs-operon(crt-bcd1-etf B2-fix B2-hbd)和adh E,构建了两个重组质粒p ETDuet-bcs和pRSFDuet-thl A-adh E,并成功转入E.coli JM109(DE3)实现异源表达,使大肠杆菌具备产丁醇能力。在半厌氧条件下进行重组菌的发酵,并研究不同培养基对产丁醇的影响。【结果】该重组菌在半厌氧条件下经摇瓶发酵丁醇产量达到25.4 mg/L,通过优化培养基后,在TB发酵培养基中丁醇产量可达到34.1 mg/L。【结论】通过构建重组共表达质粒,将糖丁基梭菌来源的丁醇途径关键酶基因在大肠杆菌中表达,成功构建产丁醇大肠杆菌。该研究提供了一株易于操作的丁醇发酵重组大肠杆菌,避免了传统梭菌发酵丁醇生产中苛刻的厌氧条件、易产孢子等限制问题。

[ Objective ] Several key genes （ thlA, bcs-operon/crt-bcdl-etfB2-fixB2-hbd and adhE ） in butanol pathway from Clostridium saccharobutylicum DSMI3864 were cloned, and a butanol-producing Escherichia coli strain was successfully constructed. [ Methods] Using genome of Clostridium saccharobutylicum DSM13864 as template, the key genes in butanol synthesis pathway were amplified, the recombinant plasmids pETDuet-bcs and pRSFDuet-thlA-adhE were constructed. Then the resultant plasmids were transformed into E. coli JM109（DE3） to obtain E. coli BUT1 for butanol production, under the semi-anaerobic condition. Effects of different mediums on butanol production were studied. [ Results] The recombinant E. coli was capable of producing butanol （25. d mg/L） under semi-anaerobic fermentation. After optimization on the fermentation medium, butanol titer reached 34. 1 mg/L. [ Conclusion ] Butanol production by recombinant E. coli harboring exogenous butanol-producing pathway from Clostridium saccharobutylicum provides a feasible solution to overcome the hurdles in traditional butanol production approach by Clostridia.