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

摘要:

摘要:【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(thlA，bcs-operon和adhE)，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thlA，bcs-operon(crt-bcd1-etfB2-fixB2-hbd)和adhE，构建了两个重组质粒pETDuet-bcs和pRSFDuet-thlA-adhE，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。

Abstract:

Abstract:［Objective］Several key genes (thlA，bcs-operon/crt-bcd1-etfB2-fixB2-hbd and adhE) in butanol pathway from Clostridium saccharobutylicum DSM13864 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 (25.4 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.