以葡萄糖为底物合成2-吡咯烷酮重组谷氨酸棒杆菌的构建及发酵研究

Recombinant Corynebacterium glutamicum produced 2-pyrrolidone from glucose

2-吡咯烷酮(2-pyrrolidone)因在纺织和制药行业的广泛应用而受到越来越多的关注。通过对合成2-吡咯烷酮途径的关键酶CoA转移酶的挖掘及功能的研究,首次探索了从谷氨酸棒杆菌(Corynebacterium glutamicum)中建立2-吡咯烷酮合成途径,为未来可持续合成2-吡咯烷酮工业指明了方向。首先通过敲除N-乙酰谷氨酸激酶基因(argB)阻断L-精氨酸合成途径,促使更多葡萄糖流向L-谷氨酸。其次,通过表达将L-谷氨酸转化为γ-氨基丁酸(γ-aminobutyric acid,GABA)的谷氨酸脱羧酶(Gad)突变体,获得合成GABA的重组菌。同时,异源表达经N-端RBS优化的丙酸厌氧菌(Anaerotignum propionicum)来源的CoA转移酶,实现了GABA向2-吡咯烷酮的转化。最终,构建的C.glutamicum EAGAN2重组菌株在5 L发酵罐补料分批发酵72 h时,积累了(8±0.3)g/L的2-吡咯烷酮。该研究首次在谷氨酸棒杆菌中建立了2-吡咯烷酮合成途径,实现了以廉价原料葡萄糖为底物一步法合成2-吡咯烷酮。

2-Pyrrolidone as a highly promising bio-based platform chemical,has received more attention due to their widely used in textile and pharmaceutical industries.2-Pyrrolidone synthesis pathway was established in Corynebacterium glutamicum by exploring the function of the key enzyme,CoA transferase,thus providing guidance for sustainable industrial synthesis of 2-pyrrolidone.Firstly,argB gene was knocked out to block the L-arginine biosynthesis pathway and increase the carbon flux to the 2-pyrrolidone synthesis pathway,resulting in a highly efficient synthesis of L-glutamate in the chassis cells.Secondly,a glutamate decarboxylase(Gad)was expressed to convert L-glutamate to GABA.Finally,CoA transferase derived from Anaerotignum propionicum was optimized for the N-terminus RBS,then recombined and expressed in C.glutamicum to convert GABA to 2-pyrrolidone.The engineered C.g EAGAN2 strain was tested in a 5 L fermenter and after 72 h of fermentation,(8±0.3)g/L 2-pyrrolidone was accumulated.The 2-pyrrolidone synthesis pathway was for the first time established in C.glutamicum.The CoA transferase could convert GABA to 2-pyrrolidone,achieving optimum biosynthesis in fed-batch cultures using glucose as a cheap carbon source.