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食品与发酵工业  2020, Vol. 46 Issue (11): 1-8    DOI: 10.13995/j.cnki.11-1802/ts.023680
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
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
马振锋, 徐美娟, 杨套伟, 张显, 邵明龙, 中西秀树*, 饶志明*
(工业生物技术教育部重点实验室(江南大学),江苏 无锡,214122)
Recombinant Corynebacterium glutamicum produced 2-pyrrolidone from glucose
MA Zhenfeng, XU Meijuan, YANG Taowei, ZHANG Xian, SHAO Minglong, NAKANISHI Hideki*, RAO Zhiming*
(Key Laboratory of Industrial Biotechnology, Ministry of Education(Jiangnan University), Wuxi 214122, China)
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摘要 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。
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马振锋
徐美娟
杨套伟
张显
邵明龙
中西秀树
饶志明
关键词:  谷氨酸棒杆菌  2-吡咯烷酮  CoA转移酶  γ-氨基丁酸  谷氨酸脱羧酶    
Abstract: 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.
Key words:  Corynebacterium glutamicum    2-pyrrolidone    CoA transferase    γ-aminobutyric acid    glutamate decarboxylase
收稿日期:  2020-02-19                出版日期:  2020-06-15      发布日期:  2020-06-24      期的出版日期:  2020-06-15
基金资助: 国家重点研发计划项目(2018YFA0900300);国家自然科学基金(31770058);江苏省自然科学基金项目(BK20181205);宁夏回族自治区重点研发计划(2019BCH01002);中组部万人计划科技创新领军人才项目资助
作者简介:  硕士研究生(中西秀树教授和饶志明教授为共同通讯作者,E-mail: hideki@jiangnan.edu.cn;raozhm@jiangnan.edu.cn)
引用本文:    
马振锋,徐美娟,杨套伟,等. 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[J]. 食品与发酵工业, 2020, 46(11): 1-8.
MA Zhenfeng,XU Meijuan,YANG Taowei,et al. Recombinant Corynebacterium glutamicum produced 2-pyrrolidone from glucose[J]. Food and Fermentation Industries, 2020, 46(11): 1-8.
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