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食品与发酵工业  2021, Vol. 47 Issue (16): 9-14    DOI: 10.13995/j.cnki.11-1802/ts.027099
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
α-法尼烯在巴斯德毕赤酵母中的生物合成
刘慧1,2, 陈胜玲1,2, 徐建中1,2*, 张伟国1,2*
1(江南大学 生物工程学院,江苏 无锡,214122)
2(工业生物技术教育部重点实验室(江南大学),江苏 无锡,214122)
Metabolic engineering of Pichia pastoris for α-farnesene production
LIU Hui1,2, CHEN Shengling1,2, XU Jianzhong1,2*, ZHANG Weiguo1,2*
1(School of Biological Engineering, Jiangnan University, Wuxi 214122,China)
2(Key Laboratory of Industrial Biotechnology of Ministry of Education (Jiangnan University), Wuxi 214122,China)
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摘要 由可再生原料微生物生产α-法尼烯是一种有前途的替代传统石油基工艺的方法。尽管已经报道α-法尼烯可以通过常规模型菌株如大肠杆菌和酿酒酵母异源产生,但是其发酵规模不易扩大。巴斯德毕赤酵母是生产类异戊二烯的良好平台且可高密度培养,具有大规模生产α-法尼烯的潜力。在该项研究中,首先确定tHmg1,IDI1,ERG19,AFSLERG20是甲羟戊酸途径和α-法尼烯合成途径的限速酶基因。然后对限速酶基因进行组合过表达并优化基因拷贝数以平衡代谢路径增大流向α-法尼烯合成的代谢通量,最终获得菌株F16,其α-法尼烯产量为(1.09±0.02) g/L。最后,通过外源添加不饱和脂肪酸促进α-法尼烯分泌到细胞外,当培养基添加20 mmol/L的油酸,在摇瓶中获得最高的α-法尼烯产量约1.40 g/L[0.32 g/g 细胞干重(dry cell weight,DCW)]。这是出发菌株F1产量的3.1倍。该研究是首次以巴斯德毕赤酵母作为底盘微生物细胞来生产α-法尼烯,并为其他倍半萜的异源生物合成提供了新的思路。
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刘慧
陈胜玲
徐建中
张伟国
关键词:  代谢工程  巴斯德毕赤酵母  甲羟戊酸途径  α-法尼烯  脂肪酸    
Abstract: Microbial production of α-farnesene from renewable feedstock is a promising alternative to traditional petroleum-based processes. Although it has been reported that α-farnesene can be heterologously produced by conventional model strains such as Escherichia coli and Saccharomyces cerevisiae, its fermentation scale is not easy to expand. Pichia pastoris is a good platform for the production of isoprene and has the potential for large-scale production of α-farnesene at high density. tHmg1, IDI1, ERG19, and AFSLERG20 were confirmed as the rate-limiting enzymes of the mevalonate pathway and the α-farnesene synthesis pathway. Then, the metabolic flux to α-farnesene synthesis was increased by combining overexpression rate limiting enzyme genes and optimizing the copy number of genes to balance the metabolic pathway. Strain F16 was obtained, and its α-farnesene yield was (1.09±0.02) g/L. Furthermore, the secretion of α-farnesene into extracellular space was promoted by adding unsaturated fatty acids, when 20 mmol/L of oleic acid was added to the medium, the highest α-farnesene titer in the shake flask was about 1.40 g/L [0.32 g/g (dry cell weight,DCW)], which was 3.1 times of that in the starting strain F1. For the first time, P. pastoris was used as a chassis microbial cell to produce α-farnesene and the results provide a new idea for the heterologous biosynthesis of other sesquiterpenes.
Key words:  metabolic engineering    Pichia pastoris    mevalonate pathway    α-farnesene    fatty acid
收稿日期:  2021-02-20      修回日期:  2021-03-02                发布日期:  2021-09-10      期的出版日期:  2021-08-25
基金资助: 国家自然科学基金资助项目 (31601459);国家轻工业技术与工程一流学科计划项目 (LITE2018-08);江苏省高等学校一流学术计划项目,111项目 (111-2-06)
作者简介:  硕士研究生(徐建中副教授和张伟国教授为共同通讯作者,E-mail:xujianzhong@jiangnan.edu.cn;zhangwg@jiangnan.edu.cn)
引用本文:    
刘慧,陈胜玲,徐建中,等. α-法尼烯在巴斯德毕赤酵母中的生物合成[J]. 食品与发酵工业, 2021, 47(16): 9-14.
LIU Hui,CHEN Shengling,XU Jianzhong,et al. Metabolic engineering of Pichia pastoris for α-farnesene production[J]. Food and Fermentation Industries, 2021, 47(16): 9-14.
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http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.027099  或          http://sf1970.cnif.cn/CN/Y2021/V47/I16/9
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