创建酿酒酵母细胞工厂发酵生产芳香精油瓦伦烯  

Construction of cell factories for production of valencene in Saccharomyces cerevisiae

作　　者：杨婷婷 王冬[2,3] 李文豪[1,2,3] 石玉松 李荣生[2,3] 马文建 戴住波 张学礼[2,3] YANG Ting-ting;WANG Dong;LI Wen-hao;SHI Yu-song;LI Rong-sheng;MA Wen-jian;DAI Zhu-bo;ZHANG Xue-li(College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457,China;Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China;Key Laboratory of Systems Microbial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院天津工业生物技术研究所,天津300308 [3]中国科学院系统微生物工程重点实验室,天津300308

出　　处：《药学学报》2023年第6期1619-1628,共10页Acta Pharmaceutica Sinica

基　　金：国家重点研发计划项目(2020YFA0908000)。

摘　　要：瓦伦烯是一种具有柑橘气味的倍半萜类化合物,常被用于化妆品和食品添加剂,以及工业合成诺卡酮。本研究拟用合成生物学技术创建酿酒酵母细胞工厂发酵生产瓦伦烯。首先,在底盘细胞YTT-T5中引入北美金柏来源的瓦伦烯合酶(Cn VS),成功获得可生产1.1 mg·L^(-1)瓦伦烯的工程菌VAL-01;采用蛋白质融合技术,不同植物来源的瓦伦烯合酶筛选,并调整了关键基因拷贝数后,瓦伦烯产量提升至436.4 mg·L^(-1)。进一步工作中,删除转录因子ROX1,瓦伦烯的产量提高了17.4%。最后,对半乳糖诱导系统进行调控,并过表达多药耐药性调控因子PDR3和调控内质网大小转录因子INO2,所得工程菌株VAL^(-1)0经高密度发酵能生产2 798.6 mg·L^(-1)瓦伦烯(产量是初始菌株的2 500倍),为瓦伦烯绿色生产提供了基础。Valencene,a kind of sesquiterpenoid with a citrus flavor,is mainly found in Valencia orange and is commonly used in cosmetics and food additives,as well as industrial synthetic nootkatone.In this study,synthetic biology was used to create a Saccharomyces cerevisiae cell factory to produce valencene.Fistly,valencene synthase gene(CnVS)from Callitropsis nootkatensis was inserted into the chromosome of the chassis strain YTT-T5.The resulting strain VAL-01 could produce 1.1 mg·L^(-1) valencene.Protein fusion technique was used,different valencene synthases were compared and the copy number of key genes was adjusted,yielding valencene to 436.4 mg·L^(-1).Then,knocking-out the transcription factor ROX1 resulted in valencene improvement by 17.4%.Moreover,the induction system of galactose was regulated,transcription factor PDR3 and INO2 were overexpressed.The engineered strain VAL^(-1)0 could produce 2798.6 mg·L^(-1) valencene by high cell density fermentation method(nearly 2500 times higher than VAL-01).This study provides a basis for green production of valencene.

关 键 词：合成生物学 酿酒酵母 倍半萜 瓦伦烯 

分 类 号：R931[医药卫生—生药学]