基于细胞色素P450单加氧酶介导的4-甲酚氧化降解途径的天麻素生物合成  

Biosynthesis of Gastrodin Based on the Cytochrome P450-Mediated Oxidative Biodegradation of 4-Cresol

作　　者：钟贝芬 杜磊 李众[2] 张伟[2] 张兴旺[2] 李菁 黄淑兰 胡翔 李盛英[2] 刘琨 ZHONG Bei-fen;DU Lei;LI Zhong;ZHANG Wei;ZHANG Xing-wang;LI Jing;HUANG Shu-lan;HU Xiang;LI Sheng-ying;LIU Kun(State Key Laboratory of Developmental Biology of Freshwater Fish,School of Life Sciences,Hunan Normal University,Changsha 410081,China;Shandong Provincial Key Laboratory of Synthetic Biology,Qingdao Institute of Bioenergy BioprocessTechnology,Chinese Academy of Sciences,Qingdao 266101,China;Hunan Yunheng Biotechnology Co.,Changsha 410005,China)

机构地区：[1]湖南师范大学生命科学学院,省部共建淡水鱼类发育生物学国家重点实验室,中国长沙410081 [2]山东省合成生物学省级重点实验室,中国科学院青岛生物能源与过程研究所,中国青岛266101 [3]湖南云恒生物科技有限公司,中国长沙410005

出　　处：《湖南师范大学自然科学学报》2018年第4期33-40,共8页Journal of Natural Science of Hunan Normal University

基　　金：山东省自然科学基金重大基础研究项目(ZR2017ZB0207); 湖南省教育厅产业化培育项目(15CY006)

摘　　要：为实现天麻素的微生物异源生产,将谷氨酸棒状杆菌中4-甲酚降解相关基因cre HIJEFD和药用植物红景天来源的尿苷二磷酸糖基转移酶基因ugt分别克隆至载体pRSFDuet-1和p ACYCDuet-1,转化大肠杆菌后获得重组菌株S1.在S1中诱导关键酶表达后,外源添加4-甲酚,生物转化后对产物进行定性和定量分析.最终以5mmol/L 4-甲酚为底物,重组菌株S1在30℃诱导培养48 h后转化产物中有天麻素生成,产量达到1.5 mmol/L,摩尔转化率为30.3%.本研究实现了从4-甲酚到天麻素的生物转化,可为芳烃化合物降解与天然药物分子生物合成相偶联提供新的思路,并且具有一定的产业化应用前景.To establish the microbial production of gastrodin, the 4-cresol biodegradation gene creHIJEFD derived from Corynebacterium glutamicum , and the uridine diphosphate glycosyltransferase encoding gene, ugt, originated from the medicinal plant Rhodiola rosea , were cloned into pRSFDuet-1 and pACYCDuet-1, respectively. These two recombinant plasmids were then transformed into Escherichia coli BL21 （DE3）, resulting in the recombinant strain S1. The expression of key enzymes was induced with isopropyl β -D-1-thiogalactopyranoside （IPTG）, after which 4-cresol was added as a substrate for biotransformation. The products were subsequently analyzed by HPLC and LC-MS. We found that the recombinant strain S1 produced 1.5 mmol/L of gastrodin over 48 h at 30 ℃ when 5 mmol/L of 4-cresol was fed, representing a molar conversion rate of 30.3%. Our results show that gastrodin could successfully be produced in E. coli by utilizing 4-cresol as the starting material. It should provide a new pathway to incorporate the biodegradation of aromatic compounds with the biosynthesis of medicinal natural products with the significant potential of applications.

关 键 词：4-甲酚 天麻素 芳烃生物降解 生物合成 细胞色素P450单加氧酶 

分 类 号：R283[医药卫生—中药学]