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

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.