摘要
异鼠李素是一种黄酮类化合物,广泛存在于银杏、沙棘、旱柳和枸杞等植物中,具有多种药理作用。植物来源的O-甲基化酶能对槲皮素羟基进行甲基化,因而构建重组菌高效表达O-甲基化酶可应用于催化槲皮素生成异鼠李素。笔者对大豆来源的O-甲基化酶SOMT-9编码基因进行了密码子优化,其密码子适应指数(CAI)从优化前的0.59,提高为0.87。将优化后的基因SysOMT-9插入表达载体pGEX-2T,构建获得重组质粒pGEX-SysOMT。将重组质粒转化大肠杆菌BL21(DE3),通过改变诱导温度及诱导剂浓度,实现了SOMT-9可溶性表达。在此基础上对其转化条件进行了优化,其最佳转化条件为:起始菌量OD600为10,转化温度为30℃,诱导剂IPTG用量为0.01mmol/L,助溶剂DMSO质量分数为1%,1%甘油,40μmol/L的S-腺苷基甲硫氨酸和1mmol/L槲皮素。在此条件下,重组菌在12h内,可将127mg/L的槲皮素,转化生成133mg/L异鼠李素,摩尔转化率为42%,是优化前的7.8倍。
As a kind of important flavonoids, isorhamnetin exists widely in many plants such as Ginkgo biloba, Sea buchthorn, Saliz matsudana and Matrimony vine. Isorhamnetin has many pharmacological activities. The hydroxyl group in quercetin can be methylated by O-methyltransferases from plants. Thus, the construction of bioconversion engineering bacteria that could catalyze the quercetin into isorhamnetin was considered as an effective approach to produce isorhamnetin. In order to improve the expression of O-methyltransferase SOMT-9, the gene of O-methyltransferase SOMT-9 was designed, optimized and synthesized based on the synonymous condon bias of Escherichia coli, and the codon adaptation index （CAI） increased from 0. 59 to 0. 87. The recombinant plasmid pGEX-SysOMT was constructed by inserting the optimal gene into pGEX-2T. The recombinant O-methyltransferase SOMT-9 was over-expressed in E. coli BL21 （DE3） by different strategies such as changing induction temperature and concentration of isopropy-β-D-thiogalactoside （IPTG）. Based on that, the optimal transformation conditions were determined. The optimal of initial OD600 value was 10; the optimal concentration of IPTG was 0.01 mmol/L; the optimal concentration of dimethyl sulphoxide （DMSO） was 1%; the optimal carbon source was glycerol with 1% concentration; the optimal concentration of S-adenosyl methionine was 40 μmol/L; and the optimal concentration of the substrate, i.e., quercetin, was 1 mmol/L. The recombinant strain was at 30℃ for 12 h. Under the optimal transformation conditions, 127 mg/L quercetin was transformed into 133 mg/L isorhamnetin within 12 h by the recombinant strain and the molar conversion rate was 42%, which was eight times higher than that before the optimization.
出处
《林业工程学报》
北大核心
2017年第3期50-57,共8页
Journal of Forestry Engineering
基金
国家林业公益性行业科研专项(201404601)
江苏省自然科学基金(BK20131423)
江苏省“青蓝工程”项目
江苏高校优势学科建设工程资助项目(PAPD)
关键词
槲皮素
异鼠李素
O-甲基化酶
重组菌
转化条件
quercetin
isorhamnetin
O-methyltransferase
recombinant bacteria
optimization
