产5α羟化紫杉二烯醇人工酵母的组合设计构建  

Combinatorial design and construction of artificial yeast for production of taxadien-5α-ol

作　　者：翟芳 宋田青 肖文海[1] 丁明珠[1] 乔建军[1] 元英进[1] 

机构地区：[1]系统生物工程教育部重点实验室,天津大学化工学院制药工程系,天津化学化工协同创新中心,天津300072

出　　处：《化工学报》2016年第1期315-323,共9页CIESC Journal

基　　金：国家高技术研究发展计划项目(2012AA02A701);高等学校全国优秀博士学位论文作者专项基金(201456)~~

摘　　要：利用工程化微生物生产天然药物如紫杉醇在近年来受到研究者广泛关注。本工作研究如何设计和构建人工酵母以生产紫杉醇生物合成途径中第一个由细胞色素P450酶催化的羟化产物——5α羟化紫杉二烯醇(taxadien-5α-ol)。利用组合设计原理,选用3种不同红豆杉来源的紫杉二烯5α羟化酶和2种不同植物源的细胞色素P450还原酶,分别对其进行N端穿膜区域的预测和截短,并对还原酶使用2种不同强度启动子进行调控,所得羟化酶模块和还原酶模块之间分别表达共产生72种组合方式。然后将其分别整合入紫杉二烯生产菌株基因组中,最终在72个组合中筛选到48个可以生产5α羟化紫杉二烯醇的菌株,最高产量67.3μg·L-1,在酿酒酵母中实现紫杉二烯C5位羟化产物的从头合成。研究结果表明,利用组合设计策略研究模块间相互作用及其与底盘间适配性将为在微生物细胞中实现细胞色素P450酶系介导的催化反应过程提供重要参考。Producing natural drugs such as Taxol by engineered microbes have attracted extensive attention in recent years. This work focused on how to design and construct artificial yeast for the production of taxadien-5α-ol, which was the first hydroxylated product catalyzed by cytochrome P450 enzymes in Taxol biosynthesis pathway. Based on combinatorial design, three hydroxylase genes derived from different yew subspecies and two reductase genes from different organisms with N-terminal truncatd with various length were tested simultaneously to investigate the fitness between the genes and the chassis. Two kinds of promoters were used for regulating the cytochrome P450 reductase genes. In total, 72 kinds of combinations were constructed and then integrated into the chromosome of the taxadiene producing strain. 48 taxadine-5α-ol producing strains were obtained out of 72 combinations by co-expression of the hydroxylase and the reductase. The highest titer was 67.3 μg·L-1. This was the first time to realize the biosynthesis of taxadine-5α-ol form glucose in Saccharomyces cerevisiae. The result provided a good reference for the research of the catalytic reaction mediated by cytochrome P450 enzymes in microbes through the interactions between modules and chassis by combinatorial design.

关 键 词：合成生物学 5α羟化紫杉二烯醇 组合设计 酿酒酵母 生物催化 发酵 

分 类 号：Q819[生物学—生物工程]