胆固醇7α-羟化酶在毕赤酵母中的异源表达  

Allogeneic Expression of Cholesterol 7α-hydroxylase in Pichia pastoris

作　　者：赵昕 杜玉瑶 殷子扬 毛淑红[1,2] ZHAO Xin;DU Yu-yao;YIN Zi-yang;MAO Shu-hong(College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457;Key Laboratory of Industrial Fementation Microbiology,Tianjin 300457)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]工业发酵微生物教育部重点实验室,天津300457

出　　处：《生物技术通报》2023年第10期304-310,共7页Biotechnology Bulletin

基　　金：国家重点研发计划(2019YFA0905300);天津市合成生物技术创新能力提升项目(TSBICIP-KJGG-009-04)。

摘　　要：胆固醇7α-羟化酶(CYP7A1)是胆固醇分解为胆汁酸的限速酶,其催化胆固醇为7α-羟基胆固醇,7α-羟基胆固醇是重要的甾体药物及中间体。以人源CYP7A1作为研究对象,首先构建了重组毕赤酵母/pPIC3.5K-CYP7A1;然后对7α-羟化酶进行分子改造,之后将酶与不同来源NADPH细胞色素氧化还原酶(CPR)适配,以提高目的产物7α-羟基胆固醇的产量。其中突变体G485A将7α-羟基胆固醇的产量提高了8.70%;5种不同来源的CPR与CYP7A1共表达毕赤酵母菌株均可提高7α-羟基胆固醇的产量,并且罗汉果(Siraitia grosvenorii)来源的CPR(SgCPR)与CYP7A1适配性最好,使7α-羟基胆固醇的产率提高了82.26%;在以上研究的基础上,构建突变体G485A与SgCPR共表达毕赤酵母菌株,最终7α-羟基胆固醇的产量提高了133.79%,其产量为0.25 mg/L。实验在毕赤酵母中成功异源表达人源胆固醇7α-羟化酶,并通过分子改造及与不同来源CPR适配的方法提高了7α-羟基胆固醇的产量。Cholesterol 7α-hydroxylase(CYP7A1)is a rate-limiting enzyme that breaks down cholesterol into bile acids,which catalyzes cholesterol to produce 7α-hydroxycholesterol,it is an important steroidal drug and intermediate.Taking human CYP7A1 as the research object,firstly,recombinant Pichia pastoris/pPIC3.5K-CYP7A1 was constructed,and then the yield of the target product 7α-hydroxycholesterol increased by site-directed mutation and adaptation to NADPH cytochrome oxidoreductase(CPR)from different sources.Among them,the mutant G485A increased the yield of 7α-hydroxycholesterol by 8.70%.The co-expression of P.pastoris with five different sources of CPR and CYP7A1 increased the yield of 7α-hydroxycholesterol,and the CPR(SgCPR)derived from Siraitia grosvenorii was the most compatible with CYP7A1,which increased the yield of 7α-hydroxycholesterol by 82.26%.Based on the above research,the co-expression of G485A and SgCPR in P.pastoris increased the yield of 7α-hydroxycholesterol by 133.79%,and the yield was 0.25 mg/L.The human cholesterol 7α-hydroxylase was successfully expressed in P.pastoris,and the yield of 7α-hydroxycholesterol increased based on molecular modification of CYP7A1 and adaptation of CPR from different sources.

关 键 词：甾体药物 胆固醇 胆固醇7Α-羟化酶 7α-羟基胆固醇 毕赤酵母 细胞色素氧化还原酶 

分 类 号：Q78[生物学—分子生物学]