电子传递链的适配提高孕酮17α羟基化生物催化效率  

Adaptation of the electron transport chain improves the biocatalytic efficiency of progesterone 17α hydroxylation

作　　者：王蓝蓝 赵昕 李杰 艾佳莹[1,2] 孙静 毛淑红[1,2] WANG Lanlan;ZHAO Xin;LI Jie;AI Jiaying;SUN Jing;MAO Shuhong(College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457,China;Key Laboratory of Industrial Fementation Microbiology,Ministry of Education Tianjin 300457,China)

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：《生物工程学报》2023年第11期4608-4620,共13页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(2019YFA0905300)。

摘　　要：17α羟化酶是转化孕酮制备各种孕激素药物中间体的关键酶。为提高该酶在生物催化中的特异性羟基化能力,本研究将来源于纤维素黏性细菌(Sorangiumcellulosum)Soce56的羟化酶CYP260A1与大肠杆菌(Escherichia coli)K-12来源的Fpr和牛肾上腺来源的Adx4-108组建成新的电子传递系统,用于孕酮的生物转化。通过对CYP260A1进行选择性突变,获得17α羟化酶活性显著提高的突变体S276I,经体外催化体系的优化设计,使17α-OH孕酮的产率达到58%。此外,利用定点突变技术探究铁氧还蛋白Adx4-108的模拟磷酸化对17α羟化酶活性的影响,结果显示,突变体Adx4-108T69E向S276I传递电子,进一步增强了对孕酮C17位的特异性,17α-OH孕酮的产率最终提高到74%。本研究为细菌来源的17α羟化酶特异性转化生产17α-OH孕酮提供了新的方案,为孕激素类药物在工业上利用生物转化法生产奠定了理论基础。17αhydroxylase is a key enzyme for the conversion of progesterone to prepare various progestational drug intermediates.To improve the specific hydroxylation capability of this enzyme in steroid biocatalysis,the CYP260A1 derived from cellulose-mucilaginous bacteria Sorangium cellulosum Soce56 and the Fpr and bovine adrenal-derived Adx4-108 derived from Escherichia coli str.K-12 were used to construct a new electron transfer system for the conversion of progesterone.Selective mutation of CYP260A1 resulted in a mutant S276I with significantly enhanced 17αhydroxylase activity,and the yield of 17α-OH progesterone reached 58%after optimization of the catalytic system in vitro.In addition,the effect of phosphorylation of the ferredoxin Adx4-108 on 17αhydroxyl activity was evaluated using a targeted mutation technique,and the results showed that the mutation Adx4-108T69E transferred electrons to S276I more efficiently,which further enhanced the catalytic specificity in the C17 position of progesterone,and the yield of 17α-OH progesterone was eventually increased to 74%.This study provides a new option for the production of 17α-OH progesterone by specific transformation of bacterial-derived 17αhydroxylase,and lays a theoretical foundation for the industrial production of progesterone analogs using biotransformation method.

关 键 词：17α羟化酶 孕酮 电子传递链 蛋白突变 

分 类 号：TQ426.97[化学工程] TQ460.1