运用BioBrick组装策略共表达CYP108N7及其氧化还原伴侣

Coexpression of CYP108N7 and its redox partner facilitated by BioBrick assembly

细胞色素P450单加氧酶(简称P450,CYP)是功能强大的单加氧酶超家族,可以催化多种化学法难以实现的氧化反应.实验室前期挖掘的P450新酶CYP108N7,能够利用菠菜铁氧化还原蛋白(Fdx)和铁氧化还原蛋白还原酶(FdR)作为非天然氧化还原伴侣进行电子传递,并在双质粒系统中实现共表达.本研究拟简化表达体系,重新构建单个质粒的全细胞表达系统.利用BioBrick组装策略,将CYP108N7、Fdx和FdR基因模块化并排列重组,成功构建了编码上述3条基因的、6种排列结构的表达质粒,将其在大肠杆菌中异源表达后获得全细胞催化剂.这6种全细胞的粗酶液的CO差谱都显示出P450酶的典型吸收特征;全细胞在催化苯甲硫醚不对称氧化反应时,都能以优异的立体选择性得到S-苯甲亚砜(>99%ee).其中,催化效率最高全细胞来自以Fdx-FdR-CYP108N7方式排列的质粒N7S6.本研究应用BioBrick组装策略极大地简化了多组分CYP108N7酶的共表达质粒的构建和优化工作,可为后续该酶的分子进化奠定基础.(图6表1参31)

Cytochrome P450 monooxygenases(P450,CYP)comprise a powerful monooxygenase superfamily that can catalyze diverse oxidation reactions,some of which are difficult or impossible to achieve by chemical approaches.In our previous study,a novel P450 enzyme,CYP108 N7,was found to accept ferredoxin(Fdx)and Fdx reductase(FdR)from spinach efficiently as surrogate redox partners after heterologous co-expression in Escherichia coli using two plasmids.In this study,we reconstructed a simplified whole-cell expression system using a single plasmid.The genes encoding CYP108 N7,Fdx,and FdR were first modularized based on the BioBrick assembly strategy and rearranged in every permutation to obtain six plasmids.Each of the six plasmids was then transformed into Escherichia coli and overexpressed,resulting in six whole-cell biocatalysts.All of these biocatalysts displayed typical CO-binding difference spectra of active P450 s when measured with crude enzyme extracts.The biocatalysts were able to catalyze the asymmetric oxidation of thioanisole,yielding(S)-sulfoxide with excellent stereoselectivity(>99%ee).Among them,the whole-cell biocatalyst transformed with plasmid N7 S6(Fdx-FdR-CYP108 N7)displayed the highest activity.This work applied the BioBrick assembly strategy to facilitate the construction and optimization of co-expression plasmids of CYP108 N7 and its redox partners,which can pave the way for future studies on the molecular evolution of CYP108 N7.