Heterologous Expression,Biochemistry And Enzyme Engineering Of Cytochrome P450 Enzymes CYP11A1s

Steroid drugs,the second largest class of pharmaceuticals after antibiotics in the world,have significant anti-allergic,anti-inflammatory,endocrine-regulating,and other effects.De novo total biosynthesis provides a new means for the industrial production of steroid drugs.However,the low enzyme activity involved in each step and the complexity of the whole synthesis process leads to low steroid production.A group of cytochrome P450 enzymes,namely,CYP11A1 isoenzymes from different organisms are capable of converting cholesterol into pregnenolone,which is a pivotal reaction in both steroid metabolism and(bio)synthetic network of steroid products.Nevertheless,the low activity of CYP11A1s greatly limits its industrial application for microbial production of steroids.Therefore,it is necessary to mine new CYP11A1 isoenzymes or perform protein engineering of CYP11A1s to improve its activity.In this study,in order to mine CYP11A1s with cholesterol side-chain cleavage activity,CYP11A1s from different origins selected by the bioinformatical analysis were performed heterologous expression in Escherichia coli,and their catalytic activity towards cholesterol was detected.Subsequently,several CYP11A1s with higher expression levels were biochemically characterized including their substrate specificity and redox partner optimization.In addition,semirational enzyme engineering of CYP11A1s based on the known crystal structures was performed to obtain cholesterol side-chain cleavage enzyme with higher catalytic activity.The main research contents and conclusions are as follows:1.Genome mining of CYP11A1s from different vertebrate sourcesExplosive genome sequencing data has led to disclosure of a large number of CYP11A1 genes.To identify more cytochrome P450 enzymes with the cholesterol side-chain cleavage activity,we BLAST searched the CYP11A1s in the Uniprot database using BtCYP11A1(Uniprot#:P00189)as a sequence probe and constructed a phylogenetic tree for the sequence homologues with the protein sequence identity greater than 45%.Based on the phylogenetic tree,ten CYP11A1s with different levels of homology and origins were selected for heterologous expression in E.coli,among which seven were derived from different mammals,two from birds,and one from fish.By detecting the catalytic activity of cell lysates expressing CYP11A1s towards cholesterol,the catalytic product pregnenolone was detected in the enzymatic reaction of mChCYP11A1(Capra hircus),mBtCYP11A1(Bos taurus),mHsCYP11A1(Homo sapiens),mSsCYP11A1(Sus scrofa),mMmCYP11A1(Mus musculus)and mTgCYP11A1(Taeniopygia guttata).The in vitro catalytic activity reconstruction of six CYP11A1s from different origins was achieved,among which mChCYP11A1,mSsCYP11A1(Sus scrofa),mMmCYP11A1(Mus musculus)and mTgCYP11A1 were expressed functionly in E.coli for the first time.2.Substrate specificity and redox partner optimization of CYP11A1s from different originsAmong the six CYP11A1s with cholesterol catalytic activity obtained in the first part,mChCYP11A1 and mSsCYP11A1 with higher expression levels were selected for further biochemical characterization,together with mBtCYP11A1 and mHsCYP11A1 as references.Firstly,protein purification and in vitro activity detection of these four CYP11A1s with membrane-bound properties were carried out,and purified proteins with in vitro catalytic activity were successfully obtained.Then,the catalytic activities of four purified CYP11A1s were compared towards cholesterol,desmosterol,β-sitosterol,campesterol,and 7-dehydrocholesterol.mSsCYP11A1 showed the highest catalytic activities towards the selected five sterols,and all four CYP11A1s had the highest catalytic activities for 7-dehydrocholesterol,which was explained by molecular docking.In addition,a suitable redox partner is critical to the catalytic activity of P450.The adrenodoxin(Adx)and adrenodoxin reductase(AdR)of mChCYP11A1,mBtCYP11A1,mHsCYP11A1,and mSsCYP11A1 were heterologously expressed in E.coli,and redox partners combination screening was performed using the purified proteins,it showed that the combination consisting of mSsAdx derived from wild boar and mHsAdR derived from human was the best.3.Enzyme engineering of CYP11A1Enzyme engineering of CYP11A1 was performed in order to improve its catalytic activity.Firstly,three different amino acids within 6A distance from the substrate were identified through protein sequence alignment,and the effect of these three amino acids on the catalytic activity of CYP11A1 was studied.Then,based on the crystal structure of mHsCYP11A1 and mBtCYP11A1,alanine scanning was performed towards amino acid residues located within the active site or at the substrate entrance channel,and a mutant mBtCYP11A1-Q377A with a 46%increase in catalytic activity was obtained.In addition,it was found that the substrate entrance channel diameter of mBtCYP11A1Q377A was widened by 34%,which partly explained the increase in catalytic activity of mutant.The obtained mutant mBtCYP11A1-Q377A provides a new candidate parent enzyme for CYP11A1 engineering and also provides a reference for the modification of other eukaryotes P450s.In summary,this study successfully reconstructed the in vitro activities of CYP11A1s from six different sources through genome mining and heterologous expression.Moreover,taking the two wellstudied mHsCYP11A1 and mBtCYP11A1 as references,the mSsCYP11A1 and mChCYP11A1 with high expression levels was biochemically characterized.Towards five selected sterol substrates with different side chain structures,S.scrofa CYP11A1 displays relatively higher activities,and all four CYP11A1 had a preference for 7-dehydrocholesterol.Through redox partners combination screening,it revealed the optimal redox partner pair of S.scrofa adrenodoxin and C.hircus adrenodoxin reductase.In addition,the semi-rational enzyme engineering for the active sites and substrate entrance channels of CYP11A1 was performed,and the mutant mBtCYP11A1-Q377A shows a 1.46 times higher activity than the wild type enzyme.These results not only demonstrate the tunability of the highly conserved CYP11A1 isoenzymes,but also lay a research foundation for the microbial production of steroidal drugs.