Mining And Functional Study Of P450 Hydroxylase Genes Putatively Related To The Biosynthesis Of Taxol From Taxus Chinensis

Taxol is an effective drug commonly used to treat various types of cancer,mainly from the medicinal plant Taxus.But the yield cannot meet the clinical demand because of low content in yew.Therefore,it is extremely urgent to look for new pathes of taxol drug source.Obtaining taxol by using synthetic biology technology will be an important means in the future.Some genes of the taxol biosynthetic pathway have been identified,but the CYP450 hydroxylases responsible for C1 hydroxylation,C4 hydroxylation and C9oxidation remain unknown,which is the hotspot and difficulty in research taxol synthetic biology technology.In this study,the established systematic information of CYP450s in Taxus chinensis were obtained by mining available transcriptome data.Based on functional annotation databases and the analysis of difference expression levels,we investigated the candidate genes that relate to the taxol biosynthesis pathway.Moreover,gene cloning and functional study were used to confirm the candidate genes.The main results are listed as follows:1)Systematic identification of cytochrome P450s in Taxus chinensisBased on available transcriptome data,a total of 118 full-length and 175 partial CYP450 genes were identified in T.chinensis.Among them,only 5 genes?TcCYP725A1,TcCYP725A2,TcCYP725A4,TcCYP725A5,TcCYP725A6?were previously reported,and,TcCYP725A3,which is highly homologous?99%?with CYP725A3 in Taxus Cuspidata.The rest of the T.chinensis CYP450 genes were obtained for the first time.The 118 full-length CYP450 were named and classified by a P450 nomenclature committee.44.1%?52 genes?of the 118 full-length CYP450s are A-type and belong to 11families.The remaining 55.9%?66 genes?CYP450s are non-A-type and are distributed to18 families and 7 clans.Two new non-A-type CYP450 families,named CYP864 and CYP947,were discovered in T.chinensis.T.chinensis CYP450s were further compared with three other typical plant species,such as angiosperms Arabidopsis thaliana and Medicago sativa and the gymnosperm Picea glauca.The results showed that distinct genetic differences exist between the gymnosperm and angiosperm,and P.glauca is a good reference for T.chinensis in comparative studies of CYP450 genes.Although T.chinensis CYP450s are highly differentiated into multiple families,they still have structurally conserved features,including PFG element,PERF motif,K-helix region and I-helix region.2)Screening candidate genes for taxol biosynthesisFunctional annotation of the identified 118 full-length T.chinensis CYP450s were made.In Blast2Go database,these CYP450s were divided into three categories:cellular components,biological processes and molecular functions,and further been subdivided into 21 functional groups.These CYP450 sequences were submitted to Swissport,Nt,Nr,COG,GO,KEGG databases for further comparison,with an e-value cutoff of 1e^-5.The results showed that the vast majority of them were annotated.Among them,15 novel CYP725s?from CYP725A9 to CYP725A23?were involved in taxol biosynthesis.Further,analysis of gene differential expression was used to select screen candidate genes for taxol biosynthesis.In the 15 novel CYP725s,the expression regularity of TcCYP725A9,TcCYP725A11,TcCYP725A16,TcCYP725A20,TcCYP725A22 and TcCYP725A23 were similar to the known taxol biosynthesis hydroxylases and consistent with the rule of taxol content.Therefore,these six novel CYP725s were the most likely to be candidate hydroxylase genes involved in taxol biosynthesis.3)Cloning and function study in yeast of T.chinese TcCYP725A22 and TcCYP725A23The candidate genes TcCYP725A22,TcCYP725A23 and the positive control T2?H were amplified and then inserted into the high copy,callus induction type saccharomyces cerevisiae expression vector pESC-TRP.The results of western blot indicated that all target proteins were expressed successfully in WAT11.Taxusin with multiple potential hydroxylation sites was used as substrate to functionally screen unknown hydroxylases.The results of LC-MS indicated that T2?H could catalyse taxusin,which was consistent with previous report.The candidate hydroxylase TcCYP725A22 transformed taxusin to its hydroxyl derivatives.However,TcCYP725A23 had no obvious catalytic activity for taxusin.4)Functional study of TcCYP725A22 in Chinese taxus cellsIt is difficult to find suitable taxane substrates in the functional inquiry system of hydroxylase in vitro.Based on the analysis of taxane metabolite fingerprints of the over-expression transgenic Taxus cell lines for the candidate CYP450s,we can identify the specific CYP450 involved in taxol biosynthesis.The results showed that,in the TcCYP725A22 overexpressed cell line,expression levels of most defined hydroxylase genes for taxol biosynthesis increased;the yield of taxanes were also increased,specially,the content of H3T,decinnamoyl taxinine J,TC and TPT significantly increased.Based on the structure characteristics of these substances and the results of in vitro functional study,we can speculate that TcCYP725A22 may be a new C2 hydroxylase gene.The study established the foundation for further determine the unknown hydroxylases in taxol biosynthesis pathway.It is helpful to fully analyze the source pathway of taxol and promote the establishment of taxol synthetic biology technology.