Synthetic Biology Research Of Taxol Intermediate Taxadiene

Taxol is an anti-cancer drug that is in great demand,but its source is very limit and is hard to meet the commercial requirements.The emergence of synthetic biology provides a new opportunity for solving the problem of supply and demand contradiction of taxol.However,a number of hydroxylase genes in the taxol synthesis pathway have not yet been determined because of the lack of substrate,limiting the progress of the use of synthetic biology to produce paclitaxel.In this study,in order to provide substrate for the subsequent study of hydroxylases and accelerate the process of paclitaxel biosynthesis by synthetic biology,we plan to construct the synthetic pathway of paclitaxel intermediate taxadiene in the eukaryotic model organism Saccharomyces cerevisiae.Main results are as follows:(1)Cloning of genes needed in the study.In the study,thmgr,erg20,bts1,upc2.1 genes from Saccharomyces cerevisiae,ggpps,tTS,hmgr,fpps genes from Taxus chinensis,promoter tdh3 p,tef1p,terminator CYC1 t were cloned.And then these gene coding protein were analyzed with various bioinformatics softwares.(2)Construction of the engineering strains.First,genes of each module were constructed into the expression vectors.Each module was in an independent expression cassette,containing separate promoter and terminator.Yeast endogenous gene modules used integrated plasmids,exogenous gene modules used free plasmids.Through combinational expression of these expression vectors,five engineering strains that may produce taxadiene were constructed.After that,PCR,qRT-PCR and western blot were used to test the genes’ expression.(3)Fermentation characteristic analysis of engineering strains.The results showed that,the growth period of each engineering strains were long and beneficial to the accumulation of secondary metabolites,the introduction of taxadiene synthesis module had a certain inhibitory effect on the growth of engineering strains,and increasing the expression of tHMGR could alleviate the growth inhibition of engineering strains,the introduction of taxadiene synthesis module increased the squalene production greatly,and inhibition of the tributary pathway was beneficial for the acquisition of the target product.In conclusion,our study has successfully cloned the enzyme gene,promoter,terminator and transcription factor needed to construct the taxadiene pathway in Saccharomyces cerevisiae,and constructed five engineering strains,Although the synthetic path of the target product needs to be further optimized,the construction of engineering strains still lay a foundation of the constructin of taxadiene pathway in the Saccharomyces cerevisiae.