Cloning And Expression Of Paclitaxel Microbial Synthesis Pathway Related Enzyme Gene

Taxol is a kind of diterpene compounds with considerable antitumor effect.It was found from yew trees and has currently been widely used in many kinds of cancer,such as ovarian cancer,cervical cancer,breast cancer etc.And the therapeutic effect of taxol is obviously.Taxol was mainly acquired by a few methods,including yew planting,chemical synthesis,cell culture,microorganism fermentation etc.Pharmaceutical shortage is a bottleneck problem that restricts the widely clinical application of taxol,And this has been an hot issue in the area.Using genetic engineering methods to construct high-yielding taxol strains is expected to significantly improve the taxol production in a short time,but the genetic background of taxol-yielding fungi is still indistinct today,therefore,it's essential to research the metabolic pathway and correlative enzyme of taxol biosynthesis in microorganism.In this study,we used strain HDF-68 which was one kind of endophytic fungi engineering strain isolated from cuspidata as our starting microorganism.some pairs of different primers were synthesized,they were designed by the gene sequences that have been published in GenBank.The genes of correlative enzyme on taxol biosynthesis pathway in HDF-68 were cloned by RT-PCR technology.The results showed that full lenth cDNA sequence of GGPP synthase,taxadiene-5 alpha hydroxylase,taxadienol acetyl transperase and taxane 10?-hydroxylase were successfully cloned.Meanwhile,Blast analysis results showed that the homology of the four cloned cDNA sequences and the genes in GenBank reach to 99%-100%.The cloned gene sequences were double digested by related restriction enzyme,and were linked to pET28a vector with the same restriction enzyme sites.Then the recombinants were transformed into E.coli BL21(DE3)to research their expression profiling.Recombinants were induced in culture-medium with 0.05mmol/L IPTG.,and their optimum inducing time points was studied during the experiment.SDS-PAGE analysis showed that three cloned genes have highly expressed successfully,including GGPP synthase gene,taxadiene-5 alpha hydroxylase gene and taxadienol acetyl transperase gene.The expressed form of protein in the recombinants was studied as well.According to SDS-PAGE results,GGPP synthase gene and taxadiene-5 alpha hydroxylase gene have expressed soluble protein.Meanwhile,the properties and structures of the expressed proteins were forecasted by bioinformation methods,which will establish theoretical basis for further search.Besides,three cloned genes were connected to pBI121-43 binary expression vector,and the constructed vectors were transformed in HDF-68 by means of ATMT methods.Then a kind of special plate solid medium was utilized to preliminarily screen the positive transformants.After extracting genome DNA of the positive transformants,PCR identification was carried out to identify the transformants.The positive transformant strains were cultivated in S-7 fermented liquid medium for 12 days,after cultivation ended,the taxol in the fermentation broth were retrieved and purified for quatitive and quantitive analysis by TLC and HPLC methods.This study established a solid foundation for isolating more taxol biosynthesis related genes from endophytic fungi and for constructing high-yield gene engineering strain.Meanwhile,it also provided us with theoretical evidence for clarifying taxol biosynthesis pathway in microorganism.