Screening And Functional Analysis Of Enzyme Genes Related To Taxol Biosynthesis In Endophytic Fungus

Taxol is a broad-spectrum anti-cancer drug used for treating ovarian cancer,cervical cancer,and lung cancer and so on.However,the now available paclitaxel production method can not be industrialized due to low paclitaxel yield.Therefore,how to improve paclitaxel yield has become a hotpot topic and using modern molecular biology method to construct genetic engineered strain become a efficacious way for improving paclitaxel yield.Although key genes involving in paclitaxel biosynthetic pathway have been successfully cloned from Taxus cell,documents reporting the genes cloned from endophytic fungi are rare.In addition,the genetic background of the endophytic fungi(Nodulisporium sylviforme)is not clear,which adds difficult to construct a high-yield engineering strain.Based on the constructed cDNA library of taxol-producing fungus N.sylviforme HDFS4-26,primers were designed according to the cDNA sequences which had similarity with the reporting sequences of taxol synthesis related enzyme-encoded genes in GenBank in this study and target genes were cloned via PCR.Consequently,two related enzyme encoded genes were obtained by blast alignment and had 99%similarity with the deposited gene sequences,taxane-13a-hydroxylase gene respectively,in GenBank.In order to validate the gene function,prokaryotic expression vectors were constructed by linking the genes to corresponding pET32a vector,and western blotting was carried out to confirm the expression of the target genes.The results showed that the taxane-13?-hydroxylase gene had full open reading frame and the target proteins were successfully expressed,this laid a good foundation for subsequent genetic engineering strain constructing.In addition,binary expression vector pBI 121-43 constructed by the laboratory,after double digestion connection,was linked with the acquired genes and the genes obtained at earlier stage,then,the obtained recombinant plasmid was transferred to strain HDFS4-26 mediated by Agrobacterium tumefaciens.The results showed that the recombinant plasmid was successfully introduced into the endophytic fungus HDFS4-26,and five paclitaxel engineering strains were obtained,they respectively were taxane-13a-hydroxylase gene contained engineering strain(HD-1),GGPP synthase gene contained engineering strain(HD-2),taxol diene 5a-ol-acetyl transport enzyme gene contained engineering strain(HD-3),and taxol-diene-5a-hydroxylase gene contained engineering strain(HD-4).Using thin layer chromatography(TLC)to quantitatively determine the constructed engineering strain,TLC results showed that the five obtained strains could be seen characteristic blue spot in the same position with taxol,while the blue spots of the strain HD1-4 was larger and darker than HDFS4-26.The study preliminarily demonstrated that GGPP synthase,taxane-13?-hydroxylase,taxol diene 5a-ol-acetyl transport enzyme and taxol-diene-5a-hydroxylase were the related enzymes involving the biosynthetic pathway of taxol.This study provided related enzyme genes for building high paclitaxel yield genetically engineered strain of endophytic fungi,as well as laid a solid foundation for constructing high paclitaxel yield strain,which would facilitate the realization of industrialization of taxol production by endophytic fungi.