Transcriptome Sequencing Of Cladosporium Cladosporioides MD2 And Overexpression Analysis Of Two Candidate Genes Related To Taxol Biosynthesis

Taxol is a highly effective and low toxic anticancer drug in clinic.Taxol-producing endophytic fungi are one of the important ways to produce taxol.Since the first report of taxol-producing endophytic fungi(Taxomyce andreanae)in 1993,more than 200 endophytic fungi of 55 genera have been reported to produce taxol,but the yield of taxol in fungi is low and unstable for industrial application.Based on directionally genetic modification of taxol metabolic pathway in fungi,construction of genetic engineering strains with stable and high taxol yield is one of effective ways to realize their industrial application.Screening and functional verification of genes related to taxol biosynthesis in fungi are pivotal to elucidate the molecular mechanism of taxol biosynthesis in fungi and construct high-yield engineering strains.In this study,the transcriptome information of Cladosporium cladosporioides MD2 was analyzed,the taxol synthesis pathways in C.cladosporioides MD2 was preliminarily speculated,and 49 unigenes related to taxol biosynthesis were obtained.Two candidate genes A05868 and A02976 were cloned and analyzed by bioinformatics method,their overexpression vectors and transgenic strains were constructed,the effects of these two candidate genes on taxanes biosynthesis in C.cladosporioides MD2 were analyzed by overexpression biotechnology.The main research results were as follows:1)The transcriptome of C.cladosporioides MD2 was sequenced by Illumina Hiseq 2500 high-through sequencing technology and analyzed with various bioinformatics methods.A total of 16603 unigenes were assembled with de novo Trinity,and were further annotated in NR,NT,PFAM,Swiss Prot,GO,KOG and KEGG databases,respectively.As a result,12479 unigenes were annotated with at least one database,and 1593 unigenes were annotated in all of seven databases.8425 unigenes were functionally annotated by GO database,which belonged to 57 functional groups of biological processes,cell components and molecular functions.3350 unigenes were annotated by KEGG database,and belonged to 262 KEGG metabolic pathways.On these basis,one potential taxol biosynthetic pathways in C.cladosporioides MD2 were speculated,including 9 unigenes involved in terpenoid bone biosynthetic pathway and 40 unigenes involved in taxane biosynthetic pathway.2)One candidate gene A05868 was cloned.The DNA and cDNA sequences of A05868 were 1425 bp and 1260 bp,respectively.The encoded protein of A05868 contained 419 amino acids,which had 57% identity to lysophosphatidic acid acyltransferase endophilin of Pseudocercospora fijiensis CIRAD86,and had a Bin Amphiphysin RVS(BAR)domain at the C-terminal.An over-expression vector pTFC-A05868 was further constructed and transformed into C.cladosporioides MD2.A total of 14 transgenic strains of A05868/MD2 were obtained through resistance screening.Southern blot was used to detect the copy numbers of target gene in each transgenic strains,and 8 transgenic strains with a single copy of exogenous genes were obtained.Four of them(A05868/MD2-A8?B1?B6?C1)were selected for qRT-PCR analysis,and the results showed that the expression level of A05868 in A05868/MD2-B6 was highest,about 3 times as much as the control(untransgenic)strain(5 days).HPLC analysis results showed that none of taxol,baccatin III,7-xylosyl-10-deacetylpaclitaxel and daxotere were detected in the transgenic strains and the control strain.The 10-deacetylbaccatin III yield(14.621 ± 0.186 ?g/g)and 10-deacetyltaxol yield(1.564 ± 1.025 ?g/g)in transgenic A05868/MD2-B6 were 20.195% and 51.323% lower than those in the control strain(18.321 ± 0.045 ?g/g,3.213 ± 0.063 ?g/g),respectively.These results preliminarily indicated that overexpression of A05868 inhibited the biosynthesis of 10-deacetylbaccatin III and 10-deacetyltaxol in C.cladosporioides MD2.3)The candidate gene of A02976 was cloned,and the DNA and cDNA sequences of A02976 were 1685 bp and 1518 bp,respectively.The encoding protein of A02976 contained 505 amino acids,which had 43% identity with trichothecene 3-O-acetyltransferase of Fusarium oxysporum and had a transferase domain at the C-terminal.An over-expression vector pTFC-A02976 was constructed and further transformed into C.cladosporioides MD2.A total of 14 A02976/MD2 transgenic strains were obtained through resistance screening.Southern blot was used to detect the copy numbers of target genes in each transgenic strain,and 10 transgenic strains with a single copy of exogenous genes were obtained.Four of them(A02976/MD2-A6,A8,C1,D8)were selected for qRT-PCR analysis,and the results showed that the expression level of A02976 in A02976/MD2-C1 was the highest,about 3.2 times as much as the control strain(10 days).HPLC analysis results showed that taxol,baccatin III and daxotere were not detected in the transgenic strains and the control strain.None of 10-deacetylbaccatin III and 10-deacetyltaxol was not detected in transgenic A02976/MD2-C1,but detected in the control strain.7-xylosyl-10-deacetylpaclitaxel was detected in transgenic A02976/MD2-C1,the yield of which was 4.253±0.017 ?g/g,but not detected in the control strain.These results preliminarily indicated that overexpression of candidate gene A02976 inhibited the biosynthesis of 10-deacetylbaccatin III and 10-deacetyltaxol and promoted the biosynthesis of 7-xylosyl-10-deacetylpaclitaxel in C.cladosporioides MD2.