Influence Of Overexpressing Of Candidate Genes A00981 And A03231 On Taxol Productivity Of Cladosporium Cladosporioides MD2

Taxol is a widely used anticancer drug in clinic.Taxol-producing endophytes are one of valuable natural resources for taxol production.Currently,more than 40 fungal genera about 200 endophytic fungi had been reported to produce taxol,however low and unstable taxol productivity in fungi restricted their industrial application.Directional genetic modification of taxol metabolic pathway in fungal is one of the main ways to obtain the genetic engineering strain with high and stable yield of taxol and meet its industrial application.Cloning of taxol-related genes from fungi and clarification of their function are prerequisites for engineered fungal strains with high taxol productivity.In this study,candidate genes were screened based on the genome and transcriptome sequencing results of one taxol-producing fungus Cladosporium cladosporioides MD2.The full-length DNA sequences of five candidate genes were cloned,and the structure and function of the five candidate genes were analyzed by bioinformatics methods.Transgenic strains with overexpression of two candidate genes A00981 and A03231 were constructed,and the effects of overexpressing candidate genes on taxanes biosynthesis of C.cladosporioides MD2 were analyzed,respectively.These results laid a ground for further function research of the candidate genes A00981 and A03231,provided references for the molecular mechanism research of taxol biosynthesis in C.cladosporioides MD2,and produced potential gene resources for construction of engineering strains with high-yield taxol.The main results of this study were as follows:(1)Five candidate genes were screened based on the genome and transcriptome sequencing of C.cladosporioides MD2,and the full-length DNA sequences of them were cloned.Through comparing of the full-length DNA sequences and the CDS sequences of candidate genes,none of introns was detected in the DNA sequences of the candidate genes A12109 and A11767,one intron was detected in the DNA sequences of A10817 and A00981 harbored,and two introns was detected in the DNA sequence of A11450.(2)The bioinformatics method was used to predict the function of the five candidate genes.There was no protein in the database has an obvious identity with the encoding protein of A12109,indicating that A12109 might be a new gene.The encoding protein of A11767 had a highest identity(37%)to the zinc ion binding of Ascochyta rabiei;and the encoding protein of A00981 had a highest identity(71%)to acyl-CoA dehydrogenase of Blastomyces gilchristii SLH14081.In addition,the encoding proteins of A10817 and A11450 had 75% identity and 59% identity to putative aryl-alcohol dehydrogenase AAD1 of Rachicladosporium antarcticum and S-2-hydroxy-acid oxidase of Aureobasidium melanogenum CBS 110374,respectively.(3)The overexpression vector of candidate gene A00981 was constructed and transformed in C.cladosporioides MD2 by Agrobacterium tumefaciens mediated transformation method,and a total of thirteen A00981-transgenic strains were obtained.Southern blot results showed that the exogenous genes of five transgenic strains(ZB,ZC,ZF,ZH and ZL)were integrated by single copy.QRT-PCR results showed that the highest expression level of candidate gene A00981 in transgenic strain ZC was two folds of that in control strain(non-transgenic strain).HPLC analysis showed that there was no significant change in the yield of taxol(1.05±0.02 ?g/mL),baccatin ?(2.69±0.05 ?g/mL)and 10-deacetyl baccatin ?(2.46±0.05 ?g/mL)of transgenic strain ZC and that(1.06±0.02 ?g/mL taxol,2.62±0.05 ?g/mL baccatin ? and 2.55±0.05 ?g/mL)of the control strain,respevtively,indicating that overexpressing of candidate gene A00981 might have no significant effect on the synthesis of taxol,baccatin ? and 10-deacetyl baccatin ? in C.cladosporioides MD2.(4)The overexpression vector of candidate gene A03231 was constructed and transformed into C.cladosporioides MD2,and nine A03231-transgenic strains were obtained.Southern blot results showed that the exogenous genes of five transgenic strains(XA,XF,XG,XH and XI)were integrated into the host genome by single copy.QRT-PCR results showed that the highest expression level of candidate gene A03231 in transgenic strain XH was two folds of that in control strain.HPLC analysis showed that the yield of taxol(0.99±0.02 ?g/mL)and baccatin ?(2.64±0.05 ?g/mL)did not significantly change in transgenic strain XH in comparison to that in the control strain,but the yield of 10-deacetyl baccatin ?(1.59±0.03 ?g/mL)in transgenic strain XH was lower than that in control strain,indicating that overexpressing of candidate gene A03231 had no significant effect on the synthesis of taxol and baccatin ?,but reduced 10-deacetyl baccatin ? yield in C.cladosporioides MD2.