| Arthrobotrys oligospora can produce a multitude of secondary metabolites, however, few studies about secondary metabolite biosynthetic genes in the fungi have been reported. Based on bioinformatic analysis of the whole genomic of A. oligospora, seven biosynthetic genes were screened out as targets in the study, which including a putative type I polyketide synthase (PKS) gene AOL_s00043g828, a putative type Ⅲ PKS gene AOL_s00043g287, a putative aromatic prenyltransferases gene AOL_s00043g400 and 4 putative P450 genes AOL_s00043g381, AOL_s00078g577, AOL_s00097g233 and AOL_s00097g384. A combination of biology technology and genetic manipulation was applied to knock out the target genes, and 7mutants were obtained. Subsequently, the mutant strains were compared with the wild type strain in colony morphology, growth rate, sporulation, nematicidal activity, secondary metabolite profiles and gene expression levels.The disruption of P450 genes AOL_s00043g381 and AOL_s00078g577, respectively, led to a significant difference in hyphal growth and sporulation. Growth rate of both mutant strains were higher than wild-type strain, the mutant ΔAOL_s00043g381 increased by from 24% to 88% when incubated on PDA plate, and the mutant ΔAOL_s00078g577 increased by from 11% to 22% incubated on YMA plate. The mutant AAOL_s00043g381 showed less dense mycelia than wild-type strain, however, the mutant AAOL_s00043g381 displayed thicker colonies. Sporulation of the mutant ΔAOL_s00043g381 decreased by 2.5 fold compared with wild-type strain, while the mutant ΔAOL_s00078g577 increased by 5.6 fold. Furthermore, The mutant ΔAOL_s00078g577 strain increased in the trap formation by 13,4,2.3 and 3 times at 12,18,24 and 36 hours, respectively, meanwhile, the nematicidal activity increased by 60,9.6 and 2.1 times at 12,18 and 24 hours, respectively, significantly higher than those of the wild-type strain. However, there were no obvious difference between phenotypes and nematicidal activity either the wild-type strain or another mutant strains.Ethyl acetate extracts of secondary metabolites in cultural broths from wild-type and all mutant strains were analyzed. Comparison of the HPLC profiles of type 1 PKS gene mutant ΔAOL_s00043g828 and aromatic prenyltransferases gene mutant ΔAOL_s00043g400 strains with that of wild-type strain revealed that 6 peaks showed higher areas in both mutant stains. Among them.4 and 3were remarkable higher in the mutant ΔAOL_s00043g828 and ΔAOL_s00043g400 strains respectively. In addition, 4 peaks missed and 5 peak values becamed smaller in mutant AAOL_s00043g400. GC-MS analysis showed that the peaks in the mutant ΔAOL_s00043g828 strain were more abundant than the wild-type strain, there were 7 peaks present only in the mutant strain while 3 only in the wild-type strain, and 3 peaks were significantly higher in areas than those of the wild-type strain. Meanwhile, GC-MS analysis of the mutant ΔAOL_s00043g400 and wild-type strains showed that 7 peaks present only in the wide-type strain. Among them,5 were determined as compounds with isopentenyl. And 7 peaks present only in the mutant strain, among them 2 compounds were determined as acrylamide and a compound with isopentenyl respectively. In addition, one compound that increased obviously in mutant strain was determined as a amino acid. GC-MS analysis also showed that the main difference compounds between the wide-type strain and the P450 mutant ΔAOL_s00043g381 and AAOL_s00097g384 strains were long-chain alkane and long-chain fatty esters. However, the metabolites profile of ΔAOL_s00043g287, ΔAOL_s00078g577 and AAOL_s00097g233 strains showed no difference with wide-type strain.Differential gene expression analysis showed that, in comparison to the wild type strain, there were 151 differentially expressed genes in type 1 PKS gene mutant ΔAOL_s00043g828,57 and 94 genes were up and down regulation respectively, meanwhile,1231 differentially expressed genes screened in P450 mutant ΔAOL_s00097g384,488 were up-regulation and 743 were down- regulation, which showed much more than the mutant ΔAOL_s00043g828.According to the the results, we found that among the 7 biosynthetic genes in A. oligospora in this study, including the type I PKS gene, the aromatic prenyltransferases gene and some of P450 genes, P450 gene showed effects the phenotypes and the nematicidal activity, and might play a very important role in strain development and secondary metabolites. For examples, AOL_s00043g381 and AOL_s00078g577 may play roles in colony growth and sporulation, AOL_s00043g381 and AOL_s00097g384 can affect the secondary metabolic process in some degree, furthermore, the disruption of AOL_s00097g384 led to more than one thousand of differentially expressed genes. In particular, AOL_s00078g577 can significantly increase trap formation and nematicidal activity. All that mentioned above suggested it’s for the first time that P450 genes were found to be involved in trap formation and nematicidal activity in fungi. In addition, the aromatic prenyltransferases gene AOL_s00043g400 may play a role in the process of isopentenyl transfer in the biosynthesis of indole type compounds. The type Ⅲ PKS gene AOL_s00043g287 and P450 gene AOL_s00097g233 perhaps be cryptic or silent under our laboratory conditions.In conclusion, although some of biosynthetic genes showed effects on the phenotypes, nematicidal activity and secondary metabolites in A. oligospora, the mechanism how these genes works were still unknown. This article provided not only valuable mutant strains for exploiting the diversity of secondary metabolites and genes but also an insight for further research on the underlying mechanism of the biosynthetic genes in A. oligospora. |
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