| This article investigates on five biosynthetic genes in nematode-trapping fungus Arthrobotrys oligospora, including a Type I polyketide synthetase (PKS) gene AOL_s00079g496, a terpenoid synthetase (TPS) gene AOL_s00079g224 and three cytochrome P450 genes AOL_s00079g225, AOL_s00043g740 and AOL_s00210g57. Six mutants with the disruption of these genes respectively, were gained through methods of homologous double-crossover recombination. The differences among mycelial morphology, conidiation, spore germination, trap formation, infection capability and secondary metabolisms were compared between six mutants and wide-type strain.Phenotype analysis revealed that the six mutants showed changes to varying de-grees compared with wild-type strain, and the disruption of PKS gene AOL_s00079g496, P450 genes AOL_s00079g225 and AOL_s00210g57, respectively led to significant differences in phenotype. The three mutants growed more fluffy aerial hypha on PDA media compared with the wild-type strain, the mutant △AOL_s00079g496-KS showed significant difference among them, and other mutants showed no obvious difference. The mutant △AOL_s00079g496-KS and △AOL_s00079g496-KR incuba-ted on plate of rich nutrient medium TYGA displayed lower growth rates than wild-type strain, another TPS gene mutant △AOL_s00079g224 incubated on plate of poor nutrition medium CMA displayed a significant reduction in colony diameter, and other P450 gene mutants AOL_s00079g225, AOL_s00043g740 and AOL_s00210g57 showed no difference. All the mutants except mutant △.OL_s00210g57 showed higher conidial yields than the wild-type strain ranging from 58% to 300%, with the mutant strain △AOL_s00079g224 as the highest. All the six mutants showed reduced spore germination rates compared with wild-type strain, especially within 2 h with range between 58% and 300% with the TPS gene mutant △AOL_s00079g224 as the most significant. The mutant △AOL_s00079g225 strain formed more adhesive networks than the wild-type strain after the nematodes were added, and the trap numbers of △AOL_s00079g225 increased respectively by 49%,35%,20% at 12,24, 36 hours. The other mutants did not show obvious difference in trap number. Furthermore, compared with wild-type strain, the mutants AAOL_s00079g496-KS and △AOL_s00079g496-KR showed reduced infection capability by 41% and 32%, respectively, while the mutants △AOL_s00079g225 and △AOL_s00210g57 showed increased capability by 200% and 300%, respectively. The other mutants showed no obvious difference in nematode-catching capability.Analysis of HPLC profiles revealed that knocking out the KS domain of PKS gene AOL_s00079g496 led to the most obvious difference in the profile of secondary metabolites in A. oligospora. In comparison to the wild-type strain, there were 8 peaks present only in the HPLC spectrum of the mutant △AOL_s00079g496-KS strain while five only in the wild-type strain, and 4 peaks changing significantly in area values. Three P450 gene mutants showed main changes in area values in its HPLC spectrum. The mutant AAOL_s00079g225 displayed seven cvarying peaks and the mutants △AOL_s00210g57 and △AOL_s00043g740 displayed eleven peaks in their HPLC spectra. GC-MS analysis revealed that six terpenoid type compounds changed in the P450 gene mutant AOL_s00079g225 compared to the wildtype strain, and nine aliphatic hydrocarbons and five terpene compounds changed in the the P450 gene mutant AOL_s00043g740. In addition, the HPLC profiles of the other PKS gene mutant AOL_s00079g496, TPS gene mutant AOL_s00079g224 and P450 gene mutant AOL_s000210g57 remains unchanged. It is worth noting that the compound 5-methyl-2-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-l-yl)benzene-1,3-diol was detected in all the cultural broths of three P450 mutants. The compound had a similar carbon skeleton of Arthrobotrisins, it is likely that the three P450 gene may be associated with the biosynthesis of Arthrosporols.Among the three different types of biosynthesis genes in A. oligospora, the disruption of type I PKS gene AOL_s00079g496 and P450 gene AOL_s00079g225, AOL_s00210g57 led to significant changes in morphology in the mutants, and the disruption of type I PKS gene AOL_s00079g496 and TPS gene AOL_s00079g224 led to obvious changes in the chemical profiles. In conclusion, the PKS gene AOL_s00079g496 played a significant role in not only the morphological traits but also secondary metabolisms, and the P450 genes AOL_s00079g225 and AOL_s00210g57 mainly displayed impact on the fungal morphology. Our result showed that some biosynthesis genes were correlated with phenotypes, infection capability nematicidal activity and secondary metabolites in A. oligospora. This article primarily revealed that Type I PKS and P450 genes could be involved in regulating the fungal mycelial morphology, trap formation and infection capability in nematode-trapping fungi. |
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