Study On The Function Of A P450Gene In Arthrobotrys Oligospora And Construction Of Two Gene Knock-out Cassettes For The PKS/NRPS Hybrid Biosynthetic Gene In Talaromyces Thermophilus

This article mainly includes two parts of the research. For the first part, a mutant was constructed for cytochrome P450gene AOL_s00110g222in nematode-traping fungus, Arthrobotrys oligospora. Themorphological traits and secondary metabolism profile of mutant AAOL_s00110g222were compared with that of the wide-type strain for the determination of the biological function of target gene AOL_s00110g222. For the second part, through homologous recombination system in yeast, two gene knock-out cassettes were constructed for the MT and NRPS domains of putative PKS/NRPS hybrid genes invloved in the biosynthesis of PKS/NRPS hybrid secondary metabolite thermolides, in the thermophilic fungus Talaromyces thermophiles.Analysis of the whole genomic information of A. oligospora by antiSMASH2.0and BLAST for potential biosynthetic gene clusters of secondary metabolites showed25biosynthetic gene clusters. The pisatin demethylase-like cytochrome P450gene AOL_s00110g222in the No.20gene cluster was chosen as the target gene. The phylogenetic analysis of the AOL_s00110g222suggested that the identity rate of the protein is47%to the pisatin demethylase in Pyronema omphalodes and82%to the hypothetic protein H072₇405in nematode-trapping fungus Dactylellinahaptotyla. The mutant△AOL_s00110g222was gained through methods of homologous double-crossover recombination.The mutant was compared with the wild-type in aspects of mycelial growth, the morphology of spore and hyphae, conidiation and germination, trap formationand predatory ability. Results indicated that the mutant△AOL_s00110g222displayed significant inhibition in the formation of trap formation and predatory ability, respectively. In the period of36hours after induction of nematode, the trap number and predatory rate of the mutant averagely decreased by36.0%and51.0%, respectively, compared to that of the wild-type strain. The result indicated that the targeted P450gene might be involved in the formation of a three-dimensional network and regulation of predatory process in A. oligospora.The GC-MS analysis of ethyl acetate extracts of PDB fermentation broths of the mutant and the wild-type strain showed that the metabolite profile of the mutant was notably different from that of the wild type strain.18peak values were present only in the profile of the wide-type strain. According to the comparison with those in database,15compounds were determined, including4phenyl compounds,5polyring aromatics,2indoles and4long-chain lipids. The GC-MS analysis also showed that14peak values were present only in the profile of the mutant AAOL_s110g222.10compounds were determined, inclucing1phenyl compound,1benzofuran,3long-chain aliphatic compounds,2cyclic peptide,2terpenoids, and1sterol. Also,6peak values of the mutant△AOL_s110g222were significantly different from that of the wide-type strain.4compounds were determined, including2phenyl compounds （e.g. phenylacetic acid）,1terpenoid, and1long-chain saturated fatty acid. However, the mutant and the wild-type showed no significant difference in the HPLC-DAD results. Especially, the mutant still produced arthrosporols. According to those results that the mutant lack alkyl fatty acids as well as unsaturated fatty acids, the targeted P450may be involved in the post-modification of alkyl fatty acid.In the study of A. oligospora, the research showed that the P450gene AOL_s110g222may be involved in biosynthesis of alkyl fatty acids, and in the formation of predatory organ, resulting in defect in predatory capability.The two gene knock-out cassettes for MT and NRPS domains in theputative PKS/NRPS gene cluster for the biosynthesis of thermolidesin T. thermophiles, were successfully constructed.