Genome Analysis Of Bio-Control Fungus Purpureocillium Lilacinum And The Biosynthesis Of Antibiotic Leucinostatins

Plant parasitic nematodes with wide host range cause huge yields and economic losses around the world annually. Biological control by fungi is increasingly popular due to nematicides’ risk against environment and human health. One of the most promising and commercialized agents, Purpureocillium lilacinum was evaluated the bio-control activity against plant nematodes in a number of studies. In addition, P. lilacinum has been reported to effectively control of Fusarium wilt, cotton aphid, leaf-cutting ant, and so on. Opportunistic infection would happen when nematode eggs encounter P. lilacinum. So a variety of hydrolytic enzymes and protease such as chitinase and serine protease play important roles in the infection by degrading nematode eggshells. P. lilacinum has strong ability for encoding secondary metabolites, which contribute a lot for its bio-control function. Leucinostatins is a family of lipopeptide antibiotics isolated from P. lilacinum, and was found significantly suppress against tumour cell, bacteria and fungi. Unfortunately, little was known about its biosynthetic genes and pathway to data. The purpose of this research is to analyze the genome features of P. lilacinum, identify the biosynthetic gene cluster of leucinostatins and depict its biosynthetic pathway, by genome sequence of two P. lilacinum strains PLBJ-1 and PLFJ-1.To carry out the research of gene function, we developed the PEG mediated protoplast transformation system. PLBJ-1 was marked with GFP and the GFP-marked P. lilacinum throw out green fluorescence by UV excitation with stability after successive transfer culture.PLBJ-1 was assembled into 144 scaffolds, with a total size of 38.14 Mb and PLFJ-1 was assembled into 163 scaffolds with a 38.53 Mb total size. A total of 11773 and 11763 gene models were predicted in both genome, respectively. About 6.07% and 6.00% repeat sequences were identified in PLBJ-1 and PLFJ-1. BLASTN analysis was performed between two genome, demonstrating that 88.12% of PLBJ-1 genome and 88.60% of PLFJ-1 genome shared high synteny. A detailed examination of the CAZymes and proteases of P.lilacinum were performed and quite a lot of genes were identified. The inferred phylogeny illustrated that the T. inflatum and T. ophioglossoides were most related to P. lilacinum. The three nematode parasitic fungi P. chlamydosporium, H. minnesotensis and P. lilacinum cluster with insect pathogen respectively, implied that nematode and insect pathogens may share a common ancestor. In total, 13 PKSs, 10 NRPSs, 2 PKS-like enzyme, 10 NRPS-like enzymes, 1 DMATS and 4 TS and 1 PKS-NRPS hybrid were identified in PLBJ-1 genome.The NRPS VFPBJ₀2539（lcsA） was determined as the only plausible candidate gene based on the structure of lecuinostatins and the secondary metabolite encoding genes in P. lilacinum genome. To verify the associations between putative lcsA and leucinostatins, a lcsA deletion mutant（ΔlcsA） was obtained by double homologous deletion. P. lilacinum（PLBJ-1） wild type and ΔlcsA mutant were cultured in PDB medium for 8 days and the ethyl acetate extracts were analyzed by HPLC-MS. Comparison with authentic standard confirmed that the missing compounds of ΔlcsA mutant were indeed leucinostatin A and B. These results demonstrated its essential role of lcsA for biosynthesis of the leucinostatins as predicted.We found that P. lilacinum produced leucinostatin A and B when cultured with lab-made PDB but not produced leucinostatins cultured in PDB-BD. qRT-PCR analysis was conducted to compare the expression pattern of genes franking lcsA when PLBJ-1 grown in the two medium, which identified 20 genes were co-expressed. To further confirming, RNA-Seq under leucinostatins inducing condition and expression patterns of genes franking the cluster in wild type PLBJ-1 and ΔlcsA mutant growing in leucinostatins inducing medium were analyzed. Thus, a serial of genes from VFPBJ₀2521 to VFPBJ₀2539, designed as lcsA to lcsT, including core enzymes, modifying enzymes and transporter enzymes coded for the biosynthesis of leucionostatins.PKS, ligase and thioesterase were reported to play important roles in the biosynthesis of lipopeptides. To figure out whether these enzymes play critical roles in the biosynthesis pathway of leucinostatins, we disrupted PKS（lcs C）, ligase（lcsD） and thioesterase（lcsE） in the cluster by homologous recombination. After culturing the fungi in PDB medium and comparative analysis the extracts by HPLC-MS with PLBJ-1 wild type and ΔlcsA, the leucinostatin A and B were disappeared in ΔlcsC, ΔlcsD and ΔlcsE as in ΔlcsA. The overexpression of lcs F indicated that pathway specific transcriptional factor lcs F was capable of regulating the entire gene cluster and biosynthesis of leucinostatins, and enhanced the production of leucinostatins. Disruption mutants allowed us to propose a putative biosynthetic pathway: A polyketide synthase was responsible for the assembly of 4-methylhex-2-enoic acid at N terminal, and an acyl-AMP ligase lcsD offering a conceivable route for connecting the fatty acid and peptide encoding by lcsA. In the biosynthesis of leucinostatin A and B, intermediate metabolite went through transamination, hydroxylation and vary extend of methylation.P. infestans and P. capsici showed growth inhibition when confronting incubation with wild type P. lilacinum, and a stronger inhibition effect on OE::lcs F, while inhibition was disappeared when they confronting incubation with ΔlcsA. The results indicated P. lilacinum inhibited growth of some oomycetes depended on the existense of leucinostatin A and B.In conclusion, the genome features of P. lilacinum were identified by sequencing of two P. lilacinum strains and comparatively analyzing with other fungi. The function of some secondary metabolite related genes were identified by homologous deletion. The biosynthetic gene of leucinostatins was identified and the biosynthetic pathway was depicted. Overexpression of lcs F was capable of regulating the entire gene cluster and enhancing the production of leucinostatins. In addition, confronting incubation of P. lilacinum and oomycetes indicated P. lilacinum inhibited growth of some oomycetes depended on the existense of leucinostatin A and B.