Study On The Functions Of 7 PKS Genes In Thermomyces Fungi

Fungi produce a wide variety of structurally diverse and biologically active polyketide compounds,exploring fungal potential polyketides and their synthesis gene clusters are valuable and significant in the study of fungal active natural products.Thermophilic fungi are the only eukaryotic organisms which can growth under high temperature conditions(45-50℃).It was reported previously by our laboratory that Thermophiles dupontii and its sister thermophilic fungi Themomyces lanuginosus 200065 could produce a novel class of potent nematocides possessing a 13-membered lactam-bearing macrolactone,thennolides.To further study the biosynthetic pathway of thennolides,we used bioinformatics methods to predict and analyze two thermophilic fungal genomes.Five polyketide synthase genes of T.dupontii were screened out as target genes,PKS12(consisting of one NRPS and one type 1 PKS),PKS25(consisting of a PKS-NRPS hybrid gene),PKS18,PKS30,and PKS31 are type 1 PKS.There are 6 PKS genes in T.lanuginosus,two PKS genes L-PKS12 and L-PKS25 are homologous to PKS12 and PKS25,which were also screened out as target genes.The biosynthesis function and biological significance of these PKS were researched based on the gene knock-out technology,the comparison results of phenotype,stress resistance,metabolic profile between the mutants and wild-type strain.The main results are as follows:1.This was for the first time that PEG-mediated protoplast transformation method was applied to T.dupontii,and a stable and efficient gene replacement system was successfully established.Five PKS gene knockout mutants,△PKS12、△PKS18、△PKS25、△PKS30、△PKS31 of T.dupontii,two PKS gene knockout mutants,△L-PKS12、△L-PKS25 of T.lanuginosus were obtained by this method.2.Compared with the wild-type strain,△PKS30 displayed significant changes in colony color,the colony of APKS30 on PDA plates was white,while the wild type strain showed brown color.On the other hand,the spore yield of APKS30 was significantly reduced 2.6 times,and the germination rate was also significantly reduced 10.8%.There were no significant differences in △PKS12、△PKS18、△PKS25、△PKS31.The results of stress resistance experiments showed that the knockout of these five PKS genes had no obvious influence on ability of the the antioxidation(H2O2),permeation resistance(NaCl).and anti-ultraviolet radiation(λ =185nm)in T.dupontii.However,in the cell wall synthesis interference(SDS,Congo red)experiment,the aerial hyphae of the mutant strains APKS12 and APKS18 displayed more dense than the wild strain on PDA medium containing 0.02%SDS.The dense mycelium indicated that knock-out of the genes PKS12 and PKS18 might increase the ability of T.dupontii to resist 0.02%SDS.On the other hand,the colony growth diameter of △PKS12,△PKS18,△PKS25 also increased compared with the wild type strain on PDA medium containing 0.2 mg/mL Congo red.Further analysis based on RNA-seq data revealed that,compared to wild-type strain,the expression levels of the 7 genes involved in cell wall synthesis of APKS12 were up-regulated.In APKS18,4 genes of these 7 genes were up-regulated.and the expression up-regulation levels were higher than the down-regulation level of these 7 genes.It indicated that the cell wall synthesis ability of APKS12 and APKS18 were improved compared with the wild type strain.3.Compared with wild-type strain,the LC-MS profiles of the ethyl acetate extracts of the YGP broth from △PKS12、△L-PKS12 indicated that several natural products were significant loss,especially the total loss of thermolides,which revealed the PKS12.L-PKS12 involved in the biosynthesis of macrolactones thermolides.The LC-MS profiles of the ethyl acetate extracts of the PDB broth from APKS30 indicated that disruption of PKS30 led to a total loss of carviolin(roseo-purpurin)A,which revealed the PKS30 involved in the biosynthesis of carviolin(roseo-purpurin)A.The LC-MS results of other three PKS mutant strains showed that there were relatively less differential peaks of them,which might be due to the low expression level of these genes or the restriction of fermentation conditions.The biosynthesis function of△PKS18、△PKS25、△PKS31 still remain to be further studied.4.RNA-Seq analysis of five PKS mutants revealed that there were more down-regulated differential express genes(DEGs)than up-regulated DEGs of all mutants(>1000,the down-regulated DEGs was 1.5 times the number of the up-regulated DEGs in △PKS30).△PKS12 had the most DEGs(2655 genes)among the five PKS mutants,while △PKS25(938 genes)and △PKS31(929 genes)had relatively less DEGs.GO enrichment and KEGG pathway enrichment of common DEGs in five PKS mutants showed that these DEGs were mainly enriched in the metabolic processes and related enzymes activities,cell structure and composition.The pathways and biosynthesis processes of these enriched DEGs participated,interactions,and their effects on other biological processes still remain to be further studied.