The Identification And Expression Analysis Of The Non-reductive Polyketone Compound Synthetic Gene Cluster Of Purpureocillium Lilacinum

Purpureocillium lilacinum is a broad spectrum of biocontrol fungi.It can secrete hydro-lases and secondary metabolites,and it has a good preventive effect on plant diseases and in-sect pests.Polyketide is a common secondary metabolite and synthesized by decarboxylation condensation of acetic acid,malonic acid or butyric acid.According to the degree of reduction ofβ-keto groups by polyketide synthase（PKS）,PKS is divided into NR-PKS,PR-PKS and HR-PKS.NR-PKS can catalyze the synthesis of monocyclic or polycyclic aromatic polyketides.Previous genomic data showed that VFPBJ₀5021,VFPBJ₀9342,VFPBJ₀9755,and VFPBJ₁0843 genes were four NR-PKS genes in Purpureocillium lilacinum.Acremoxanthone C is a polyketide produced by Purpureocillium lilacinum,which is a xanthone-hetero-dimer and belongs to calmodulin inhibitor that can be used to study cal-cium-mediated signal transduction way and play an important role in the discovery of new drugs.At present,there is no report on the molecular biology of Acremoxanthone C.This study,Purpureocillium lilacinum Beijing（BJ）strain was used as experimental material,the transcriptome datas were analyzed,then the expression of four NR-PKS gene clusters were verified by qPCR.The VFPBJ₀9756 gene was over-expressed,then the products of wild-type and over-expressing strains were compared.The specific results are as follows:The transcriptomes of VFPBJ₀5021,VFPBJ₀9342,VFPBJ₀9755,and VFPBJ₁0843gene clusters were analyzed,then total RNA was extracted and reverse transcribed into cDNA.qPCR was performed to verify the expression of these four gene clusters.The results showed that the transcriptome expression trends of the four gene clusters of VFPBJ₀5021,VFPBJ₀9342,VFPBJ₀9755,and VFPBJ₁0843 were basically consistent with the qPCR expression trends.The VFPBJ₀5021,VFPBJ₀9342,and VFPBJ₀9755 gene clusters had a clustered co-expression trend,the gene expression in the clusters was higher than that in the extra-cluster.However,the genes expression in the VFPBJ₁0843 gene cluster were very low,especially the synthetase gene in the cluster was hardly expressed,but the extra-cluster gene had a high expression level.Therefore,it was preliminarily determined that VFPBJ₀5021,VFPBJ₀9342,VFPBJ₀9755 of gene clusters might be the candidate synthetases of Acremoxanthone C.Knockout vector construction was performed on the VFPBJ₀5021,VFPBJ₀9342,and VFPBJ₀9755 genes.There were VFPBJ₁1784 transcription factor in VFPBJ₀9342 gene cluster,VFPBJ₁1786 and VFPBJ₀9756 transcription factor in VFPBJ₀9755 gene cluster,and VFPBJ₁1784,VFPBJ₁1786 and VFPBJ₀9756 genes overexpression vectors were constructed.Knockout vectors（VFPBJ₀5021,VFPBJ₀9342,and VFPBJ₀9755 genes）and overexpression vectors（VFPBJ₁1784,VFPBJ₁1786 and VFPBJ₀9756 genes）were trans-formed with protoplasts.The protoplasts quality were poor due to high concentration spores of Purpureocillium lilacinum and long incubation times,and time was limited,which resulted in only VFPBJ₀9756 overexpressed transformants was obtained.qPCR comparative analysis of wild-type strains and VFPBJ₀9756 overexpression strains,the results showed that the ex-pression levels of VFPBJ₀9756（encoding fungal zn（2）-Cys（6）binuclear cluster）and VFPBJ₀9755（encoding polyketide synthase）genes were obviously up-regulated in VFPBJ₀9756 overexpression strains.Meanwhile,the VFPBJ₀9752（encoding monocarboxylate permease-like proteins）,VFPBJ₀9754（encoding the metallo-βlactamase superfamily protein）,VFPBJ₀9761（encoding aflatoxin regulatory protein do-main-containingprotein）andVFPBJ₀9762（encodingNAD-dependent epimerase/dehydratase）gene expression are also obviously up-regulated,but the expression level of most genes outside the VFPBJ₀9755 gene cluster did not increase.The HPLC-QTOF-MS was used to compare and analyze the crude extract isolated from the wild type strain and the VFPBJ₀9756 overexpression strain in ten kinds of medium such as barley,MYIZ,MYOG,corn,MYM,V8,carrot,tomato,LP and TSSM.The results showed that in V8 medium,the peaks of VFPBJ₀9756 overexpression strains were obviously higher than those of wild type strains at 7.8 min,8.25 min,9 min,and 9.52 min.The mass spectra of these four time periods were extracted and found the mass-to-charge ratio of the substance（m/z[M+H]⁺）was 1234.8,which corresponded to the Leucinostain K at 7.8 min.The mass-to-charge ratio of the substance（m/z[M+H]⁺）was 1204.8,which might be Leucinostain B at 8.25 min.The mass-to-charge ratio（m/z[M+H]⁺）of the substance was 1104.8,which might be Leucinostain A at 9 min.In the barley culture medium,it was found that the peak of the VFPBJ₀9756 overexpression strain was higher than that of the wild type strain at 8.15min,and the corresponding mass/charge ratio（m/z[M+H]⁺）of the substance was 1204.8,probably be Leucinostain B.In MYOG medium,the peak of VFPBJ₀9756 overexpression strain was higher than that of wild type strain at 7.7 min and 8.22 min,and the corresponding mass-to-charge ratio（m/z[M+H]⁺）of the substance was 1190.8 at 7.7 min,probably be Leucinostain C,the corresponding mass-to-charge ratio（m/z[M+H]⁺）of the substance was1204.8 at 8.22 min,the substance probably be Leucinostain B.However,in several other cul-ture media,the wild-type strains and the VFPBJ-09756 over-expressing strains showed little difference in their products.In summary,the VFPBJ₀5021,VFPBJ₀9342,and VFPBJ₀9755 gene clusters might be synthetic gene cluster of Acremoxanthone C,and VFPBJ₀9756 gene can regulate the ex-pression of the VFPBJ₀9755 gene cluster.