Effect Of Histone Deacetylases On Secondary Metabolism Of Sea Cucumber Associated Penicillium Sclerotiorum

Because of antibiotic abuse,the occurrence of drug resistance in pathogenic bacteria increased sharply in recent years.However,the development of anti-infective drugs enters a bottleneck period.There are rich secondary metabolite biosynthetic gene clusters in fungal genomes,which show great potential for producing abundant secondary metabolites.But most gene clusters are silent under laboratory conditions.How to activate these silent gene clusters and discover novel secondary metabolites is one of the hotspots and difficulties in fungal secondary metabolism research.Histone acetylation is an important epigenetic modification.The acetylation of histone lysine residues leads to an open chromatin structure,which facilitates gene activation and transcription.Thus,histone acetylation is an important strategy to improve the diversity of microbial natural products.In this study,sea cucumber associated Penicillium sclerotiorum SD-36 was chosen for the research of the effects of histone deacetylases(HDACs)on secondary metabolism.Bioinformatics analysis of functional gene clusters and HDACs in P.sclerotiorum SD-36 was carried out and 51 functional gene clusters were found in its genome,belonging to T1 PKS,NRPS,Terpene and other types.However,the corresponding secondary metabolites were not found in the fermentation products of the wild-type strain(WT),which shows that most of the gene clusters were silent.Seven HDAC homologous genes were screened from the genome of P.sclerotiorum SD-36,and three HDAC genes(PsHos2,PsRpd3,and PsHos3)were knocked out respectively using CRISPR-Cas9 and CRISPR-Cpf1 gene editing technologies established in this research.The CRISPR-Cpf1 system using Pgpda promoter for the expression of cr RNA had a high editing efficiency for P.sclerotiorum SD-36 and was suitable for subsequent experiments.The phenotype of ?PsHos2 mutant showed great difference with that of WT,and the high performance liquid chromatography(HPLC)analysis spectrum of its fermentation exhibited many new peaks compared with that of WT,which indicated that the deletion of PsHos2 led to the activation of secondary metabolite biosynthetic gene clusters.Six compounds(A1-A6)were obtained from ?PsHos2 mutant,most of which are azaphilones.Compared with WT,the yield of sclerotiorin(A1)in ?PsHos2 mutant increased 40-fold.A1 showed weak antibacterial activity,while isochromophilone III(A2)and sclerotioramine(A5)exhibited broad-spectrum antibacterial activity and have potential usage as antibacterial drugs.Comparative transcriptome analysis between ?PsHos2 and WT identified 4,975 differentially expressed genes,including 350 secondary metabolite biosynthetic genes,which distributed in 50 gene clusters.Among them,43 genes were core biosynthetic genes.In ?PsHos2,24 core biosynthetic genes were up-regulated.Besides,HDAC genes,PsHos3 and PsClr3 were also up-regulated by 2.722 and 1.903-fold,respectively.The above results indicate that PsHos2 has a global impact on the secondary metabolism of P.sclerotiorum SD-36,and different HDACs may have compensatory functions.HDACs PsRpd3,PsHos3 and PsClr3 also showed significant effects on the phenotype,growth rate,sporulation ability and secondary metabolite biosynthesis of P.sclerotiorum SD-36.In addition,double knockout mutants ?PsHos2?PsRpd3 and?PsHos2?PsHos3 were constructed based on the ?PsHos2 mutant.The phenotype of double knockout mutants showed significant difference with those of WT and ?PsHos2,and the growth rate of double knockout mutants decreased.However,the production of secondary metabolites was significantly increased in double knockout mutants.In this study,a simple and efficient gene knockout method was successfully established in P.sclerotiorum SD-36,and three HDAC single knockout mutants and two HDAC double knockout mutants were constructed.Silent gene clusters were activated,six differentially synthesized compounds were obtained from ?PsHos2 mutant,and antibacterial activity of the six compounds were tested.The effects of PsHos2,PsRpd3,PsHos3 and PsClr3 on the growth and secondary metabolism of P.sclerotiorum SD-36 were analyzed.The results provide research foundation for further exploration of secondary metabolites in P.sclerotiorum SD-36.