Epigenetic Regulation Genes Of Huperzine A Biosynthesis In Theendophytic Fungi From Huperzia Serrata

Huperzine A(HupA)is a kind of efficient acetylcholinesterase inhibitor,can be applied to the treatment of mild-to-moderate alzheimer's disease.Huperziaceae plants such as the Lycopodium serratum Thunb.is the major natural sources of HupA,but due to the shortage of resources and the difficulty of tissue culture,the complex synthetic steps and high cost limit the development of HupA industry.Endophytic fungi have gradually become the focus of research on the synthesis of plant secondary metabolites due to their short growth cycle,easy cultivation and low cost,which provides a new idea for HupA biosynthesis research.With the development of epigenetic research in recent years,epigenetic regulation has shown great potential in the field of fungal research.Epigenetics refers to the inheritable changes in gene expression without alteration of chromosomal DNA sequence.From the point of view of molecular mechanism,epigenetic regulation is a chemical modification of genetic material in biological cells caused by environmental factors,including DNA methylation,histone modification,RNA interference and so on,These regulation modes are very vulnerable to environmental impact.As a secondary metabolite of Colletortrichum gloeosporiodes,HupA production is regulated by environmental factors,it is of great scientific significance to study the influence of epigenetics on the synthesis of HupA by C.gloeosporiodes and elucidate the molecular mechanism of yield accumulation.At present,the research methods of fungi are mainly the knockout or overexpression of related epigenetic modification genes by genetic engineering or the use of small molecular epigenetic inhibitor.In our previous work,we isolated HupA-producing endophytic fungus Cg01 from Huperzia serrata,subsequently,the genome and transcriptome sequencing and gene function annotation of the strain were completed.We obtained epigenetic regulatory genes for instance histone lysine methyltransferase(HMT),histone acetyltransferase(HAT),histone deacetylase(HDAC).In this study,we explored the regulation of epigenetics on HupA biosynthesis by endophytic fungi through small molecular epigenetic inhibitors,gene knockout,RNA-seq,chromatin immunoprecipitation combined with high-throughput sequencing(Chip-seq),and laid the foundation for HupA biosynthesis in vitro.The main results are as follows:1.Two small molecule epigenetic inhibitors were added in the fermentation process of endophytic fungi,respectively 5-azacytidine(5-azacytidine,5-ac),a DNA methyltransferase inhibitor;Histones to acetylation enzyme inhibitors-valproic acid Sodium,Sodium Valproate,SV),Nicotinamide(Nicotinamide,NIC),Sodium butyrate(Sodium butyrate,SB),song of ancient liu cephalosporins(TrichostatinA,TSA)to explore the affect Hup A Cg01 biomass and biosynthesis.The yield of SV and NIC added HupA was significantly reduced,while that of SB group was significantly increased,and the rest had no effect.2.PEG-mediated homologous recombination and transformation method was adopted to knock out and supplement the four candidate genes related to epigenetics,and real-time fluorescence quantitative PCR(rt-qpcr)was used to detect the corresponding gene expression amount in the complement strain and the yield of huperine a in the mutant strain detected by HPLC.The results showed that the production of hupA in the mutant strain CgMst2 had no effect,and the production of hupA in the mutant strain CgClr4,CgSir2 and CgClr3 decreased by 21.3%,16.6% and 24.7%,respectively.Three genes affecting hup A were supplemented,and the mutant strain's production of hup A was restored to the level of the wild type under the condition that there was no significant difference between the corresponding gene expression and that of the wild type.3.The functions of CgClr4 and NIC were identified by Western blot and enzyme activity assay.It was found that both of them could effectively reduce the histone methylation H3K9me2 level of Cg01 and the methyltransferase activity at H3K9 site.It was shown that dimethylation at Cg01 H3K9 site was an important methylation modification method,and that NIC and CgClr4 regulated the biosynthesis of hupA by reducing methylation at this site.4.The transcription level of related genes was explored by rna-seq and the possible genes related to hup Abiosynthesis were obtained by chip-seq analysis : such as cg01-09994(ethanol dehydrogenase)and cg01-03750(cytochrome c peroxidase).