The Biosynthetic Genes Fuction And Regulatory Mechanism Of Cordycepin And Ergothioneine In Cordyceps Militaris

Cordyceps militaris belongs to Clavicipitaceae of Ascomycota,is a model species of Cordyceps,also knows as an edible and medicinal fungus.Cordyceps militaris contains lots of bioactive compounds,some of them,such as cordycepin and ergothioneine,perform valuable pharmacological functions.cordycepin has the highest share of the secondary metabolite production of C.militaris.With the pharmacological research development of cordycepin,the demand for producing this compound is getting growth.Nowadays,the mainly method to produce cordycepin is doing extraction of the fermentative cell or medium of C.militaris.Although there are many studies focus on the optimization of fermentative production,its intensive studies on metabolic pathways at molecular-level have not been reported.Besides,cordycepin is a structural analog of 2-deoxyadenosine,the unique synthesis mechanism of cordycepin has attracted much attention.But the expression of cordycepin synthase still faces many obstacles,which makes its mechanism study slow.Another bioactive compound,ergothioneine,has attracted attention because of its excellent antioxidant properties.Although ergothioneine can be isolated and purified from the fruiting body of C.militaris,its biosynthesis pathway is not clear.The further in-depth study of C.militaris and its bioactive compounds require the assistance of an efficient genomic editing technology.However,there is no suitable technology reported even in Ascomycota.The traditional gene-editing technology is inefficiency and time-consuming,developing highthroughput molecular manipulation tools are urgently needed in the future.Thanks to the development of high-throughput sequencing technology and bioinformatics,it provides new ideas for the study of metabolic pathways and heterologous expression of proteins.Also,the development and widespread application of CRISPR technology offers an opportunity for various species to establish high-throughput geneediting technologies.Therefore,based on the analysis of transcriptome data and the prediction of the structure and function of genes and proteins,this thesis performed the regulatory mechanism study of cordycpin in C.militaris,and the biosynthetic genes along with its fuction research of cordycepin and ergothioneine.1)Transcriptome sequencing and analysis of cordycepin biosynthesis pathway in C.militaris.The effects of adding L-alanine,Amp and cytidine on the cordycepin production were verified.Then,the mycelia with double yield of cordycepin due to the addition of Lalanine were selected for transcriptome sequencing analysis.Total 511 up-regulated and 472down-regulated genes were obtainted,there were 94 putative transcription factors among these differentially expressed genes.Based on the analysis results of activated cordycepinrelated differential expression genes,a map of the global metabolism network of cordycepin was drawn.This network led to the conclusion that the main reasons for the overproduction of cordycepin were the transcription level of genes,which participated in energy synthesis and amino acidtransforming biological pathways,were activated and up-regulated.Innovatively start at the molecular level,analyze high-throughput data to draw metabolic networks associated with inducers and cordycepin,reveal the flexibility of cordycepin metabolism networks,and provide more precise editing targets for improving yields of cordycepin or other valuable medicinal metabolites.2)The functional verification of cordycepin related transcription factors CmTf1 and CmTf2.Two transcription factors CmTf1 and CmTf2 related to cordycepin production were excavated from the significantly differentially expressed genes,their positive function of increasing cordycepin content was further verified by constructing the over-expressing strains.The over-expression did not cause any obviously change of grown rate and morphology of mycelium.The accumulation of pigment also mainrain the same between the wild type and over-expressed mutation.These all suggest these two transcription factors regulated specific secondary metabolite but not induce polymerase chain reactions to increase the cordycepin production.3)The efficient expression of cordycepin synthetase Cns1 and Cns2 in E.coli.Drived by tetracycline induced promoter and fused with biotin affinity tag,the high-efficiency expression and purification of Cns2 was performed in E.coli.The expressed obstacle of Cns1 was solved by testing mutipul conditions,such as constructions of transmembrane regions and precursor peptide knockout mutants,supplement of ferrous ion and iron sulfur cluster carrier,optimization of fermentative oxygen content and coexpression of chaperonin.The soluable expression of Cns1 was ultimately solved by anaerobic fermentation and chaperonins co-expression.The exploration of optimizing protein expression implement a significant reference for the construction of high-yield engineering strains of cordycepin in yeast or other species,and at the same time provide multiple solutions for the expression of eukaryotic proteases in E.coli.4)Excavate the candidates of ergothioneine synthases and in vivo rebuild the synthesis pathway of ergothioneine.The bioinformatics BLAST analysis revealed the synthetic pathway of ergothioneine in C.militaris is similar to that in filamentous fungi,and the ergothioneine synthases in C.militaris are CmEgt1 and CmEgt2.After reconstructing CmEgt1 to CmE1 B by deleting the N-terminal domain to improve its expression in E.coli,further with the help of Egt D,the in vitro reaction of CmE1 B and CmEgt2 was performed.The reaction achieved the synthesis of ergothioneine,which extends the understanding of the synthesis pathway of ergothioneine in Ascomycete,and lays the foundation for the construction of industrial strains,which with reduced sensitivity to oxygen stress,by using overexpressed ergothioneine to solve the problem of strain degradation.5)Establish a CRISPR/Cas9 gene editing system in C.militaris.The expression elements were cloned from C.militaris,which increased the availability of molecular tools for C.militaris editing.A gene knocked out was achieved by transforming a pre-synthesized sg RNA along with its homologous ss DNA into a pre-built stably Cas9 expressed C.militaris,which implement this study has built an efficient CRISPR knockout system.This is the first report of building a successful CRISPR system in Ascomycetes.The successful application of this editing technology provides a convenient tool for the study of strain degradation,and it will greatly improve the process of new mutation breeding for secondary metabolite production.To conclude,this study analyzed the cordycepin synthesis and metabolism pathways of C.militaris to draw a comprehensive metabolic map,to solve the expression of cordycepin synthase expression in E.coli,and to discover the synthesis pathway of ergothioneine.This study also implemented the application of CRISPR editing technology in C.militaris,laying the cornerstone for various further studies of Cordyceps sp.