Function Study Of Polyketone Synthase And Lysine Decarboxylase Gene In The Endophytic From Huperzia Serrata

Huperzine A(Hup A)is a clinical drug for the treatment of Alzheimer's disease which is mainly extracted from lycopodium such as plant resources H.serrata.However,the yield of Hup A is restricted because of the shortage of plant resources,difficulty of tissue culture and cumbersome steps of artificial synthesis.In recent years,researchers have isolated endophytic fungi that produce Hup A.Pass through endophytic fungi fermentation to produce Hup A is a good choice,because the endophytic fungi have the advantages of fast growth,easy cultivation and low cost.In this study,an endophytic fungus fungal Colletotrichum gloesporioides(C.gloesporioides)is capable of producing Hup A,which was isolated from H.serrata,and the genome and transcriptome of this fungus was sequenced.The C.gloesporioides that low yield of Hup A before induction and the higher yield of Hup A after induction were sequenced and the gene function was annotated.Lysine decarboxylase and polyketone synthase related genes were annotated in Hup A biosynthesis pathway.In order to study the function of Lysine decarboxylase and polyketonase,this study made following content:1.HPLC method was established for determination the content of Hup A and Hup B in endophytic fungi fermentation broth.2.The elicitors which could induce endophytic fungi C.gloesporioides to increase the content of secondary metabolites Hup A were screened,which provided the basis for the functional study of candidate genes.3.Homologous transformation by PEG mediated was used to knockout five candidate PKS genes in C.gloesporioides.The effect of gene knockout on mycelium biomass and Hup A content was detected.The results showed that knockout CgPKS-06762 gene had no effect on the content of Hup A.When CgPKS-02152/12940 genes were knockout,respectively,the mycelium biomass was significantly increased,but the content of Hup A in fermentation broth and the yield of Hup A in unit mycelium had no significant effect.When CgPKS-03512 gene was knockout,content of Hup A in fermentation broth had no significant effect,however,the mycelium biomass and yield of Hup A in unit mycelium was decreased significantly.The mycelium biomass was decreased significantly;however,the content of Hup A in fermentation broth and the yield of Hup A in unit mycelium had no significant effect when CgPKS-00749 was knocked out.The results showed that all of the PKS gene were important,in which CgPKS-03512 was more important to the synthesis of Hup A.4.The CgLDC genes of C.gloesporioides were knockout.In this study,the mycelium biomass increased significantly after knocking out CgLDC-03647 and CgLDC-04099,respectively.However,the content of Hup A in fermentation broth and the yield of Hup A in unit mycelium had no significant effect.After knockout the genes of CgLDC-03647 and CgLDC-04099 simultaneously,there was no significant difference in biomass of mycelium,however,the content of Hup A in fermentation broth and the yield of Hup A in unit mycelium was increased significantly.It was found that the simultaneous knockout of CgLDC-03647 and CgLDC-04099 genes had an effect on the growth rate,sporulation ability and pigment.