| Huperzine A（HupA）is a natural alkaloid extracted from Huperzia serrata（Thunb.）Trev.It has been approved for clinical treatment of Alzheimer’s disease as a highly effective and low toxic targeted inhibitor of acetylcholinesterase.As the main medicinal plant producing HupA,H.serrata contains low content of HupA and is difficult of artificial cultivation.This has lead to the increasingly prominent contradiction between supply and demand,and shortage of natural resources.It was found that some endophytic fungi of H.serrata produce HupA,which provided a new possibility of solving the shortage of HupA.In this work,endophytic fungi were isolated from H.serrata in order to search for high yield strains of HupA.Based on the Colletotrichum gloeosporioides with high yield of HupA,we identified possible gene sequences encoding key enzymes for HupA synthesis and performed bioinformatics analyses on them,including lysine decarboxylase（LDC）and copper amine oxidase（CAO）.The relationship between candidate genes and HupA synthesis was further explored by gene knockout experiment,which provided a new basis for analyzing the synthesis pathway of HupA.The results of the study are as follows:（1）The 46 endophytic fungi were isolated from different parts of H.serrata,which were preliminarily classified into 12 species by morphological identification.After analysis by LC-MS,it was confirmed that strain No.4（NWUHS004）produces HupA.The strain was identified a Fusarium sp.by ITS sequence analysis.The per unit yield of HupA of NWUHS004 was 1.396μg/g,which was significantly lower than that of C.gloeosporioides（5.036μg/g）.Therefore,subsequent studies were carried out based on the high-yield strain.（2）Based on the genomic data of C.gloeosporioides,two LDC and 12 CAO genes were identified.The LDC2151,LDC9857 and CAO3097 were identified as candidate genes by homology comparison with other LDC and CAO homologous genes of HupA-producing fungi.Bioinformatics analysis showed that LDC2151 and LDC9857 encoded similar proteins sharing similar properties and spatial structure.It is supposed that LDC2151 and LDC9857may have similar biological functions.（3）By homologous recombination,we successfully constructed a p UC-CAO3097 knockout vector,and obtained the CAO3097 gene knockout mutant.The mycelial biomass and the yield of HupA of the wild-type strains and knock-out strains were measured.Results showed that the mycelial biomass and HupA yield of mutant strains had no significant differences.（4）Using CRISPR/Cas9 gene editing,a recombinant knockout vector p FC-LDC2151 was constructed and transformed into C.gloeosporioides by PEG-Ca Cl2-mediated protoplast transformation,but positive transformants were not identified successfully.In conclusion,an endophytic fungus producing HupA was isolated and identified in this study.Three genes that may be involved in the upstream of the HupA synthesis pathway were screened.The mutant of CAO3097 had no significant effect on the synthesis of HupA based on molecular and physiological analyses,suggesting that the fungi might have other family genes acting as substitutes for CAO3097.This research provided the basis for the study of HupA synthesis pathway and the establishment of subsequent gene editing system in C.gloeosporioides. |
