Cloning And Functional Verification Of The Key Enzyme Genes CgCAO And CgLDC In The Synthetic Pathway Of The Endophytic Fungus Colletorichum Gloeosporioides ES026

Huperzine A,which belongs to the lycodine type of lycopodium alkaloids,has been used as an anti-Alzheimer's disease drug candidate and derived from traditional Chinese medicine plant Huperzia serrata.H.serrata,the original source of Hup A,actually possesses a very low content of Hup A.It belongs to a club moss,has a very limited distribution,and grows very slowly.It takes at least 15 years from spore germination through a gametophyte stage to finally reach a mature sporophyte stage,thus facing that,the natural production of Hup A has been greatly limited.With the demand of the Hup A increases sharply in recent years,the H.serrata plant are in danger of extinction in China due to extensive collection for Hup A production.To protect the H.serrata from extinction and to support efforts to produce Hup A,several studies have confirmed that endophytic fungi and the host plant have the same route for synthesis of secondary metabolites and can produce the same or identical physiologically active metabolites.Thus,in recent years much attention to search for a potential microbial source producing Hup A has been paid,Gene ontology and Kyoto Encyclopedia of Genes and Genomes assignments allowed annotation of lysine decarboxylase(LDC)and copper amine oxidase(CAO)for their conversion of L-lysine to 5-aminopentanal during Hup A biosynthesis.In order to verify the function of lysine decarboxylase and copper amine oxidase,this study mainly made a few aspects of the content:1.The Cg LDC and the CgCAO sequences were cloned from C.gloeosporioides ES026,the Cg LDC total length of 769 bp,encoding 256 amino acids,and the CgCAO sequence was 2072 bp in length,encoding 690 Amino acids.2.The Cg LDC protein and the CgCAO protein in vitro heterologous expression and protein purification.Build a heterologous expression vectors : p ET28a-Cg LDC and p ET28a-CgCAO,expressed in E.coli BL21(DE3).Protein expression was optimized conditions,and by using the optimized method for purification of protein purification after Cg LDC protein and CgCAO proteins in vitro enzymatic reaction activity and reaction products by LC-MS analysis,revealed that Cg LDC catalyzed the conversion of L-lysine to cadaverine,and CgCAO converted cadaverine to 5-aminopentanal.3.C.gloeosporioides ES026 CgCAO and Cg LDC genes in the body to express According to methods used for Agrobacterium transformation,C.gloeosporioides ES026 was transformed using the 10 plasmids,and a randomly selected transformant was confirmed by PCR.Quantification by q RT-PCR of Cg LDC and CgCAO expression during fermentation indicated that the Pagd A-Cg LDA and Palc A-CgCAO transformants exhibited the highest expression levels...