Heterologous Expression Of The Enzyme Genes Related To Biosynthesis Of Huperzine A And Isolation Of Endophytic Fungi

Huperzine A(Hup A)is a plant-derived lycopodium alkaloid that can be used to treat Alzheimer’s disease.Currently,industrial production of Hup A relies primarily on direct extraction from Huperziaceae plants or chemical synthesis.However,because plants grow slowly and the yield of chemical synthesis is low,bioproduction of Hup A is gradually becoming an alternative to plant extraction and chemical synthesis.Unfortunately,the biosynthetic pathway and mechanism of Hup A remain poorly understood.Thus,this project aimed to explore the biosynthetic pathway of Hup A and advance its biosynthesis in microbes.To begin with,three key enzymes in Hup A biosynthesis including lysine decarboxylase,copper amine oxidase and type III polyketide synthase,were successfully expressed in Escherichia coli.The key precursor Δ1-piperidine in the biosynthesis of Hup A was successfully detected by L-lysine as a substrate in shake flask fermentation.Next,the experiment was carried out to build a eukaryotic expression system for Hup A biosynthesis.Firstly,the adaptability of Saccharomyces cerevisiae INVSc1 and p YES2 plasmid was verified.Secondly,the feasibility of expressing key enzyme genes in Pichia pastoris and S.cerevisiae was explored,and the related enzymes were simply functionally verified.In addition,in view of the fact that endophytic fungi can produce medicinally active analogs,we isolated 14 endophytic fungi from the leaves of Huperzia serrata.The results of fungal species identification showed that most of the isolated endophytic fungi belonged to Colletotrichum sp.This work provides basis for subsequent screening of endophytic fungi that can produce Hup A.Lastly,fermentation experiments were carried out on the isolated endophytic fungi.We analyzed the fermentation products and proved that one of the isolated endophytic fungi could produce Hup A.The discovery of this strain provides important clues for the later exploration of key enzymes in biosynthetic pathway of Hup A.This may contribute largely to complete biosynthesis of Hup A.