Study On Biotransformation Of Endophytic Fungi In The Formation Of Metabolic Components Of Ephedra Sinica

The bio-transformation of endophytic fungi,such as bio-synthesis,bio-decomposition,glycosylation,and epoxidation,has important effects on plant metabolism,and has become an important factor in the study of plant metabolic formation.Microbial bio-catalysis and bio-transformation have characteristics of high efficiency,green,and sustainability.Screening endophytic fungi with bio-transformation activity from medicinal plants has become a new direction in searching for new resources,and has attracted great attention.In this study,firstly,endophytic fungi were isolated from the roots and stems of the plant Ephedra sinica,and their ecological characteristics and growth adaptability were investigated.Secondly,the effect of endophytic fungi on the overall metabolism of host tissues was studied using liquid fermentation co-culture method.Finally,endophytic fungi with bio-transformation activity for intermediates of biosynthesis pathways were screened.The results of the study are summarized as follows:1.In this study,146 and 113 endophytic fungi were isolated from the root and stem tissue blocks of E.sinica.According to the morphological differences of fungi,they merged into 25、7 strains,and belonged to 19 and 6 species,involving a total of 23 species.Studies have shown that they have significant tissue specificity,and there are significant differences in the composition of endophytic fungi in roots and stems.Among them,the representative genera of fungi in root include Purpuricillium,Penicillium,Parachaetomium,Hydeomycos,Herpotrichia,Fusarium,Clonostachys,Camarosporidiella,Aspergillus,Talaromyces and Microascus;The stem are Alternaria,Didymella,Talaromyces and Microascus.The phylogenetic tree analysis constructed by the Neighbor-Joining method showed that the endophytic fungi from root and stem had both genetic associations and micro-evolutionary trends.The former were mainly distributed in Pleosporales,Eurotiales,Microascales,Hypocreases,and Sordariales;The latter are mainly distributed in Pleosporales,Eurotiales,and Microascales.Studies on the ecological adaptability of microbial communities have shown that the biggest factor affecting the selective colonization of endophytic fungi is the external environmental factors(such as temperature and humidity)for roots and stems,followed by the nutritional components of roots and stems.2.In order to explore the effect of endophytic fungi on the metabolism of E.sinica,HPLC-Q/TOF-MS metabonomics was used to study the bio-transformation of endophytic fungi on the overall metabolites of E.sinica tissue.The results showed that the bio-transformation of 8 representative fungi caused changes in 45 metabolic components in the stems of E.sinica,indicating that endophytic fungi have an important impact on the formation of metabolites of E.sinica.The most influential components are alkaloid metabolites,involving 14 compounds such as Actinidine,Cathinone,Dopamine,Ephedrine,and Ephedroxane;9 aromatic acid compounds,such as Benzoic acid,Ferulic acid,Galate,Nepodin and Quinaldic acid;8 flavonoids,such as(+)-Catechin,Afzelechin,Apigenin and Isoliquiritigenin,Naringenin chalcone;4 coumarin compounds,such as Esculetin,Scoparone,Xanthotoxin,and Xanthotoxol;10 other compounds,such as1-Phenylethanol,6-Deoxyphystrol,Adenine,Cinnamaldehyde and Cinnamyl alcohol.By analyzing the metabolic pathways of these metabolites,it was found that endophytic fungi mainly affect the pathways of phenylpropanoid biosynthesis,flavonoid biosynthesis and flavone and flavonol biosynthesis.3.In order to explore the bio-transformation of metabolic intermediates by endophytic fungi,studies on the bio-transformation of metabolic intermediates from E.sinica by endophytic fungi were carried out.In the bio-transformation experiment of 32 strains endophytic fungi on individual compounds,one strain with practical application value was finally obtained,which can transform 2’-hydroxychalcone,a representative intermediate in the flavonoids and flavonols biosynthesis pathway,into4,2’,4’-trihydroxychalcone.This fungus is an endophytic fungus Aspergillus alliaceus(No.EDCS M032)from roots.This discovery provides a scientific evidence for in-depth research on effects of endophytic fungi on the formation of metabolites from E.sinica,as well as the possibility of industrial production of isoliquiritigenin.Under laboratory conditions,the optimal transformation conditions for this strain are: temperature 18.2℃,time 2.9 d,and inoculation rate 5.8%.