Dissection Of The Biosynthetic Pathway And Heterologous Synthesis Of C-Glycosylation Flavonoids From Stenoloma Chusanum

Flavonoid C-glycosides are important secondary metabolites in plants that have a C-C or C-O bond between the aglycone and the sugar moiety.Compared with O-glycosides,flavonoid C-glycosides contain the rigid C-C bond,are highly stable against gastrointestinal hydrolytic metabolism and contain potent bioactivity in mammals,including antioxidant,antiviral,anti-diabetes,anti-tumor,and antihypertensive activities.These compounds also play an important role in protecting plants from herbivores,rice-blast fungi,drought stress,and ultraviolet radiation.Currently,commercial C-glycosyl flavones are mainly extracted from plants,limiting their availability and sustainability.Chemical C-glycosylation remains challenging because of the structural complexity of these components.Therefore,dissecting the biosynthesis of flavonoid C-glycosides and understanding the characteristics of related enzymes,especially the chalcone synthase and C-glycosyltransferase,will be crucial for future biotechnological applications.At present,such enzymes have been mainly studied in angiosperms but have not been reported in non-seed plants.Hence,mining key enzymes in ferns has become an urgent problem to be solved.Stenoloma chusana belongs to the Stenoloma genus of the Lindsaeaceae family.In folk medicine,its prescription is considered effective in cancer treatments,traumatic bleeding,dysentery,and pesticide poisoning,which results in its reputation as an "all-purpose antidote".Previous research indicated that it contains abundant flavonoids,particularly flavone Cglycosides,such as vitexin,orientin,and their derivatives.Therefore,in the present investigation,several key enzymes in the biosynthesis pathway of flavone C-glycosides in S.chusana were studied,including functional characterization,crystallographic structural analysis,and synthetic biology.The main contents are as follows:(1)Functional characterization and crystallographic study of chalcone synthase from S.chusanaOne putative chalcone synthase(CHS)sequence was recognized and tentatively designated as ScCHS1 from the S.chusanum transcriptional sequence database.Multiple sequence alignment results showed that ScCHS1 aligned well with the established homologs from other plants.Phylogenetic analysis indicated that ScCHS1 was clustered together with two CHS from other ferns,and located in the root of the CHS sub-branch from seed plants.Enzymatic assay results showed that ScCHS1 could catalyze p-coumaroyl-CoA,cinnamoylCoA,caffeoyl-CoA,and feruloyl-CoA to form naringenin,pinocembrin,eriodictyol,and homoeriodictyol,respectively.Different from the most reported CHSs,ScCHS1 exhibits a significant substrate preference for feruloyl-CoA,which contains a methoxy substitution on the benzene ring.Moreover,kinetics studies revealed that the catalytic efficiency of ScCHSl for feruloyl-CoA was highest,about 13 times that of p-coumaroyl-CoA.To understand its catalytic mechanism,the crystal structures of ScCHS1 in apo and in complex with the reaction products(pinocembrin,naringenin,and eriodictyol)and CoA were solved,and all their structures were determined within 2.2 A.Crystallographic and site-directed mutagenesis experiments revealed that four amino acid residues,located in the substrate-binding pocket close the B-ring of products,could exert a significant effect on the unique catalytic activity of ScCHS1.This work explains the selectivity of ScCHS1 to substrate from the perspective of structure and provides new insight for the study of CHSs.(2)The application of ScCHS1 in flavonoid synthetic biologyTwo 4CL-encoding genes were identified from the S.chusanum transcriptomic database,termed Sc4CL1 and Sc4CL2,to construct the biosynthetic pathway of flavanones.Sequence alignment and phylogenetic analysis demonstrated that Sc4CLs shared high homology with seed plant 4CLs and were classified into different groups.The results of enzyme activity showed that Sc4CL1 was able to efficiently convert p-coumaric acid,caffeic acid,cinnamic acid,and ferulic acid into their corresponding CoA esters,whereas Sc4CL2 displayed activity only towards caffeic acid,cinnamic acid,and ferulic acid.Therefore,Sc4CL1 was selected as the candidate gene,which is co-expressed with ScCHS1 in E.coli,and ferulic acid was used as the substrate to achieve a two-step synthesis of homoeriodictyol.Through optimization of fermentation conditions,the maximum conversion rate of ferulic acid reached above 98%.Additionally,Sc4CL1 and ScCHS1 were used in combination with other key genes(CHI and FNS I),heterologous biosynthesis of B-ring flavonoids containing ortho-dihydroxy or methoxy-substituted in E.coli.(3)Excavation and functional characterization of C-glycosyltransferase from S.chusanumBy analyzing the distribution of flavone C-glycosides and the results of the expression levels of these candidate genes in different tissues,four putative CGT genes were screened out from the transcriptome database of S.chusanum.Biochemical analyses revealed that ScGT1 showed the C-glycosylation activity against phloretin,2-hydroxynaringenin,and 2hydroxyeeriodictyol,resulting in the formation of corresponding C-glucosides.Intriguingly,although ScCGT1 and type I CGTs both belonged to an orthologous group of enzymes,they were not closely grouped.In contrast,ScCGTl and type Ⅱ CGTs grouped closer in the phylogenetic tree.Different from the characterized type Ⅰ CGTs in seed plants,the DPFXL motif conserved in 2-hydroxyflavanone CGTs was absent in ScCGT1.Homology-based protein modeling and site-directed mutagenesis indicated that the P164T mutation increased C-glycosylation activity by forming a hydrogen bond with the sugar donor.The research on introducing plant F2Hs and CGTs into E.coli to produce flavone C-glycosides are extremely limited.Currently,only two relevant studies have been reported,and both take a truncated F2H gene coupled with a P450 reductase CPR to co-express in E.coli,which provides the reaction intermediates for the catalysis of CGT.In this study,the soluble protein CjFNS I/F2H and ScCGT1-P164T were combined in E.coli to lead a high conversion from naringenin to vitexin and isovitexin,which also simplified the biosynthesis pathway of C-glycosides.This is the first time that C-glycosyltransferase has been characterized from fern species and provides a candidate gene and strategy for the efficient production of bioactive C-glycosides using enzyme catalysis and metabolic engineering.In summary,this thesis identified a novel chalcone synthase(ScCHS1)and the first nonseed plant C-glycosyltransferase(ScCGT1)from S.chusana.The catalytic functions and mechanisms of ScCHS1 and ScCGT1 were investigated comprehensively.This study provides a reference for the evolution of key enzymes for the synthesis of plant secondary metabolites,filling a gap in the field of synthetic pathways of these active components in nonseed plants.