Functional Characterization And Synthetic Biology Research Of Flavonoid UDP-glycosyltransferases From Liverworts

Flavonoids are plant secondary metabolites that are widely distributed in the plant kingdom,and most of them usually exist in their decorated forms catalyzed by glycosylation.Glycosylation is a common post-modification reaction in the biosynthesis of flavonoids,which can increase the water solubility,stability and chemical properties of the aglycones.Glycosylation in plants is mostly catalyzed by uridine diphosphate dependent glycosyltransferases(UGTs)of the family 1 glycosyltransferases,which can catalyze the transfer of glycosyl moieties from UDP sugars to a wide range of acceptor molecules.Flavonoid glycosides show excellent biological activities in medicine and food.In recent years,with the rapid development of metabolic engineering and synthetic biology technology,progress has been made in the synthesis of plant-derived flavonoid glycosides by model microorganisms.At present,there are few reports on the biosynthesis of flavonol 7-O-glucosides,and the production of flavone 7-O-glucosides(apigenin 7-O-glucoside and luteolin 7-O-glucoside)reported are relatively low.Therefore,it is of great significance to screen candidate flavonoid UGTs with efficient 7-O-glycosylation function,clarify their catalytic activity,and apply them to the research of synthetic biology of flavonoid glucosides.So far,flavonoid UGTs have been discovered in a wide range of plant species,including crops and medicinal plants such as Arabidopsis,maize,soybean and tea.In order to adapt to the land environment,the liverworts species Plagiochasma appendiculatum and Marchantia polymorpha synthesize an abundance of metabolites including bibenzyls,terpenes,lignans and flavonoids.The genes encoding the key enzymes involved in flavonoids synthesis in liverworts have been isolated and characterized over the past few years.However,those encoding the enzymes responsible for the flavonoids glycosylation have yet to be identified.In the present investigation,we explored the UGTs cloned from the liverwors P.appendiculatum and M.polymorpha,and carried out research on their functional characterization,enzymatic analysis and synthetic biology.1.Isolation and characterization of multifunction UGTs from P.appendiculatumExamining the Swissprot annotation of the transcriptomic sequences derived from the liverworts species P.appendiculatum,four putative P aUGTs encoded a flavonoid UGT were screened out and termed PaUGT1 through PaUGT4.Multiple sequence alignment revealed that the 4 UGTs showed low identity with UGTs from other plants,and shared a conserved domain with the PSPG(Plant Secondary Product Glycosyltransferase)motif near their C-terminal domains.Phylogenetic analysis showed that the PaUGTs were clustered into the 7-O-glycosylation clade.Enzymatic assay results indicated that PaUGTl and PaUGT2 showed a broad substrate specificity.PaUGTl and PaUGT2 recombinant proteins could catalyze flavonols,flavones,flavanones and dihydrochalcones to form new products comparing with their corresponding negative controls.The kinetic parameters of enzyme activity showed that PaUGT1 and PaUGT2 have comparable catalytic activity to flavonols,but PaUGT1 has significantly higher catalytic activity to flavones than PaUGT2,and could be used as a candidate gene for biosynthesis of flavone 7-O-glucosides.This is the first time the flavonoid UDP-glycosyltransferases were characterized in liverworts.2.Potential of PaUGTl for synthesizing flavonoid 7-O-glucosides in vivoIn an effort to identify key amino acids responsible for the catalytic activity of PaUGT1,a series of site-directed mutagenesis was performed based on the homology modeling and docking analysis using VvGT1(2clz)from Vitis vinifera as a template.A mutated form PaUGT1-Q19A encoded an enzyme exhibiting a higher catalytic efficiency than did the wild type form.When expressed in E.coli fed with flavonol aglycones,maximum bioconversion rate corresponded to over 70%of 7-O-glucosides.Co-expression of PaUGTl-Q19A with the upstream flavone synthase ? PaFNS I-1 from the same species proved able to convert the flavanone aglycones naringenin and eriodictyol into the higher-yield flavone glucosides apigenin 7-O-glucoside(A7G)and luteolin 7-O-glucoside(L7G).Also,the yield of both A7G and L7G in E.coli cultures could be dramatically boosted by metabolically engineering the sugar donor synthesis pathway while at the same time co-expressing PaFNS I-1 and PaUGT1-Q19A.In the end,the maximum concentration of 35.0 mg/L(81.0 ?M A7G and 39.6 mg/L(88.6 ?M)L7G was achieved under optimized conditions,respectively.The key outcome of the research has been to identify some candidate genes harbored by a basal land plant showing the potential to efficiently and economically produce biologically useful flavonoid glucosides.3.Identification of two UGTs involved in 3-OH site glycosylation in M.polymorpha18 distinct putative UGTs were screened from M polymorpha genome sequencing database and two MpUGTs named MpUGTl and MpUGT2 respectively were further functional identified in this study.The phylogenetic tree analysis showed that these two UGTs were clustered into one clade with flavonoid 3-OH glycosylation.The ORFs of MpUGT genes were cloned from the cDNA of M.polymorpha and then constructed into prokaryotic expression vectors.Enzymatic activity showed that MpUGT1 and MpUGT2 are flavonol 3-O-glycosyltransferases that catalyze flavonol aglycones(quercetin and kaempferol)to form their 3-O-glucosides.In order to further research the function of MpUGTs in vivo,MpUGT1 and MpUGT2 were constructed into plant expression vector and transformed into Arabidopsis thaliana,respectively.