Functional Characterization Of O-methyltransferases And Glycosyltransferases For Flavonoids From Liverworts

Bryophytes are the most primitive terrestrial taxa.In order to adapt to dry environment,an abundance of secondary metabolites is accumulated,including bibenzyls,terpenoids,phenylpropanoids,coumarins and flavonoids.Flavonoids possess a wide spectrum of physiological activities and play a very important role in response to biotic and abiotic stress.Various substitution reactions can occur on flavonoids such as hydroxylation,methylation and glycosylation.Different types of flavonoids have been isolated and identified including methylated and glycosylated flavonoidsin bryophytes.Methylation of flavonoids is catalyzed by O-methyltransferases,transferring methyl group from S-adenosyl-L-methionine to hydroxyl groups.OMTs are divided into two classes:Class I OMTs and Class II OMTs.Class I OMTs,caffeoyl CoA O-methyltransferases,are typically 23-29 KDa and require the involvement of Mg2+.Class I OMTs are considered to methylate caffeoyl CoA.The subclass CCoAOMT-likes are able to catalyze various coumarins and flavonoids.Class II OMTs have larger molecular weight.They are not cation dependant and referred to as caffeic acid O-methyltransferases.Their substrates include caffeic acid,coumarins,alkaloids and various flavonoids.In this study,four distinct putative COMTs were screened from Plagiochasma appendiculatum transcriptome sequencing database,named PaF4'OMT,PaCOMT2,PaCOMT3 and PaCOMT4 respectively.Through sequence alignment,we found that PaF4'OMT was 27 amino acids longer than other COMTs from higher plants.The truncated version of the ORF was designated PaCOMTl-Tr.Full length of PaCOMT2 was obtained by 3' RACE.A phylogenetic analysis showed that all four COMTs belong to Class ? OMTs.They were located in the root of flavonoid OMTs.We cloned genes from plants.Proteins were expressed and purified by Ni column.Enzymatic activities showed that PaF4'OMT could catalyze various flavonoids to their 4'-O-methylether while failed to convert phenylpropanoids.Its optimal substrate was apigenin,which was converted to acacetin.PaCOMT1-Tr protein displayed a much lower level of substrate selectivity and activity.Acacetin is an important chemical that has medicinal value.We employed an approach of methylating apigenin for the production of acacetin.By constructing genetically engineered strain harboring PaF4'OMT,acacetin was produced via fermentation.This is the first time that a Class? OMT was characterized in liverworts.Two Class I OMTs,MpOMT and PaF60MT,were characterized from Marchantia paleacea and P.appendiculatum in previous study.Results of sequence alignment and phylogenetic tree showed that they shared a low similarity with classic OMTs while a higher identity with catechol OMTs.In order to determine their homologous genes in other liverworts and study evolutionary relationships,a search was performed using MpOMT as the query against the transcriptomic sequences derived from Marchantia emarginata and Haplomitrium mnioides.Two putative CCoAOMTs were found and designated as MeOMT and HmOMT.A phylogenetic analysis showed that they clustered with MpOMT and PaF60MT.Enzymatic assays showed that MeOMT,HmOMT and MpOMT had higher catalytic activities and broad substrate selectivity.Esculetin was their favorite substrate.In contrast,PaF60MT showed a substrate preference for 6-OH of baicalein and scutellarein.MpOMT,MeOMT,HmOMT and PaF60MT shared a high sequence similarity with different catalytic specialities.To investigate the influence on substrate preference of key amino acids,site-directed mutagenesis was carried out on MpOMT and PaF60MT.Results showed that Arg-59 in PaF60MT was beneficial for methylating 6-OH of baicalein and scutellarein.In vivo feeding experiment indicated that cloning site of genes,concentration of substrates could both affect the conversion of substrates.It recommends a new way of producing oroxylin A and scopolamine.Glycosylated flavonoids are catalyzed by Uridine diphosphate glycosyltransferases(UGTs),transferring an activated donor sugar onto flavonoids.Glycosylation is known to be beneficial for their transportation and metabolism.Three UGTs have been screened and designated as PaUGTl,PaUGT2 and PaUGT3 from P.appendiculatum transcriptome sequencing database in this study.Sequence alignment showed that they shared low idendity with UGTs from other plants.However,PSPG motif and key amino acids were highly conserved.In the phylogenetic tree,three UGTs were clustered into one clade of flavonoid 7-O-GTs.We subcloned genes,ligated them into prokaryotic expression vector and tested their catalytic activity using UDP-glucose.Results showed that PaUGT1 is a flavonoid 7-O-glucosyltransferasethat can catalyze apigenin,luteolin,quercetin and kaempferol into 7-O-glucosides,respectively.