Functional characterization and key residues engineering of a regiopromiscuity O-methyltransferase involved in benzylisoquinoline alkaloid biosynthesis in Nelumbo nucifera

Lotus(Nelumbo nucifera),an ancient aquatic plant,possesses a unique pharmacological activity that is primarily contributed by benzylisoquinoline alkaloids(BIAs).However,only few genes and enzymes involved in BIA biosynthesis in N.nucifera have been isolated and characterized.In the present study we identified the regiopromiscuity of an O-methyltransferase,designated NnOMT6,isolated from N.nucifera;NnOMT6 was found to catalyze the methylation of monobenzylisoquinoline 6-O/7-O,aporphine skeleton 6-O,phenylpropanoid 3-O,and protoberberine 2-O.We further probed the key residues affecting NnOMT6 activity via molecular docking and molecular dynamics simulation.Verification using site-directed mutagenesis revealed that residues D316,N130,L135,N176A,D269,and E328 were critical for BIA O-methyltransferase activities;furthermore,N323A,a mutant of NnOMT6,demonstrated a substantial increase in catalytic efficiency for BIAs and a broader acceptor scope compared with wild-type NnOMT6.To the best of our knowledge,this is the first study to report the O-methyltransferase activity of an aporphine skeleton without benzyl moiety substitutions in N.nucifera.The study findings provide biocatalysts for the semisynthesis of related medical compounds and give insights into protein engineering to strengthen O-methyltransferase activity in plants.

Basic leucine zipper gene VvbZIP61 is expressed at a quantitative trait locus for high monoterpene content in grape berries

The widely appreciated muscat flavor of grapes and wine is mainly attributable to the monoterpenes that accumulate in ripe grape berries.To identify quantitative trait loci(QTL)for grape berry monoterpene content,an F1 mapping population was constructed by a cross between two grapevine genotypes,one with neutral aroma berries(cv.‘Beifeng’)and the other with a pronounced muscat aroma(elite Vitis vinifera line‘3–34’).A high-density genetic linkage map spanning 1563.7 cM was constructed using 3332 SNP markers that were assigned to 19 linkage groups.Monoterpenes were extracted from the berry of the F1 progeny,then identified and quantified by gas chromatography–mass spectrometry.Twelve stable QTLs associated with the amounts of 11 monoterpenes in berries were thus identified.In parallel,the levels of RNA in berries from 34 diverse cultivars were estimated by RNA sequencing and compared to the monoterpene content of the berries.The expression of five genes mapping to stable QTLs correlated well with the monoterpene content of berries.These genes,including the basic leucine zipper VvbZIP61 gene on chromosome 12,are therefore considered as potentially being involved in monoterpene metabolism.Overexpression of VvbZIP61 in Vitis amurensis callus through Agrobacterium-mediated transformation significantly increased the accumulation of several monoterpenes in the callus,including nerol,linalool,geranial,geraniol,β-myrcene,and D-limonene.It is hypothesized that VvbZIP61 expression acts to increase muscat flavor in grapes.These results advance our understanding of the genetic control of monoterpene biosynthesis in grapes and provide important information for the marker-assisted selection of aroma compounds in grape breeding.