A genomic approach to isoflavone biosynthesis in kudzu (Pueraria lobata)

Abstract

Roots of kudzu (Pueraria lobata) are a rich source of isoflavone O- and C-glycosides. Although O-glycosylation of (iso)flavonoids has been well characterized at the molecular level, no plant isoflavonoid C-glycosyltransferase genes have yet been isolated. To address the biosynthesis of kudzu isoflavonoids, we generated 6,365 high-quality expressed sequence tags (ESTs) from a subtraction cDNA library constructed using RNA from roots that differentially accumulate puerarin. The ESTs were clustered into 722 TCs and 3,913 singletons, from which 15 family I glycosyltransferases (UGTs) were identified. Hierarchical clustering analysis of the expression patterns of these UGTs with isoflavone synthase (IFS) in a range of tissues identified UGTs with potential functions in isoflavone glycosylation. The open reading frames of these UGTs were expressed in E. coli for functional analysis, and one was shown to preferentially glycosylate isoflavones at the 7-O-position. In addition, ESTs corresponding to chalcone synthase, chalcone reductase, chalcone isomerase (CHI) and 2-hydroxyisoflavanone dehydratase were identified. Recombinant CHI proteins had high activities with both 6'-deoxy- and 6'-hydroxy chalcones, typical of Type II CHIs. Establishment of this EST database and identification of genes associated with kudzu isoflavone biosynthesis and glycosylation provide a new resource for metabolic engineering of bioactive kudzu isoflavones.

Involvement of three putative glucosyltransferases from the UGT72 family in flavonol glucoside/rhamnoside biosynthesis inLotus japonicusseeds

“…GT04F14 from P. montana var. lobata and UGT73F2 from G. max displayed broad substrate specificity toward isoflavones, flavones, and flavonol aglycones, including daidzein, genistein, quercetin, and apigenin (Dhaubhadel et al , 2008; He et al , 2011). Therefore, UG72AF1 and UGT72V3 appear to be quite typical in terms of their broad substrate preference in vitro as compared to UGT proteins of other legume species.…”

“…lobata , C. arietinum, and P. vulgaris (Supplementary Table S4), but the UGT proteins from these species either did not show any activities toward flavonoids, such as GT01K01 from P. montana var. lobata (He et al , 2011), or are only predicted as putative anthocyanidin glucosyltransferases on the basis of sequence similarity. Therefore, these UGT proteins may be redundant genes or they may utilize substrates other than flavonoid aglycones.…”

Improving Secondary Metabolite Production in Tissue Cultures

“…Biotechno-logical production of isofl avones, mainly from the family Fabaceae, is based on suspension cultures of Pueraria sp. (Goyal and Ramawat 2008a , b ;Sharma et al 2009 ;He et al 2011 ), Psoralea sp. (Shinde et al 2009a , b ), and Glycine max (Federici et al 2003 ;Terrier et al 2007 ).…”

Expression and Functional Analysis of a Transgenic Cytochrome P450 Monooxygenase in Pueraria mirifica

“…In Pueraria, the whole processes of isoflavonoids biosynthesis include two main stages (Dhaubhadel 2011;Franzmayr et al 2012;He et al 2011). The first stage is the biosynthesis of the common precursor of isoflavonoid, the 4-coumaroyl-CoA, which is synthesized orderly from phenylalanine by phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (C4H) and 4-coumarate: CoA ligase (4CL).…”