Functional Characterization of Proanthocyanidin Pathway Enzymes from Tea and Their Application for Metabolic Engineering    

Abstract

Tea (Camellia sinensis) is rich in specialized metabolites, especially polyphenolic proanthocyanidins (PAs) and their precursors. To better understand the PA pathway in tea, we generated a complementary DNA library from leaf tissue of the blister blightresistant tea cultivar TRI2043 and functionally characterized key enzymes responsible for the biosynthesis of PA precursors. Structural genes encoding enzymes involved in the general phenylpropanoid/flavonoid pathway and the PA-specific branch pathway were well represented in the library. Recombinant tea leucoanthocyanidin reductase (CsLAR) expressed in Escherichia coli was active with leucocyanidin as substrate to produce the 2R,3S-trans-flavan-ol (+)-catechin in vitro. Two genes encoding anthocyanidin reductase, CsANR1 and CsANR2, were also expressed in E. coli, and the recombinant proteins exhibited similar kinetic properties. Both converted cyanidin to a mixture of (+)-epicatechin and (2)-catechin, although in different proportions, indicating that both enzymes possess epimerase activity. These epimers were unexpected based on the belief that tea PAs are made from (2)-epicatechin and (+)-catechin. Ectopic expression of CsANR2 or CsLAR led to the accumulation of low levels of PA precursors and their conjugates in Medicago truncatula hairy roots and anthocyanin-overproducing tobacco (Nicotiana tabacum), but levels of oligomeric PAs were very low. Surprisingly, the expression of CsLAR in tobacco overproducing anthocyanin led to the accumulation of higher levels of epicatechin and its glucoside than of catechin, again highlighting the potential importance of epimerization in flavan-3-ol biosynthesis. These data provide a resource for understanding tea PA biosynthesis and tools for the bioengineering of flavanols.

Flavan-3-ols Are an Effective Chemical Defense against Rust Infection

“…Two distinct enzymes, LAR and ANR, are involved in catalyzing the last steps of the pathway to flavan-3-ol monomers in PA-producing plants (Bogs et al, 2005;Pang et al, 2013;Liao et al, 2015). Genes encoding LAR and ANR can occur as single genes, for example in Arabidopsis (Xie et al, 2004), or as multigene families, for example in grapevine (Vitis vinifera; Figure 8.…”

“…DW, Dry weight; pCambia, vector control; WT, wild type. Bogs et al, 2005) and tea (Camellia sinensis; Pang et al, 2013). Analysis of the P. trichocarpa genome revealed three loci encoding LAR proteins and two loci encoding ANR proteins.…”

“…The biosynthesis of flavan-3-ols and PAs has been studied in many plant species (Xie et al, 2004;Bogs et al, 2005;Paolocci et al, 2007;Pang et al, 2013). The last steps of monomer biosynthesis are catalyzed by two distinct enzymes.…”

“…For the biosynthesis of 2,3-trans-(6)-flavan-3-ols (e.g. catechin), leucoanthocyanidins are reduced directly to the corresponding flavan-3-ol (Tanner et al, 2003;Pang et al, 2013) by leucoanthocyanidin reductase (LAR). For the biosynthesis of the 2,3-cis-type compounds (e.g.…”

Identification and characterization of <i>Camellia sinensis</i> glucosyltransferase, UGT73A17: A possible role in flavonol glucosylation

“…A wide variety of flavonoid-class specialized metabolites, such as flavan-3-ols and flavonols are accumulated in the fresh leaves and processed tea products, and some have been shown to have beneficial activities for human health (Lin et al 2003). Flavonols, a major subclass of flavonoids, are biosynthesized by a 2-oxoglutarate-Fe(II) dioxygenase superfamily enzyme, flavonol synthase (Lin et al 2007) and accumulated as O-glycosylated forms (flavonol glycosides represented by rutin (quercetin 3-O-β-glucopyranosyl-6-O-α-rhamnoside)) via glycosylation in C. sinensis, whereas flavan-3-ols (catechins and theaflavins) are present either in free forms or as gallic acid esters (Liu et al 2012;Pang et al 2013). Glycosylation of small molecules, such as specialized metabolites, alters their reactivity and stability, thereby regulating the cellular transport and storage of metabolites.…”