Arabidopsisglycosyltransferases as biocatalysts in fermentation for regioselective synthesis of diverse quercetin glucosides

Abstract

Regioselectivity of glycosyltransferases offers an important means to overcome the limitations of chemical synthesis of small molecule glycosides. In this study we explore a large multigene family of UDP-glucose:glycosyltransferases of Arabidopsis for their potential as novel biocatalysts for in vitro synthesis and whole-cell catalysis. We used quercetin as a substrate for this study because the flavonol and its glycosides have important medicinal properties and the metabolite provides a complex structure for regioselective glucosylation. We analyzed the activity of 91 recombinant enzymes for in vitro activity toward quercetin and discovered 29 that are capable of glucosylating the substrate. We demonstrate the first enzymic synthesis of a range of glucosides in vitro, including the 3-O-, 7-O-, 3'-O-, and 4'-O-monoglucosides, 3,7-di-O-glucoside, and 7,3'-di-O-glucoside. We also show that the regioselectivity of glucosylation can be maintained when the enzymes are used as whole-cell biocatalysts in Escherichia coli.

Mutational Analysis of the Medicago Glycosyltransferase UGT71G1 Reveals Residues That Control Regioselectivity for (Iso)flavonoid Glycosylation

“…Recently, there has been increasing interest in biocatalytic synthesis of glucosides of quercetin (27,37) and other phenolic compounds (38) due to their potential therapeutic value in cardiovascular disease and cancer, and their antimicrobial properties. Specific quercetin mono-or diglucosides have been successfully produced in an E. coli fermentation system employing regiospecific glycosyltransferases (27,37) The majority of the soluble glucoside products are secreted to the medium that facilitates their purification. We have now demonstrated that new glycosyltransferases with altered regioselectivity for quercetin can be obtained through targeted mutagenesis.…”

“…Reactions were stopped with 5 l of trichloroacetic acid (240 mg/ml) and extracted with 250 l of ethyl acetate. The extracted residues were resuspended in methanol and analyzed by reverse-phase HPLC (Hewlett Packard 1100 system) on a The glycosylated products were determined according to their relative retention times (27), UV spectra, and comparison to authentic standards. For kinetic studies, reaction mixtures contained 50 mM Tris-HCl (pH 7.0), 14 mM 2-mercaptoethanol, 10 M UDP-[U-…”

“…Eleven of the UGTs produced two products, glycosylating the 7-and 3-hydroxyls, 7-and 3Ј-hydroxyls, 7 and 4Ј-hydroxyls, or 3-and 4Ј-hydroxyls. The remaining enzymes produced three or four monoglucosides substituted on different hydroxyl groups (27). Some of these UGTs also glycosylated additional substrates such as caffeic acid (UGT71C1) (28), indole-3-acetic acid (UGT84B1) (29), or salicylic acid (UGT74F1) (24).…”

Redox-Dependent Modulation of Anthocyanin Biosynthesis by the TCP Transcription Factor TCP15 during Exposure to High Light Intensity Conditions in Arabidopsis

“…1). Notably, genes encoding enzymes involved in the synthesis of isoflavonoids, flavonols, and proanthocyanidins, which share metabolic precursors with anthocyanins, did not show changes in expression or were repressed, as in the case of the flavonol biosynthetic genes FLS2 (Owens et al, 2008;Guo et al, 2014), UGT78D1 (Jones et al, 2003), and UGT71D1 (Lim et al, 2004) (Supplemental Table S1). …”

Glucosylation of hydroxyflavones by glucosyltransferases from Phytolacca americana

“…The amino acid sequence of PaGT3 is homologous to that of flavonol 3-O-glucosyltransferase from A. thaliana (identity 56%) [25] and Fragaria x ananassa (identity 57%) [26], anthocyanin 3'-O-glucosyltransferase from Gentiana triflora (identity 56%) [27], and flavonol 7-O-glucosyltransferase from A. thaliana (identity 54%) [25]. Sequence identity between PaGT3 and PaGT2 is 27%, and PaGT2 is related to hydroquinone glucosyltransferase from Rauvolfia serpentine (identity 58%) [28] and coniferyl alcohol 4-O-glucosyltransferase from A. thaliana (identity 39%) [29].…”