Identification of a Saccharomyces cerevisiae Glucosidase That Hydrolyzes Flavonoid Glucosides

Abstract

Baker's yeast (Saccharomyces cerevisiae) whole-cell bioconversions of naringenin 7-O-␤-glucoside revealed considerable ␤-glucosidase activity, which impairs any strategy to generate or modify flavonoid glucosides in yeast transformants. Up to 10 putative glycoside hydrolases annotated in the S. cerevisiae genome database were overexpressed with His tags in yeast cells. Examination of these recombinant, partially purified polypeptides for hydrolytic activity with synthetic chromogenic ␣-or ␤-glucosides identified three efficient ␤-glucosidases (EXG1, SPR1, and YIR007W), which were further assayed with natural flavonoid ␤-glucoside substrates and product verification by thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC). Preferential hydrolysis of 7-or 4-O-glucosides of isoflavones, flavonols, flavones, and flavanones was observed in vitro with all three glucosidases, while anthocyanins were also accepted as substrates. The glucosidase activities of EXG1 and SPR1 were completely abolished by Val168Tyr mutation, which confirmed the relevance of this residue, as reported for other glucosidases. Most importantly, biotransformation experiments with knockout yeast strains revealed that only EXG1 knockout strains lost the capability to hydrolyze flavonoid glucosides.Glycoside hydrolases, in particular glucosidases (EC 3.2.1.-), are widespread in pro-and eukaryotic organisms and play a pivotal role in many biological processes, such as the metabolism of oligosaccharides or the degradation of endogenous and exogenous glycosides. Beta-glucosidases (␤-GHs) are among the oldest classes of enzymes, and microbial ␤-GHs have been identified often as molecular factors indispensable for growth; for example, they enable phytopathogenic fungi to colonize host plant tissues by hydrolyzing plant fungitoxic glucosides to less toxic or less soluble aglyca (5). They are also important for cleaving cellulose and fulfill essential functions in the sporulation of yeast cells (27,38). In plants, ␤-GHs are involved in crucial growth processes, such as the degradation of endosperm cell walls during germination or the formation of intermediates in cell wall lignification, as well as in the activation of defense compounds and the formation of phytohormones (references 49 and 58 and references therein). Moreover, plant ␤-GHs are essential for turnover of flavonoid glucosides, which are exclusively found in ␤ configuration, as had been reported for malonylglucosides of the isoflavones genistein and daidzein in soybean (21) or isoflavone 7-O-␤-glucosides in chickpea (20). Even the endophytic bacterium Pseudomonas strain ZD-8 (61), as well as cell-associated ␤-GHs (bglH and yckE) from Bacillus subtilis natto, used for fermentation of soy products (28), were shown to metabolize apigenin 7-O-␤-glucoside or glucosides and malonylglucosides of genistein and daidzein. The capacity of multiple Bifidobacterium strains of human origin to digest isoflavonoid glucosides (32, 41) is particularly noteworthy because of their releva...

Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5)

“…Surprisingly, the periplasmic Exg1 protein from Schizosaccharomyces pombe was demonstrated to be an endo -β-1,6-glucanase (EC 3.2.1.75), while the membrane-anchored protein Exg2 protein was shown to produce cell wall material when over-expressed [52]. Two of the characterized GH5_9 exo -β-1,3-glucanases have been also described as β-glucosidases (EC 3.2.1.21) [53]. …”

Metagenomic Evidence for H2 Oxidation and H2 Production by Serpentinite-Hosted Subsurface Microbial Communities

“…No sequences encoding acetyl-CoA synthase were detected at Lost City, but the phylogeny of ACS sequences from WHC2b is consistent with the presence of clostridial acetogens in very low abundance. Acetogens are known to be capable of producing H 2 and harboring a wide diversity of [FeFe]-hydrogenases (Kellum and Drake, 1984; Schmidt et al, 2010, 2011), so determining the role of these Clostridia in the H 2 budget of these systems will require physiological and biogeochemical investigations. Only three ACS sequences were recovered from WHC2b, however, and none of these were assembled into contigs.…”

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

“…β-Glucosidases can also be used for debittering of fruit juices by hydrolysis of bitter compounds such naringenin and oleuropein [215,216]. Another important application of β-glucosidase is cassava detoxification, a carbohydrate rich food and a staple food in tropical countries.…”