Enzymatic Synthesis of Novel Phloretin Glucosides

Abstract

A UDP-glycosyltransferase from Bacillus licheniformis was exploited for the glycosylation of phloretin. The in vitro glycosylation reaction confirmed the production of five phloretin glucosides, including three novel glucosides. Consequently, we demonstrated the application of the same glycosyltransferase for the efficient whole-cell biocatalysis of phloretin in engineered Escherichia coli. P hloretin is a dihydrochalcone, an intermediate of the biosynthetic pathway of flavonoids in plants, which is abundantly present in the peel of apple (1, 2) and in strawberries (3). They occur in different glycosidic forms, such as naringin dihydrochalcone, phlorizin, and phloretin-4=-O-glucoside, in the different parts of the plants, contributing to various physiological properties of the plants, as well as to their color. Phloretin and its glycosides have been determined to have beneficial biological activities. Studies have uncovered that phloretin has inhibitory activity against glucose cotransporter 1 (4, 5), antioxidant activity (6), and activity to suppress the tumor necrosis factor alpha-induced inflammatory response, ameliorate inflammation of the colon, positively affect body weight loss (7), modulate Ca 2ϩ -activated K ϩ channels, and increase endothelial nitric oxide production, which might help to protect against atherosclerosis (8). Importantly, phloretin has other biological functions, like anticarcinogenic (9) and estrogenic activities (10) and inhibition of cardiovascular disease (11, 12). Irrespective of their diverse physiological and pharmacological activities, the use of most of the natural polyphenols as drugs and food additives has been limited because of their water insolubility and low absorbability. Glycosylation enhances the bioavailability and pharmacological properties of compounds by increasing their solubility and stability (13,14). Importantly, the sugar moieties of the glycosides often participate in the specific recognition of their biological targets and help to determine their efficacy in drug development (14, 15). According to the CAZy database (http://www .cazy.org/) (16,17,18), glycosyltransferase family 1 (GT1) proteins contain the UDP-glycosyltransferases that are common in all domains of life (19) and predominantly recognize small molecules as the sugar acceptors. A recent report showed that YjiC, a Bacillus licheniformis UDP-glycosyltransferase that falls in the GT1 family of proteins, can glycosylate at different hydroxyl positions of geldanamycin analogs (20). Here, we report the use of this glycosyltransferase for the biosynthesis of diverse phloretin glucosides in vitro and the subsequent application of YjiC for in vivo production of phloretin glucosides in an Escherichia coli mutant generating a cytoplasmic pool of UDP-glucose, since the YjiC-homologous glycosyltransferases from other Bacillus species were found to have flexible glycosyltransferase activities toward different flavonoid groups of compounds. Moreover, we found that by reversing the glycosylation reaction, the enz...

Enzymatic Biosynthesis of Novel Resveratrol Glucoside and Glycoside Derivatives

“…However, an unexpected result was that the concentration of resveratrol 3-O-␤-D-glucoside was higher after a 1-h incubation period and lower concentrations were detected after 2 h and 3 h of incubation (data not shown). This decrease in the amount of product initially formed could be due to the instability of the product, or the product may be readily converted to resveratrol and UDP-D-glucose in a reverse-glycosylation reaction mediated by YjiC as previously reported for phloretin glycosylation (56). In addition, the product However, resveratrol 3,5,4=-O-␤-D-triglucoside was not detected, and this may be due to a lack of excess UDP-D-glucose in the reaction mixture.…”

“…AAU40842.1) from Bacillus licheniformis DSM 13, with a flexible donor and acceptor substrate profile was selected in order to synthesize glycodiversified resveratrol derivatives. YjiC has been shown to accept geldanamycin analogues (52), epothilone A (53), various flavonoids (54,55), chalcone (56), and isoflavonoids (57) as substrates for glucosylation. In this study, resveratrol was used as an acceptor substrate and various rare nucleotide diphosphate sugars were used as sugar donor substrates to produce glycodiversified resveratrol derivatives that have not been previously described.…”

Biotransformation of phloretin by amylosucrase yields three novel dihydrochalcone glucosides

“…Phloretin (Calliste et al, 2001;Chao and Henry, 2010;Rezk et al, 2002;Sukhorukov et al, 2001) and its most abundant and best characterized glycoside, phloretin 2'-␤-d-glucopyranoside or phlori(d)zin (Boccia et al, 1999;Ehrenkranz et al, 2005;Veeriah et al, 2006), are known to exert a wide variety of biological effects. Other characterized phloretin glycosides that have either been isolated from natural sources or have been chemically or enzymatically synthesized are the ribose analog of phloridzin (Gupte and Buolamwini, 2009), a 2'-␤-xylogalactoside (Lu and Foo, 1997), the 4'-␤-d-glucoside (Farkas et al, 1965;Gosch et al, 2010;Jorio, 1959;Pandey et al, 2013), a 4'-glucorhamnoside (Jorio, 1959), a number of 4'-␤-linked disaccharides (Kamiya et al, 1975), the 4,4'-, 4,2'-and 4,2',4'-␤-d-glucosides (Pandey et al, 2013) and the 3',5'-␤-d-glucoside (Ito et al, 2014;Sato et al, 2006). We now report the first synthesis of phloretin glycosides in which the sugar is attached in the ␣ configuration.…”

Stimulation of Erythrocyte Death by Phloretin

“…Effects of Phloretin include decrease of oxidative stress [11], inhibition of inflammation [12], increase endothelial nitric oxide production and vasodilation [13,14], downregulation of endothelial adhesion molecules [15], and inhibition of platelet activation [15]. Some of the effects are due to estrogenic activity of the substance [13,14,16].…”