Metabolic Engineering of Escherichia coli for Hyperoside Biosynthesis

Abstract

Hyperoside (quercetin 3-O-galactoside) exhibits many biological functions, along with higher bioactivities than quercetin. In this study, three UDP-dependent glycosyltransferases (UGTs) were screened for efficient hyperoside synthesis from quercetin. The highest hyperoside production of 58.5 mg·L−1 was obtained in a recombinant Escherichia coli co-expressing UGT from Petunia hybrida (PhUGT) and UDP-glucose epimerase (GalE, a key enzyme catalyzing the conversion of UDP-glucose to UDP-galactose) from E. coli. When additional enzymes (phosphoglucomutase (Pgm) and UDP-glucose pyrophosphorylase (GalU)) were introduced into the recombinant E. coli, the increased flux toward UDP-glucose synthesis led to enhanced UDP-galactose-derived hyperoside synthesis. The efficiency of the recombinant strain was further improved by increasing the copy number of the PhUGT, which is a limiting step in the bioconversion. Through the optimization of the fermentation conditions, the production of hyperoside increased from 245.6 to 411.2 mg·L−1. The production was also conducted using a substrate-fed batch fermentation, and the maximal hyperoside production was 831.6 mg·L−1, with a molar conversion ratio of 90.2% and a specific productivity of 27.7 mg·L−1·h−1 after 30 h of fermentation. The efficient hyperoside synthesis pathway described here can be used widely for the glycosylation of other flavonoids and bioactive substances.

Isolation, identification, and biochemical characterization of a novel bifunctional phosphomannomutase/phosphoglucomutase from the metagenome of the brown alga Laminaria digitata

“…Similarly, the PGM from Thermococcus kodakaraensis has also recently been used in a recombinant E. coli vector system for the production of another food sweetener, namely D-tagatose from maltodextrin using whole-cell catalysts (Dai et al, 2022); while a thermostable PGM from T. kodakaraensis has been used in the production of myo-inositol, an important human dietary supplement, from starch (Fujisawa et al, 2017). A recombinant PGM from E. coli has also been used to enhance UDP-galactose derived synthesis of the bioactive compound hyperoside (quercetin 3-O-galactoside) in a metabolically engineered E. coli strain (Li et al, 2022). In addition, PGM from T. kodakarensis has recently been heterologously expressed in B. subtilis thereby allowing for the recombinant enzyme to be produced at high levels in highdensity fermentations for subsequent potential synthesis of inositol from starch (Ye et al, 2022).…”

“…The biochemical characteristics of 10L6AlgC may have useful industrial biotechnological applications. In particular, the temperature (45°C) and pH (8.0) optima may be of greater interest than currently utilized enzymes (C. thermocellum [70°C; pH 8.8] (Wang and Zhang, 2010); T. kodakaraensis [90°C, pH 7.0] (Rashid et al, 2004); engineered E. coli [20°C] (Li et al, 2022)). Thus, it is clear that 10L6AlgC possesses a unique biochemical profile when compared to previously characterized PMM/PGM bifunctional enzymes and may find utility in enzyme-based production of biochemicals with different potential industrial applications, in which other bacterial PMM/PGMs have previously been used.…”

Enhancing Glycosylation of Flavonoids by Engineering the Uridine Diphosphate Glucose Supply in Escherichia coli

“…With the blocking of the glycolytic and UDPG branching pathways, screening and overexpression of UDPG pathway genes, and stimulation of the UDPG cycle, the supply of glycosyl donors was significantly enhanced. To further expand the platform strain, not only were UDP rhamnose and UDP galactose synthesis pathways constructed, but rhamnosyltransferase (GtfC) 26 and galactosyltransferase (PhUGT) 27 were also introduced to synthesize afzelin, quercitrin, hyperoside, and trifolin. In a 5 L bioreactor containing apigenin, luteolin, kaempferol, and quercetin as glycosyl acceptors, glycosylation products vitexin (17.2 g/L), orientin (36.5 g/L), afzelin (5.2 g/L), quercitrin (14.1 g/L), hyperoside (6.4 g/L), and trifolin (11.4 g/L) reached their highest titers reported to date (Table S5 of the Supporting Information).…”

Hyperoside prevents doxorubicin‐induced cardiotoxicity by inhibiting NOXs/ROS/NLRP3 inflammasome signaling pathway

“…Traditional Chinese medicine has attracted increasing attention in cardiovascular medicine research due to its potential ability to target a number of signaling pathways (Ferenczyova et al, 2020), and the beneficial effects of natural compounds against Dox‐induced cardiotoxicity have received substantial interest in recent years (Tabrizi et al, 2022; Yarmohammadi et al, 2021). Hyperoside (quercetin 3‐O‐galactoside) is a natural product isolated from variety of herb medicines, such as Semen Cuscutae, hawthorn and artemisia capillaris (Li et al, 2022). The potential role of hyperoside in the anti‐inflammatory (Kim et al, 2011), hepato‐protective (Wang et al, 2021) and cardiac‐protective (Wang et al, 2018) effects have been reported recently.…”

“…In myocardial infarction, hyperoside presented beneficial effects by inhibiting the NLRP1 inflammasome (Yang et al, 2021). Clinically, hyperoside is the main active ingredient of Xinxuening tablets and Xin'an capsules, two kinds of Chinese patent medicines that are mainly used in coronary heart disease, angina pectoris, and hypertension (Li et al, 2022). However, the role of hyperoside in Dox‐induced cardiotoxicity and its potential mechanism has yet to be elucidated.…”