Biosynthesis of flavone C-glucosides in engineered Escherichia coli

Abstract

Two plant-originated C-glucosyltransferases (CGTs) UGT708D1 from Glycine max and GtUF6CGT1 from Gentiana triflora were accessed for glucosylation of selected flavones chrysin and luteolin. Uridine diphosphate (UDP)-glucose pool was enhanced in Escherichia coli cell cytosol by introducing heterologous UDP-glucose biosynthetic genes, i.e., glucokinase (glk), phosphoglucomutase (pgm2), and glucose 1-phosphate uridylyltransferase (galU), along with glucose facilitator diffusion protein from (glf) from different organisms, in a multi-monocistronic vector with individual T promoter, ribosome binding site, and terminator for each gene. The C-glucosylated products were analyzed by high-performance liquid chromatography-photodiode array, high-resolution quadruple time-of-flight electrospray ionization mass spectrometry, and one-dimensional nuclear magnetic resonance analyses. Fed-batch shake flask culture showed 8% (7 mg/L; 16 μM) and 11% (9 mg/L; 22 μM) conversion of chrysin to chrysin 6-C-β-D-glucoside with UGT708D1 and GtUF6CGT1, respectively. Moreover, the bioengineered E. coli strains with exogenous UDP-glucose biosynthetic genes and glucose facilitator diffusion protein enhanced the production of chrysin 6-C-β-D-glucoside by approximately 1.4-fold, thus producing 10 mg/L (12%, 24 μM) and 14 mg/L (17%, 34 μM) by UGT708D1 and GtUF6CGT1, respectively, without supplementation of additional UDP-glucose in the medium. The biotransformation was further elevated when the bioengineered strain was scaled up in lab-scale fermentor at 3 L volume. HPLC analysis of fermentation broth extract revealed 50% (42 mg/L, 100 μM) conversion of chrysin to chrysin 6-C-β-D-glucoside at 48 h upon supplementation of 200 μM of chrysin. The maximum conversion of luteolin was 38% (34 mg/L, 76 μM) in 50-mL shake flask fermentation at 48 h. C-glucosylated derivative of chrysin was found to be more soluble and more stable to high temperature, different pH range, and β-glucosidase enzyme, than O-glucosylated derivative of chrysin.

Pathway enzyme engineering for flavonoid production in recombinant microbes

“…The sugar units for disaccharides are rutinose, sophorose, and sambubiose. Some studies have shown the biotransformation of flavonoid glycosides in recombinant E. coli or other microbial cells (Koirala et al., 2014; Pandey et al., 2016; Ruprecht et al., 2019; Shrestha et al., 2018; Wang et al., 2016a). In addition, several nucleotide sugar biosynthetic pathways are engineered in E. coli for regioselective biotransformation and high titer production of flavonoid glycosides (Leonard et al., 2008; Yan et al., 2008).…”

Combinatorial approach for improved cyanidin 3-O-glucoside production in Escherichia coli

“…Although several glucose transporter genes are known in E. coli system, we choose glf - glk system for this study as it is well-characterized and readily available in our lab. Additionally, the working efficiency of this system has been already demonstrated to produce plant polyphenol glycosides and other polyketides in the previous studies [31, 40, 41]. This system increased UDP- d -glucose production, thereby increasing the donor pool for subsequent glycosylation by the At 3GT gene to produce C3G.…”

A Synthetic Approach for Biosynthesis of Miquelianin and Scutellarin A in Escherichia coli

“…Using this approach, several flavonoid glycosides are produced from E. coli. For example, 3-O-rhamosyl quercetin, 3-O-rhamnosyl kaempferol, and 3-O-allosyl quercetin [27], astragalin [28], quercetin-3-O-4-deoxy-4-formamido-L-arabinose [29], quercetin 3-O-N-acetylglucosamine [30], quercetin-3-O xyloside [31] and quercetin 3-O-arabinoside [32], flavonol 3-O-rhamnosides [33,34], flavone 6-C-glucosides [35] are some representative flavonoid glycosides produced from E. coli. Using a similar approach, several polyketide glycosides have been produced from engineered microbial hosts [36].…”