Development of an in vivo glucosylation platform by coupling production to growth: Production of phenolic glucosides by a glycosyltransferase of Vitis vinifera

Abstract

Glycosylation of small molecules can significantly alter their properties such as solubility, stability, and/or bioactivity, making glycosides attractive and highly demanded compounds. Consequently, many biotechnological glycosylation approaches have been developed, with enzymatic synthesis and whole-cell biocatalysis as the most prominent techniques. However, most processes still suffer from low yields, production rates and inefficient UDP-sugar formation. To this end, a novel metabolic engineering strategy is presented for the in vivo glucosylation of small molecules in Escherichia coli W. This strategy focuses on the introduction of an alternative sucrose metabolism using sucrose phosphorylase for the direct and efficient generation of glucose 1-phosphate as precursor for UDP-glucose formation and fructose, which serves as a carbon source for growth. By targeted gene deletions, a split metabolism is created whereby glucose 1-phosphate is rerouted from the glycolysis to product formation (i.e., glucosylation). Further, the production pathway was enhanced by increasing and preserving the intracellular UDP-glucose pool. Expression of a versatile glucosyltransferase from Vitis vinifera (VvGT2) enabled the strain to efficiently produce 14 glucose esters of various hydroxycinnamates and hydroxybenzoates with conversion yields up to 100%. To our knowledge, this fast growing (and simultaneously producing) E. coli mutant is the first versatile host described for the glucosylation of phenolic acids in a fermentative way using only sucrose as a cheap and sustainable carbon source.

Metabolic engineering of Escherichia coli into a versatile glycosylation platform: production of bio-active quercetin glycosides

“…To this end, the wild type (WT) strain was grown on minimal sucrose medium containing different concentrations of quercetin (0, 0.15 and 1.5 g/L). The specific growth rates (h −1 ) (0.96 ± 0.06, 0.92 ± 0.05 and 0.87 ± 0.06, respectively) were not significantly different (p ANOVA = 0.12) nor from the one previously determined for the WT [ 45 ] ( p = 0.69, p = 0.98 and p = 0.68, respectively). On the other hand, the optical density at 600 nm after 24 h incubation (6.36 ± 0.12, 5.18 ± 0.16 and 4.77 ± 0.20, respectively) was lower (about 20 %) when quercetin was added ( p = 0.0002 and p = 0.0001).…”

“…To tackle these problems, we previously developed an efficient platform for the glucosylation of small molecules in E. coli W [ 45 ]. Through metabolic engineering, a mutant was created which couples the production of glucosides to growth, using sucrose as a cheap and sustainable carbon source.…”

Diversity of phosphorylases in glycoside hydrolase families

“…UDP-Glc was prepared using engineered E. coli cells expressing sucrose phosphorylase as the catalysts (Weyler and Heinzle 2015). De Bruyn et al (2015) introduced the sucrose phosphorylase gene in E. coli to increase fermenting production of phenolic glucosides using glucosyltransferase and enhancing the intracellular pool of UDP-Glc.…”

Lignin valorization meets synthetic biology

“…Vanillin glucoside was produced from ferulic acid by coexpression of VpScVAN and AtUGT72E2 in S. cerevisiae . β‐Glucogallin was obtained when gallic acid, a intermediate of lignin degradation, was glycosylated by overexpression of a glucosyltransferase gene VvGT 2 from Vitis vinifera in E. coli . These studies suggest the lignin‐derived aromatics such as p ‐coumaric acid, ferulic acid, and p HBA could be converted to value‐added bio‐active substances such as flavonoids, stilbenoids, and coumarins and their glycosylated products by building artificial pathway.…”