Synthetic Sugar Cassettes for the Efficient Production of Flavonol Glycosides in Escherichia coli

A multi-monocistronic synthetic vector system was employed to construct robust genetic circuits for the production of valuable flavonoid glycosides in Escherichia coli. Characterized functional genes involved in the uridine diphosphate (UDP)-glucose and thymidine diphosphate (TDP)-rhamnose biosynthesis from various microbial sources; and glucose facilitator diffusion protein (glf) and glucokinase (glk) from Zymomonas mobilis were assembled in a synthetic multi-monocistronic vector. The newly generated nucleotide diphosphate (NDP)-sugars biosynthesis circuits along with regiospecific glycosyltransferases from plants were introduced in E. coli BL21 (DE3) to probe the bioconversion of fisetin, a medicinally important polyphenol produced by various plants. As a result, approximately 1.178 g of fisetin 3-O-glucoside and ~1.026 g of fisetin 3-O-rhamnoside were produced in TDP-rhamnose and UDP-glucose systems, respectively under optimized conditions in 48 h of incubation in 3 Liter fermentor while supplementing 0.750 g of fisetin representing ~99 % substrate conversion. Moreover, both systems efficiently biotransformed other flavonols (quercetin, kaempferol, myricetin) to respective glycosides reaching more than 95% substrate conversion except in morin.

Key words: Sugar cassettes, Glycosyltransferase, multi-momocistronic vector, flavonol glycosides.