Abstract
BACKGROUND: A multi-monocistronic synthetic vector was used to assemble multiple genes of a nucleotide diphosphate (NDP)-sugar biosynthetic pathway to construct robust genetic circuits for the production of valuable flavonoid glycosides in Escherichia coli. Characterized functional genes involved in the biosynthesis of uridine diphosphate (UDP)-glucose and thymidine diphosphate (TDP)-rhamnose from various microbial sources along with glucose facilitator diffusion protein (glf) and glucokinase (glk) from Zymomonas mobilis were assembled and overexpressed in a single synthetic multi-monocistronic operon. RESULTS: The newly generated 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 UDP-glucose and TDP-rhamnose biosynthesis systems respectively, after 48 h of incubation in 3 L fermentor while supplementing 0.9 g of fisetin. These yields of fisetin glycosides represent ~99% of bioconversion of exogenously supplemented fisetin. The systems were also found to be highly effective in bio-transforming other flavonols (quercetin, kaempferol, myricetin) into their respective glycosides, achieving over 95% substrate conversion. CONCLUSION: The construction of a synthetic expression vector for bacterial cell factory followed by subsequent re-direction of metabolic flux towards desirable products have always been revolutionized the biotechnological processes and technologies. This multi-monocistronic synthetic vector in a microbial platform is customizable to defined task and would certainly be useful for applications in producing and modifying such therapeutically valued plant secondary metabolites.