Abstract
BackgroundA 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.ResultsThe 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.ConclusionThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0261-1) contains supplementary material, which is available to authorized users.
Highlights
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
The UDP-glucose system consists of three UDPglucose biosynthesis genes: glucokinase which catalyzes the addition of phosphate groups into the 6-hydroxyl position of d-glucose immediately after entry into the cell, phosphoglucomutase which synthesizes glucose 1-phosphate from glucose-6-phosphate, and glucose 1-phosphate uridylyltransferase which transfers the uridine diphosphate (UDP) group to make UDPglucose
In addition to the above three genes, glucose facilitator diffusion protein, which helps in the internalization of extracellular glucose present in the medium into the cell to increase the pool of UDPglucose and the regiospecific flavonol 3-O-glycosyltransferase (UGT78K1; 1,344 bp), which catalyzes the transfer of glucose moiety from activated UDP-glucose into the acceptor flavonols at the 3-hydroxyl position, were assembled in the same vector to generate a 7,312 bp UDP-glucose system
Summary
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. Most flavonoid drugs (quercetin 3-O-rutinoside, daidzein 8-C-glucoside) presently in clinical applications are in the form of glycosides [1]. These significance have attracted considerable interest in current research trend to produce them rationally in industrial scale. Microbial biotransformation is one of the biotechnological approach for the production of such bioactive flavonoids and their glycosides in desirable scale [1, 9]. Biotransformation of flavonoids could be achieved from many microorganisms including species of Aspergillus, Bacillus, Saccharomyces, Streptomyces, Escherichia coli [1, 10]
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