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

Abstract

BackgroundFlavonoids are bio-active specialized plant metabolites which mainly occur as different glycosides. Due to the increasing market demand, various biotechnological approaches have been developed which use Escherichia coli as a microbial catalyst for the stereospecific glycosylation of flavonoids. Despite these efforts, most processes still display low production rates and titers, which render them unsuitable for large-scale applications.ResultsIn this contribution, we expanded a previously developed in vivo glucosylation platform in E. coli W, into an efficient system for selective galactosylation and rhamnosylation. The rational of the novel metabolic engineering strategy constitutes of the introduction of an alternative sucrose metabolism in the form of a sucrose phosphorylase, which cleaves sucrose into fructose and glucose 1-phosphate as precursor for UDP-glucose. To preserve these intermediates for glycosylation purposes, metabolization reactions were knocked-out. Due to the pivotal role of UDP-glucose, overexpression of the interconverting enzymes galE and MUM4 ensured the formation of both UDP-galactose and UDP-rhamnose, respectively. By additionally supplying exogenously fed quercetin and overexpressing a flavonol galactosyltransferase (F3GT) or a rhamnosyltransferase (RhaGT), 0.94 g/L hyperoside (quercetin 3-O-galactoside) and 1.12 g/L quercitrin (quercetin 3-O-rhamnoside) could be produced, respectively. In addition, both strains showed activity towards other promising dietary flavonols like kaempferol, fisetin, morin and myricetin.ConclusionsTwo E. coli W mutants were engineered that could effectively produce the bio-active flavonol glycosides hyperoside and quercitrin starting from the cheap substrates sucrose and quercetin. This novel fermentation-based glycosylation strategy will allow the economically viable production of various glycosides.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0326-1) contains supplementary material, which is available to authorized users.

Engineering Saccharomyces cerevisiae with the deletion of endogenous glucosidases for the production of flavonoid glucosides

“…The glycosylation of flavonoids using GTs as the biocatalyst is of great general interest due to the synthesis of the desired compounds with high stereo- and regio-selectivity under mild conditions [ 4 ]. In recent studies, the exploration of GTs mainly focused on enzymology and the semi-synthesis of glucosides using engineered E. coli [ 4 , 13 , 22 ]. The in vitro recombinant enzyme provided a good model to study the enzymatic characteristics, and the in vivo biosynthesis of flavonoid glucosides using endogenous sugar donors could reduce the expense in engineered whole-cell E. coli system.…”

“…The combined strategy of deletion of glucosidases and the introduction of GTs plays pivotal roles in generating flavonoid glucosides. Plant GTs expressed in E. coli have been reported for the glycosylation of flavonoids [ 13 , 22 ]. Based on the expression of the GTs in E. coli , the titre of the flavonoid glucosides was less than 100 mg/L in the culture broth [ 25 , 26 ].…”

“…The two main factors for the biosynthesis of the desired products might be related to the availability of intracellular UDP-sugars and low activity of the GTs. Recently, a number of studies have primarily focused on enhancing endogenous UDP-sugars by over-expressing genes that are part of the biosynthetic pathway of UDP-sugars and on exploring efficient GTs [ 22 , 31 – 33 ]. In this study, the successful deletion of glucosidases and the expression of the S. baicalensis flavonoid GT in the engineered yeast for the whole-cell biocatalysis facilitated the bioconversion of aglycones and significantly improved the production of scutellarein 7- O -glucoside.…”

Glycan Phosphorylases in Multi-Enzyme Synthetic Processes

“…The produced UDP-glucose was converted to UDP-galactose or UDP-rhamnose by introducing UDP-glucose-4-epimerase ( GalE ) or UDP-rhamnose synthase ( MUM4 ) genes, respectively, into the E. coli strain. UDP-galactose and rhamnose were then used for galactosylation and rhamnosylation of quercetin, respectively (Figure 7 ) [ 49 ]. The advent of such in vivo glycosylation platform provides a great alternative to the extraction of these glycosylated compounds from their original sources, which are laborious and low yielding processes.…”

“… Metabolic engineering of E. coli for in vivo glycosylation platform of a flavonol molecule [ 49 ]. Underlined genes were introduced into E. coli for overexpression of the enzymes that are essential for production of desirable sugar metabolites, whereas erased genes were deleted from E. coli genome to prevent the breakdown of α-D-Glc-1-phosphate and UPD-Glc.…”