Abstract
BackgroundGlycosylation of flavonoids is a promising approach to improve the pharmacokinetic properties and biological activities of flavonoids. Recently, many efforts such as enzymatic biocatalysis and the engineered Escherichia coli biotransformation have increased the production of flavonoid glucosides. However, the low yield of flavonoid glucosides can not meet the increasing demand for human medical and dietary needs. Saccharomyces cerevisiae is a generally regarded as safe (GRAS) organism that has several attractive characteristics as a metabolic engineering platform for the production of flavonoid glucosides. However, endogenous glucosidases of S.cerevisiae as a whole-cell biocatalyst reversibly hydrolyse the glucosidic bond and hinder the biosynthesis of the desired products. In this study, a model flavonoid, scutellarein, was used to exploit how to enhance the production of flavonoid glucosides in the engineered S.cerevisiae.ResultsTo produce flavonoid glucosides, three flavonoid glucosyltransferases (SbGTs) from Scutellaria baicalensis Georgi were successfully expressed in E. coli, and their biochemical characterizations were identified. In addition, to synthesize the flavonoid glucosides in whole-cell S. cerevisiae, SbGT34 was selected for constructing the engineering yeast. Three glucosidase genes (EXG1, SPR1, YIR007W) were knocked out using homologous integration, and the EXG1 gene was determined to be the decisive gene of S. cerevisiae in the process of hydrolysing flavonoid glucosides. To further enhance the potential glycosylation activity of S. cerevisiae, two genes encoding phosphoglucomutase and UTP-glucose-1-phosphate uridylyltransferase involved in the synthetic system of uridine diphosphate glucose were over-expressed in S. cerevisiae. Consequently, approximately 4.8 g (1.2 g/L) of scutellarein 7-O-glucoside (S7G) was produced in 4 L of medium after 54 h of incubation in a 10-L fermenter while being supplied with ~3.5 g of scutellarein.ConclusionsThe engineered yeast harbouring SbGT with a deletion of glucosidases produced more flavonoid glucosides than strains without a deletion of glucosidases. This platform without glucosidase activity could be used to modify a wide range of valued plant secondary metabolites and to explore of their biological functions using whole-cell S. cerevisiae as a biocatalyst.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0535-2) contains supplementary material, which is available to authorized users.
Highlights
Glycosylation of flavonoids is a promising approach to improve the pharmacokinetic properties and biological activities of flavonoids
The results showed that each enzyme only selectively accepted uridine diphosphate (UDP)-Glu as a sugar donor
These three SbGTs catalysed glucosyl transfer to scutellarein and produced a single glycosylated product, which was clearly identified as scutellarein 7-O-glucoside based on the comparison of its LC spectrum, MS and MS/MS fragments with authentic sample and the NMR spectra (Additional file 1: Supplemental results)
Summary
Glycosylation of flavonoids is a promising approach to improve the pharmacokinetic properties and biological activities of flavonoids Many efforts such as enzymatic biocatalysis and the engineered Escherichia coli biotransformation have increased the production of flavonoid glucosides. Biocatalytic efforts to synthesize of flavonoid glucosides have largely focused on enzymatic synthesis and metabolic engineering of E. coli. The former is typically used to explore novel GTs and to characterize their new functions, and the latter automatically synthesizes sugar donors and directly catalyses the glycosylation of the target substrates supplied in the culture medium [10, 13, 14]. There are very few studies on the engineered yeast for the biosynthesis of flavonoid glucosides
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