Indirect and direct routes to C-glycosylated flavones in Saccharomyces cerevisiae

Katherina Garcia Vanegas,Michael Eichenberger,Arésu Bondrup Larsen,Uffe Hasbro Mortensen,David Fischer,Michael Naesby

doi:10.1186/s12934-018-0952-5

Katherina Garcia Vanegas, Michael Eichenberger + Show 4 more

Open Access

https://doi.org/10.1186/s12934-018-0952-5

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Abstract

BackgroundC-glycosylated flavones have recently attracted increased attention due to their possible benefits in human health. These biologically active compounds are part of the human diet, and the C-linkage makes them more resistant to hydrolysis and degradation than O-glycosides. In contrast to O-glycosyltransferases, few C-glycosyltransferases (CGTs) have so far been characterized. Two different biosynthetic routes for C-glycosylated flavones have been identified in plants. Depending on the type of C-glycosyltransferase, flavones can be glycosylated either directly or indirectly via C-glycosylation of a 2-hydroxyflavanone intermediate formed by a flavanone 2-hydroxylase (F2H).ResultsIn this study, we reconstructed the pathways in the yeast Saccharomyces cerevisiae, to produce some relevant CGT substrates, either the flavanones naringenin and eriodictyol or the flavones apigenin and luteolin. We then demonstrated two-step indirect glycosylation using combinations of F2H and CGT, to convert 2-hydroxyflavanone intermediates into the 6C-glucoside flavones isovitexin and isoorientin, and the 8C-glucoside flavones vitexin and orientin. Furthermore, we established direct glycosylation of flavones using the recently identified GtUF6CGT1 from Gentiana triflora. The ratio between 6C and 8C glycosylation depended on the CGT used. The indirect route resulted in mixtures, similar to what has been reported for in vitro experiments. In this case, hydroxylation at the flavonoid 3′-position shifted the ratio towards the 8C-glucosylated orientin. The direct flavone glycosylation by GtUF6CGT1, on the other hand, resulted exclusively in 6C-glucosides.ConclusionsThe current study features yeast as a promising host for production of flavone C-glycosides, and it provides a set of tools and strains for identifying and studying CGTs and their mechanisms of C-glycosylation.

Highlights

C-glycosylated flavones have recently attracted increased attention due to their possible benefits in human health
Construction of yeast strains for flavanone and flavone production In order to test C-glycosylation of both flavanones and flavones we created the four strains NAR1, ERI1, API1, and LUT1 producing the flavanones naringenin and eriodictyol, and the corresponding flavones apigenin and luteolin, respectively
The cassettes for construction of the Homologous Recombination Technology (HRT) plasmid were assembled in the following order: a URA3 marker cassette for selection, an ARS4/CEN6 cassette, two non-coding “stuffer sequence” cassettes, the AtCPR1 gene cassette, and the EZ closing linker, which is used to close the plasmid as it fuses to pEVE4012 and pEVE4730 (Additional file 1: Figure S3)

Summary

Introduction

C-glycosylated flavones have recently attracted increased attention due to their possible benefits in human health These biologically active compounds are part of the human diet, and the C-linkage makes them more resistant to hydrolysis and degradation than O-glycosides. Vanegas et al Microb Cell Fact (2018) 17:107 a b unlike the O–C bond, is very resistant to acid hydrolysis and enzymatic glycosidase action [11, 12] This has spurred an increased interest in C-glycosides for human health applications, including those related to metabolic syndrome [13, 14], since these molecules are expected to be more resistant to degradation in the human gastrointestinal system, and more orally bioavailable. The most commonly found C-glycosides are the mono-glucosides vitexin, isovitexin, orientin, and isoorientin derived from the common precursor naringenin (Fig. 1b)

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