Indirect and Direct Routes to C-Glycosylated Flavones In

Vanegas et al. Microb Cell Fact (2018) 17:107 https://doi.org/10.1186/s12934-018-0952-5 Microbial Cell Factories RESEARCH Open Access Indirect and direct routes to C‑glycosylated favones in Saccharomyces cerevisiae Katherina Garcia Vanegas1, Arésu Bondrup Larsen2, Michael Eichenberger2, David Fischer2, Ufe Hasbro Mortensen1 and Michael Naesby2* Abstract Background: C-glycosylated favones have recently attracted increased attention due to their possible benefts 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 diferent biosynthetic routes for C-glycosylated favones have been identifed in plants. Depending on the type of C-glycosyltransferase, favones can be glycosylated either directly or indirectly via C-glycosylation of a 2-hydroxyfavanone intermediate formed by a favanone 2-hydroxylase (F2H). Results: In this study, we reconstructed the pathways in the yeast Saccharomyces cerevisiae, to produce some rel- evant CGT substrates, either the favanones naringenin and eriodictyol or the favones apigenin and luteolin. We then demonstrated two-step indirect glycosylation using combinations of F2H and CGT, to convert 2-hydroxyfavanone intermediates into the 6C-glucoside favones isovitexin and isoorientin, and the 8C-glucoside favones vitexin and orientin. Furthermore, we established direct glycosylation of favones using the recently identifed GtUF6CGT1 from Gentiana trifora. 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 favonoid 3′-position shifted the ratio towards the 8C-glucosylated orientin. The direct favone glycosylation by GtUF6CGT1, on the other hand, resulted exclusively in 6C-glucosides. Conclusions: The current study features yeast as a promising host for production of favone C-glycosides, and it pro- vides a set of tools and strains for identifying and studying CGTs and their mechanisms of C-glycosylation. Keywords: Vitexin, Isovitexin, Orientin, Isoorientin, Glycosyl C-transferase, Flavanone 2-hydroxylase Background favonoid scafold is synthesized by condensation of two Flavones constitute a subclass of favonoids, found in precursors derived from two diferent pathways of the fruits and vegetables [1], which has been associated with primary metabolism, p-coumaroyl-CoA from the phe- a range of human health-related benefts [2]. Te basic nylalanine pathway and malonyl-CoA, an intermediate favone scafold comprises a three ring-skeleton (Fig. 1a) of fatty acid biosynthesis, to yield the common favanone with three functional groups: a C4 ketone, a conjugated precursor naringenin [2, 4] (Additional fle 1: Figure S1). C2–C3 double bond and, depending on the favone, Flavones are normally derived from the favanones by the various numbers of hydroxyl groups [2, 3]. In plants the action of favone synthase type I (FNS I), a 2-oxoglutar- ate dependent dioxygenase [5], or type II (FNS II) [6], a cytochrome P450 oxidase (CYP450) which introduce a *Correspondence: [email protected] C2–C3 double bond in the heterocyclic C-ring (Fig. 1b). 2 Evolva SA, Duggingerstrasse 23, 4153 Reinach, Switzerland Full list of author information is available at the end of the article Various modifcations of the favone backbone result in a high degree of chemical diversity, resulting in diferent © The Author(s) 2018, corrected publication July 2018. This article is distributed under the terms of the Creative Commons Attri- bution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Vanegas et al. Microb Cell Fact (2018) 17:107 Page 2 of 10 a b Fig. 1 a Structures of some important favones. b Predicted biosynthesis for C-glycosylated favones from the common naringenin precursor. FNSI/ II, favone synthase 1 or 2; F2H, favanone-2-hydroxylase; F3′H, favanone-3′-hydroxylase CGT, C-glycosyltransferase. Broken line arrows represent hypothetical steps not demonstrated in this study. Equilibrium arrows indicate 2-hydroxylfavanones equilibrium with its open-circular form. Light grey arrows indicate the indirect C-glycosylation pathway and dark grey arrows shows the direct C-glycosylation pathway biological activities [2, 7]. One of the most common moiety to the favone scafold determines which type it modifcations is glycosylation, which can improve the is [10]. In C-glycosylation the linkage occurs directly biological activity and the solubility of the favone [8, 9]. between the glycosyl moiety and one of the carbon atoms Tere are two main types of glycosylation, O-glycosyla- of the favone backbone [2]. C-glycosylation results in tion and C-glycosylation, and the linkage of the glycosyl very stable molecules because the C–C bond linkage, Vanegas et al. Microb Cell Fact (2018) 17:107 Page 3 of 10 unlike the O–C bond, is very resistant to acid hydroly- and the current study reports the reconstruction of full- sis and enzymatic glycosidase action [11, 12]. Tis has length pathways to the four basic C-glucosides isovitexin, spurred an increased interest in C-glycosides for human vitexin, isoorientin and orientin. health applications, including those related to metabolic syndrome [13, 14], since these molecules are expected to Methods be more resistant to degradation in the human gastro- Chemicals intestinal system, and therefore more orally bioavailable. Chemical standards for detection and quantifcation of In addition, these compounds are being investigated for phloretic acid, p-coumaric acid, naringenin, luteolin, prevention of certain cancers [15, 16]. apigenin, vitexin, isovitexin, orientin, isoorientin and C-glycosylated favones are widespread in nature, and eriodictyol, were purchased from Sigma-Aldrich (St. natural sources include cereals like rice, wheat, and maize Louis, Missouri, USA). We acquired standards for all the where these glucosides are among the most abundant expected compounds (see Fig. 1 for details), except for favonoids [11, 17, 18]. Additional sources of a variety of the two 2-hydroxyfavanones, 2-hydroxynaringenin and favone C-glycosides are for example bamboo [19], buck- 2-hydroxyeriodictyol. Neither of the two 2-hydroxy com- wheat [20], and fax [21]. Te most commonly found pounds was available for purchase from reliable suppliers C-glycosides are the mono-glucosides vitexin, isovitexin, and were anyway expected to be unstable due to sponta- orientin, and isoorientin derived from the common pre- neous conversion into favones by dehydration [22, 30]. cursor naringenin (Fig. 1b). Te biosynthesis of favone C-glycosides was studied Strains and culture conditions in cereals, and somewhat surprisingly it was found that Escherichia coli (E. coli) XL10 Gold (Agilent, Santa Clara, favones themselves are not the direct substrate of C-gly- California, USA) competent cells were used for sub- cosylation [11, 20, 22]. Instead, the substrate was shown cloning of genes. After transformation E. coli cells were to be the 2-hydroxyfavanone intermediate formed by a cultured at 37 °C for 12 h on Luria Broth (LB) plates pre- class of FNS II related favanone 2-hydroxylases, belong- pared with 25 g/L of LB Broth with agar (Miller) and sup- ing to the CYP93 family of enzymes [23]. Glycosylation is plemented with 100 μg/mL ampicillin. Plasmid rescue proposed to happen on an open form of the 2-hydroxy- cultivations were prepared using liquid LB media pre- favanone and a dehydratase has been implied to catalyse pared with 25 g/L LB Broth (Miller) and supplemented the leaving of the 2-hydroxy group [11, 20, 23]. with 100 μg/mL ampicillin. Other natural sources of favone C-glucosides are Yeast strains used in this study were all direct descend- dicots like the gentians [24] and passion fruit [25], which ants of S. cerevisiae S288C strain NCYC 3608 (NCYC, contain high amounts of isoorientin. Very recently, Sasaki Norwich, United Kingdom). One descendant, the BG and co-workers identifed a C-glycosyltransferase from strain described earlier [31], was the basis of strains Gentiana trifora which catalyses the direct C-glycosyla- used in this study (Table 1). Te BG strain was modi- tion of favones, including apigenin and luteolin [26]. fed to replace the non-functional gal2 gene with a No other enzyme has so far been reported to do this functional allele from S. cerevisiae SK1 strain NCYC reaction. 3615 (NCYC). Further, the ARO3 gene was deleted and Despite the potential human benefts of favone C-glu- replaced by mutant ARO4 and ARO7 genes, encoding cosides, there are currently few reports of industrial scale de-regulated versions of these enzymes [32] resulting in production of these molecules from natural sources. strain EYS4988. To prevent degradation of precursors Tis probably stems from the classical challenge of plant of the heterologous favonoid pathway, the host genes raw materials containing the desired compounds in low ARO10, PAD1, and

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