Production of 3-O-xylosyl quercetin in Escherichia coli

Quercetin, a flavonol aglycone, is one of the most abundant flavonoids with high medicinal value. The bioavailability and pharmacokinetic properties of quercetin are influenced by the type of sugars attached to the molecule. To efficiently diversify the therapeutic uses of quercetin, Escherichia coli was harnessed as a production factory by the installation of various plant and bacterial UDP-xylose sugar biosynthetic genes. The genes encoding for the UDP-xylose pathway enzymes phosphoglucomutase (nfa44530), glucose-1-phosphate uridylyltransferase (galU), UDP-glucose dehydrogenase (calS8), and UDP-glucuronic acid decarboxylase (calS9) were overexpressed in E. coli BL21 (DE3) along with a glycosyltransferase (arGt-3) from Arabidopsis thaliana. Furthermore, E. coli BL21(DE3)/?pgi, E. coli BL21(DE3)/?zwf, E. coli BL21(DE3)/?pgi?zwf, and E. coli BL21(DE3)/?pgi?zwf?ushA mutants carrying the aforementioned UDP-xylose sugar biosynthetic genes and glycosyltransferase and the galU-integrated E. coli BL21(DE3)/?pgi host harboring only calS8, calS9, and arGt-3 were constructed to enhance whole-cell bioconversion of exogeneously supplied quercetin into 3-O-xylosyl quercetin. Here, we report the highest production of 3-O-xylosyl quercetin with E. coli BL21 (DE3)/?pgi?zwf?ushA carrying UDP-xylose sugar biosynthetic genes and glycosyltransferase. The maximum concentration of 3-O-xylosyl quercetin achieved was 23.78 mg/L (54.75 μM), representing 54.75 % bioconversion, which was an ~4.8-fold higher bioconversion than that shown by E. coli BL21 (DE3) with the same set of genes when the reaction was carried out in 5-mL culture tubes with 100 μM quercetin under optimized conditions. Bioconversion was further improved by 98 % when the reaction was scaled up in a 3-L fermentor at 36 h.     

The flavonoids of ferns in the isolated genera Loxsoma and Loxsomopsis

Kaempferol and quercetin 3-O-glucosides and 3-O-rhamnoglucosides are common to both Loxsoma cunninghamii and Loxsomopsis costaricensis, but in the former the new flavonoid glycosides, kaempferol and quercetin 3-O-glucoside-7-O-arabinoside have also been identified. The data are consistent with the proposed taxonomic relationship between these geographically isolated genera. Comparative flavonoid chemistry indicates that the Loxsomaceae may be a primitive family, not closely related to the Hymenophyllaceae or the Cyatheaceae.     

Biosynthesis of a rosavin natural product in Escherichia coli by glycosyltransferase rational design and artificial pathway construction

Phytochemicals are rich resources for pharmaceutical and nutraceutical agents. A key challenge of accessing these precious compounds can present significant bottlenecks for development. The cinnamyl alcohol disaccharides also known as rosavins are the major bioactive ingredients of the notable medicinal plant Rhodiola rosea L. Cinnamyl-(6′-O-β-xylopyranosyl)-O-β-glucopyranoside (rosavin E) is a natural rosavin analogue with the arabinopyranose unit being replaced by its diastereomer xylose, which was only isolated in minute quantity from R. rosea. Herein, we described the de novo production of rosavin E in Escherichia coli. The 1,6-glucosyltransferase CaUGT3 was engineered into a xylosyltransferase converting cinnamyl alcohol monoglucoside (rosin) into rosavin E by replacing the residue T145 with valine. The enzyme activity was further elevated 2.9 times by adding the mutation N375Q. The synthesis of rosavin E from glucose was achieved with a titer of 92.9 mg/L by combining the variant CaUGT3^T145V/N375Q, the UDP-xylose synthase from Sinorhizobium meliloti 1021 (SmUXS) and enzymes for rosin biosynthesis into a phenylalanine overproducing E. coli strain. The production of rosavin E was further elevated by co-overexpressing UDP-xylose synthase from Arabidopsis thaliana (AtUXS3) and SmUXS, and the titer in a 5 L bioreactor with fed-batch fermentation reached 782.0 mg/L. This work represents an excellent example of producing a natural product with a disaccharide chain by glycosyltransferase engineering and artificial pathway construction.     

Biosynthesis of two quercetin <Emphasis Type="Italic">O</Emphasis>-diglycosides in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Various flavonoid glycosides are found in nature, and their biological activities are as variable as their number. In some cases, the sugar moiety attached to the flavonoid modulates its biological activities. Flavonoid glycones are not easily synthesized chemically. Therefore, in this study, we attempted to synthesize quercetin 3-O-glucosyl (1→2) xyloside and quercetin 3-O-glucosyl (1→6) rhamnoside (also called rutin) using two uridine diphosphate-dependent glycosyltransferases (UGTs) in Escherichia coli. To synthesize quercetin 3-O-glucosyl (1→2) xyloside, sequential glycosylation was carried out by regulating the expression time of the two UGTs. AtUGT78D2 was subcloned into a vector controlled by a Tac promoter without a lacI operator, while AtUGT79B1 was subcloned into a vector controlled by a T7 promoter. UDP-xyloside was supplied by concomitantly expressing UDP-glucose dehydrogenase (ugd) and UDP-xyloside synthase (UXS) in the E. coli. Using these strategies, 65.0 mg/L of quercetin 3-O-glucosyl (1→2) xyloside was produced. For the synthesis of rutin, one UGT (BcGT1) was integrated into the E. coli chromosome and the other UGT (Fg2) was expressed in a plasmid along with RHM2 (rhamnose synthase gene 2). After optimization of the initial cell concentration and incubation temperature, 119.8 mg/L of rutin was produced. The strategies used in this study thus show promise for the synthesis of flavonoid diglucosides in E. coli.     

Regioselective synthesis of flavonoid bisglycosides using Escherichia coli harboring two glycosyltransferases

Regioselective glycosylation of flavonoids cannot be easily achieved due to the presence of several hydroxyl groups in flavonoids. This hurdle could be overcome by employing uridine diphosphate-dependent glycosyltransferases (UGTs), which use nucleotide sugars as sugar donors and diverse compounds including flavonoids as sugar acceptors. Quercetin rhamnosides contain antiviral activity. Two quercetin diglycosides, quercetin 3-O-glucoside-7-O-rhamnoside and quercetin 3,7-O-bisrhamnoside, were synthesized using Escherichia coli expressing two UGTs. For the synthesis of quercetin 3-O-glucoside-7-O-rhamnoside, AtUGT78D2, which transfers glucose from UDP-glucose to the 3-hydroxyl group of quercetin, and AtUGT89C1, which transfers rhamnose from UDP-rhamnose to the 7-hydroxyl group of quercetin 3-O-glucoside, were transformed into E. coli. Using this approach, 67 mg/L of quercetin 3-O-glucoside-7-O-rhamnoside was synthesized. For the synthesis of quercetin 3,7-O-bisrhamnoside, AtUGT78D1, which transfers rhamnose to the 3-hydroxy group of quercetin, and AtUGT89C1 were used. The RHM2 gene from Arabidopsis thaliana was coexpressed to supply the sugar donor, UDP-rhamnose. E. coli expressing AtUGT78D1, AtUGT89C1, and RHM2 was used to obtain 67.4 mg/L of quercetin 3,7-O-bisrhamnoside.     

Biosynthesis of bioactive O-methylated flavonoids in Escherichia coli

Two bioactive O-methylflavonoids, sakuranetin (7-O-methylnaringenin) and ponciretin (7-O-methylnaringenin), were synthesized in Escherichia coli. Sakuranetin inhibits germination of Magnaporthe grisea, and ponciretin is a potential inhibitor of Helicobacter pylori. To achieve this, we reconstructed the naringenin biosynthesis pathway in E. coli. First, the shikimic acid pathway, which leads to the biosynthesis of tyrosine, was engineered in E. coli to increase the amount of available tyrosine. Second, several genes for the biosynthesis of ponciretin and sakuranetin such as tyrosine ammonia lyase (TAL), 4-coumaroyl CoA ligase (4CL), chalcone synthase (CHS), and O-methyltransferase (OMT) were overexpressed. In order to increase the supply the Coenzyme A (CoA), one gene (icdA, isocitrate dehydrogenase) was deleted. Using these strategies, we synthesized ponciretin and sakuranetin from glucose in E. coli at the concentration of 42.5 mg/L and 40.1 mg/L, respectively.     

Exploration of glycosylated flavonoids from metabolically engineered <Emphasis Type="Italic">E. coli</Emphasis>

Flavonoids glycosylated with UDP-glucuronic acid and UDP-xylose are spatially distributed in nature. To produce these glycosides, E. coli was engineered to overexpress biosynthetic gene clusters of UDP-sugars (galU from E. coli K12, UDP-glucose dehydrogenase (calS8), and UDP-glucuronic acid decarboxylase (calS9) from Micromonospora echinospora spp. calichensis). Flavonoids were glycosylated by overexpression of the glycosyltransferase gene (atGt-5) from Arabidopsis thaliana. Finally, metabolically engineered host E. coli (US89Gt-5) was generated. Production of flavonoid glycosides was observed in a biotransformation system consisting of flavonoids (naringenin and quercetin) exogenously fed to host cells. The glycosylated derivatives 7-O-glucuronyl naringenin (m/z⁺ 449), 7-O-xylosyl naringenin (m/z⁺ 405), and 7-O-glucuronyl quercetin (m/z⁺ 479) were detected and confirmed by ESI-MS/MS, ESI-MS/MS and LC/MS-MS analysis, respectively.     

Biological synthesis of quercetin 3-<Emphasis Type="Italic">O</Emphasis>-<Emphasis Type="Italic">N</Emphasis>-acetylglucosamine conjugate using engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing <Emphasis Type="Italic">UGT78D2</Emphasis>

Biotransformation of flavonoids using Escherichia coli harboring nucleotide sugar-dependent uridine diphosphate-dependent glycosyltransferases (UGTs) commonly results in the production of a glucose conjugate because most UGTs are specific for UDP-glucose. The Arabidopsis enzyme AtUGT78D2 prefers UDP-glucose as a sugar donor and quercetin as a sugar acceptor. However, in vitro, AtUGT78D2 could use UDP-N-acetylglucosamine as a sugar donor, and whole cell biotransformation of quercetin using E. coli harboring AtUGT78D2 produced quercetin 3-O-N-acetylglucosamine. In order to increase the production of quercetin 3-O-N-acetylglucosamine via biotransformation, two E. coli mutant strains deleted in phosphoglucomutase (pgm) or glucose-1-phosphate uridylyltransferase (galU) were created. The galU mutant produced up to threefold more quercetin 3-O-N-acetylglucosamine than wild type, resulting in the production of 380-mg/l quercetin 3-O-N-acetylglucosamine and a negligible amount of quercetin 3-O-glucoside. These results show that construction of bacterial strains for the synthesis of unnatural flavonoid glycosides is possible through rational selection of the nucleotide sugar-dependent glycosyltransferase and engineering of the nucleotide sugar metabolic pathway in the host strain.     

Identification and characterization of a rhamnosyltransferase involved in rutin biosynthesis in Fagopyrum esculentum (common buckwheat)

Rutin, a 3-rutinosyl quercetin, is a representative flavonoid distributed in many plant species, and is highlighted for its therapeutic potential. In this study, we purified uridine diphosphate-rhamnose: quercetin 3-O-glucoside 6″-O-rhamnosyltransferase and isolated the corresponding cDNA (FeF3G6″RhaT) from seedlings of common buckwheat (Fagopyrum esculentum). The recombinant FeF3G6″RhaT enzyme expressed in Escherichia coli exhibited 6″-O-rhamnosylation activity against flavonol 3-O-glucoside and flavonol 3-O-galactoside as substrates, but showed only faint activity against flavonoid 7-O-glucosides. Tobacco cells expressing FeF3G6″RhaT converted the administered quercetin into rutin, suggesting that FeF3G6″RhaT can function as a rhamnosyltransferase in planta. Quantitative PCR analysis on several organs of common buckwheat revealed that accumulation of FeF3G6″RhaT began during the early developmental stages of rutin-accumulating organs, such as flowers, leaves, and cotyledons. These results suggest that FeF3G6″RhaT is involved in rutin biosynthesis in common buckwheat.     

Flavonoid chemistry ofBlennospermatinae, a trans-Pacific disjunct subtribe ofSenecioneae (Asteraceae)

The flavonoid profiles of seven species ofAbrotanella and one species ofIschnea have been shown to be based upon kaempferol 3- and quercetin 3-O-glycosides and a delphinidin glycoside. Glucosides, glucuronides, arabinosides, diglucosides, and rutinosides of the flavonols were identified. The profile ofIschnea consisted solely of quercetin 3-O-glucoside and 3-O-arabinoside whereas the profiles of theAbrotanella species were more varied. Although infraspecific variation was not investigated in this study, the flavonoid chemistry of the two genera is in accordance with the flavonoid variation described for other members ofSenecioneae which are primarily flavonol producers. Based on the known phylogeny and biogeography, the flavonoid distribution from the perspective of long-distance dispersals across the Pacific is discussed. Such events should lead to genetic bottle-neck situations and depauperate flavonoid profiles. A summary of current flavonoid knowledge in theSenecioneae is supplied.     

Production of kaempferol 3-O-rhamnoside from glucose using engineered Escherichia coli

Flavonoids are ubiquitous phenolic compounds and at least 9,000 have been isolated from plants. Most flavonoids have been isolated and assessed in terms of their biological activities. Microorganisms such as Escherichia coli and Saccharomyces cerevisiae are efficient systems for the synthesis of flavonoids. Kaempferol 3-O-rhamnoside has notable biological activities such as the inhibition of the proliferation of breast cancer cells, the absorption of glucose in the intestines, and the inhibition of the self-assembly of beta amyloids. We attempted to synthesize kaempferol 3-O-rhamnoside from glucose in E. coli. Five flavonoid biosynthetic genes [tyrosine ammonia lyase (TAL), 4-coumaroyl CoA ligase (4CL), chalcone synthase (CHS), flavonol synthase (FLS), and flavonol 3-O-rhamnosyltransferase (UGT78D1)] from tyrosine were introduced into E. coli that was engineered to increase tyrosine production. By using this approach, the production of kaempferol 3-O-rhamnoside increased to 57 mg/L.     

A new ginsenosidase from Aspergillus strain hydrolyzing 20-O-multi-glycoside of PPD ginsenoside

A ginsenosidase specifically hydrolyzing multi-20-O-glycosides of protopanaxadiol type ginsenosides such as ginsenoside Rb₁, Rb₃, Rb₂ and Rc, named ginsenosidase type II, was isolated and purified from Aspergillus sp.g48p strain. The molecular weight of the enzyme was 60 kDa. Ginsenosidase type II was demonstrated to hydrolyze multi-20-O-glycoside of protopanaxadiol type ginsenoside Rb₁, Rb₃, Rb₂ and Rc; i.e. the ginsenosidase type II hydrolyzes 20-O-β-glucoside of the ginsenoside Rb₁, 20-O-β-xyloside of ginsenoside Rb₃, 20-O-α-arabinoside(p) of ginsenoside Rb₂ and α-arabinoside(f) of ginsenoside Rc to produce mainly ginsenoside Rd, and small amount of Rg₃. However, it did not hydrolyze 3-O-β-glucosides of ginsenoside Rb₁, Rb₃, Rb₂ and Rc which was different with the ginsenosidase type I previously reported, either did not hydrolyze the glycosides of protopanaxatriol type ginsenoside such as ginsenoside Re, Rf and Rg₁, showing significant difference from all previously described glycosidases.     

Flavonoids in translucent bracts of the Himalayan<Emphasis Type="Italic"> Rheum nobile</Emphasis> (Polygonaceae) as ultraviolet shields

UV-absorbing substances were isolated from the translucent bracts of Rheum nobile, which grows in the alpine zone of the eastern Himalayas. Nine kinds of the UV-absorbing substances were found by high performance liquid chromatography (HPLC) and paper chromatography (PC) surveys. All of the five major compounds are flavonoids, and were identified as quercetin 3-O-glucoside, quercetin 3-O-galactoside, quercetin 3-O-rutinoside, quercetin 3-O-arabinoside and quercetin 3-O-[6-(3-hydroxy-3-methylglutaroyl)-glucoside] by UV, ¹H and ¹³C NMR, mass spectra, and acid hydrolysis of the original glycosides, and direct PC and HPLC comparisons with authentic specimens. The four minor compounds were characterised as quercetin itself, quercetin 7-O-glycoside, kaempferol glycoside and feruloyl ester. Of those compounds, quercetin 3-O-[6-(3-hydroxy-3-methylglutaroyl)-glucoside] was found in nature for the first time. The translucent bracts of R. nobile accumulate a substantial quantity of flavonoids (3.3–5 mg per g dry material for the major compounds). Moreover, it was clarified by quantitative HPLC survey that much more of the UV-absorbing substances is present in the bracts than in rosulate leaves. Although the flavonoid compounds have been presumed to be the important UV shields in higher plants, there has been little characterisation of these compounds. In this paper, the UV-absorbing substances of the Himalayan R. nobile were characterised as flavonol glycosides based on quercetin.     

Cloning and characterization of a putative UDP-rhamnose synthase 1 from Populus euramericana Guinier

L-Rhamnose is a constituent of plant primary cell wall polysaccharides including rhamnogalacturonan-I, rhamnogalacturonan-II, and other natural plant-based compounds. UDP-rhamnose serves as a rhamnose donor whose synthesis is catalyzed by UDP-rhamnose synthase (RHM). A RHM gene, PRHM was cloned from Populus euramericana Guinier. PRHM contains two domains: the NAD dependent epimerase/dehydratase family domain and the RmlD (dTDP-keto-rhamnose-4-keto-reductase) substrate-binding domain. Because the recombinant PRHM did not demonstrate any activity during an in vitro assay, complementation with an Escherichia coli mutant was carried out. The rfbD (dTDP-4-dehydrorhamnose reductase), which encodes an enzyme catalyzing the conversion of dTDP-4-keto-rhamnose to TDP-rhamnose, was mutated in E. coli. The mutant strain B-rfbD was transformed with PRHM gene and a flavonoid rhanmosyltransferase gene, AtUGT78D1. The resulting transformant was able to convert quercetin into quercetin 3-O-rhamnoside in a manner similar to that by the wild type E. coli strain harboring AtUGT78D1. This result indicated that PRHM catalyzed the conversion of UDP-glucose into UDP-rhamnose.     

Further studies of flavonols of Clibadium (compositae)

The flavonoids of an additional eight species of Clibadium have been determined. The compounds are derivatives of kaempferol, quercetin and quercetagetin. O-Methylated quercetagetin derivatives were found in several taxa with the possibility that 6-methoxykaempferol may also exist in one collection. Kaempferol and quercetin exist as 3-O-glucosides, galactosides, rhamnosides, rutinosides and diglucosides although not all glycosides occur in each taxon. Quercetagetin derivatives occur as 7-O-glucosides. Observations on these newly investigated species confirm previous work in the genus that three types of flavonoid profiles exist: (1) kaempferol and quercetin 3-glycosides; (2) kaempferol and quercetin 3-glycosides plus quercetagetin 7-glucoside; and (3) kaempferol and quercetin 3-glycosides plus quercetagetin 7-glucoside and O-methylated derivatives of quercetagetin.     

The kinetic basis for age-associated changes in quercetin and genistein glucuronidation by rat liver microsomes

The dietary bioavailability of the isoflavone genistein is decreased in older rats compared to young adults. Since flavonoids are metabolized extensively by the UDP-glucuronosyltransferases (UGTs), we hypothesized that UGT flavonoid conjugating activity changes with age. The effect of age on flavonoid glucuronidation was determined using hepatic microsomes from male F344 rats. Kinetic models of UGT activity toward the flavonol quercetin and the isoflavone genistein were established using pooled hepatic microsomal fractions of rats at different ages, and glucuronidation rates were determined using individual samples. Intrinsic clearance (V_max/K_m) values in 4-, 18- and 28-month-old rats were 0.100, 0.078 and 0.087 ml/min/mg for quercetin-7-O-glucuronide; 0.138, 0.133 and 0.088 for quercetin-3′-O-glucuronide; and 0.075, 0.077 and 0.057 for quercetin-4′-O-glucuronide, respectively. While there were no differences in formation rates of total quercetin glucuronides in individual samples, the production of the primary metabolite, quercetin-7-O-glucuronide, at 30 μM quercetin concentration was increased from 3.4 and 3.1 nmol/min/mg at 4 and 18 months to 3.8 nmol/min/mg at 28 months, while quercetin-3′-O-glucuronide formation at 28 months declined by a similar degree (P≤.05). At 30 and 300 μM quercetin concentration, the rate of quercetin-4′-O-glucuronide formation peaked at 18 months at 0.9 nmol/min/mg. Intrinsic clearance values of genistein 7-O-glucuronide increased with age, in contrast to quercetin glucuronidation. Thus, the capacity for flavonoid glucuronidation by rat liver microsomes is dependent on age, UGT isoenzymes and flavonoid structure.     

Flavonoid glycosides from the leaves of Aphananthe aspera (Thunb.) Planch. (Cannabaceae) and their chemotaxonomic significance

From the leaves of Aphananthe aspera (Thunb.) Planch. (Family: Cannabaceae), six flavonol glycosides, such as quercetin 3-O-β-glucopyranoside (1), kaempferol 3-O-β-glucopyranoside (2), quercetin 3-O-rutinoside (3), kaempferol 3-O-rutinoside (4), quercetin 3-O-neohesperidoside (5) and kaempferol 3-O-neohesperidoside (6) were isolated and identified. Structure elucidation of these compounds was performed on the basis of NMR spectral data. All these compounds were isolated for the first time from the genus Aphananthe. Chemotaxonomic significance and distribution of these flavonoid derivatives among the genera of Cannabaceae are explained in detail.     

Flavonoids of the primitive liverwort Takakia and their taxonomic and phylogenetic significance

The flavonoid chemistry of Takakia is described for the first time. T. lepidozioides, thought to be amongst the most primitive of extant liverworts, contains a high level and wide variety of flavone C- and O-glycosides, many of which are unique. New flavonoids include the 8-O-glucuronide and 8-O-xylosylglucoside of takakin (8-hydroxyacacetin), luteolin 6-C-arabinoside-8-C-pentoside, kaempferol 3-O-glucoside-7-O-xyloside and a number of tricetin C-glycosides. The only other known Takakia species, T. ceratophylla, contains the same 4 major constituents but significantly lacks flavonols. The often suggested relationship of Takakia with the order Calobryales is not supported by the available flavonoid data. Biochemical affinities of Takakia with all major liverwort orders are noted and the flavonoid data are interpreted as supporting the concept of Takakia as an isolated branch among the ancestors of modern bryophytes.     

Flavonoid pattern and systematics of the genus Leucocyclus

The flavonoid pattern of the monotypic Turkish genus Leucocyclus consists of C-glycosylflavones (isovitexin; isoorientin and derivatives; several di-C-glycosylapigenins; schaftoside, isoschaftoside and vicenin-3; lucenin-2), of flavonol 3-O-glycosides (quercetin and kaempferol 3-O-rhamnoglucoside) and trace amounts of luteolin 7-O-rhamnoglucoside. The systematic significance of the flavonoid diversification within Leucocyclus as well as possible relationships to other genera of the Anthemideae are discussed.     

On the reported molluscicidal activity from Tephrosia vogelii leaves

The rotenoids deguelin and tephrosin were isolated from leaves of Tephrosia vogelii, together with three flavonol glycosides, rutin, isoquercitrin and quercetin 3-O-arabinoside. Although T. vogelii leaves are reportedly toxic to aquatic snails, deguelin and tephrosin were found to have no significant molluscicidal activity.