Structural and Functional Characterization of a Gene Cluster Responsible for Deglycosylation of C-glucosyl Flavonoids and Xanthonoids by Deinococcus aerius.

Plant C-glycosylated aromatic polyketides are important for plant and animal health. These are specialized metabolites that perform functions both within the plant, and in interaction with soil or intestinal microbes. Despite the importance of these plant compounds, there is still limited knowledge of how they are metabolized. The Gram-positive aerobic soil bacterium Deinococcus aerius strain TR0125 and other Deinococcus species thrive in a wide range of harsh environments. In this work, we identified a C-glycoside deglycosylation gene cluster in the genome of D. aerius. The cluster includes three genes coding for a GMC-type oxidoreductase (DaCGO1) that oxidizes the glucosyl C3 position in aromatic C-glucosyl compounds, which in turn provides the substrate for the C-glycoside deglycosidase (DaCGD; composed of α+ß subunits) that cleaves the glucosyl-aglycone C-C bond. Our results from size-exclusion chromatography, single particle cryo-electron microscopy and X-ray crystallography show that DaCGD is an α2ß2 heterotetramer, which represents a novel oligomeric state among bacterial CGDs. Importantly, the high-resolution X-ray structure of DaCGD provides valuable insights into the activation of the catalytic hydroxide ion by Lys261. DaCGO1 is specific for the 6-C-glucosyl flavones isovitexin, isoorientin and the 2-C-glucosyl xanthonoid mangiferin, and the subsequent C-C-bond cleavage by DaCGD generated apigenin, luteolin and norathyriol, respectively. Of the substrates tested, isovitexin was the preferred substrate (DaCGO1, Km 0.047 mM, kcat 51 min-1; DaCGO1/DaCGD, Km 0.083 mM, kcat 0.42 min-1).

Xanthones Isolated from Cratoxylum cochinchinensis Reduced Oxidative Stress in Periodontal Ligament Stem Cells.

Xanthone compounds from Cratoxylum cochinchinensis (C. cochinchinensis) have demonstrated antioxidant effects and potency in treating many inflammatory diseases. However, the efficiency of the three xanthone extracts isolated from the young fruit of this plant, i.e., two geranyloxy xanthones (F6, F8) and one 1,3,7-hydroxy xanthone (F137), as antioxidants and therapeutics for periodontal disease has not been evaluated. The aim of this study was to investigate the antioxidant effects of three xanthones isolated from C. cochinchinensis on periodontal ligament stem cells (PDLSCs) and their osteogenic differentiation. The antioxidant activity of the aqueous extracts was determined using a DPPH assay, and their cytotoxicity was evaluated using an MTT assay. H2O2 was used to induce intracellular stress, and the scavenging effect of the isolated compounds against reactive oxygen species (ROS) was analyzed with a fluorescence assay. The expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was evaluated, and the effects of the three compounds on PDLSCs osteogenic differentiation were investigated. The isolated compounds reduced both extracellular and intracellular ROS in a dose-dependent manner and induced the expression of Nrf2 and HO-1 in PDLSCs. Under redox conditions, these compounds potentiated PDLSCs osteogenic differentiation. Our study demonstrated that the hydroxy xanthones from C. cochinchinensis had antioxidant effects on the Nrf2/HO-1 pathway and might be effective therapeutic substrates for damage prevention and the regeneration of damaged periodontal tissues in periodontitis patients.

Xanthone synthetic derivatives with high anticandidal activity and positive mycostatic selectivity index values.

With the current massive increases in drug-resistant microbial infection as well as the significant role of fungal infections in the death toll of COVID-19, discovering new antifungals is extremely important. Natural and synthetic xanthones are promising derivatives, although only few reports have demonstrated their antifungal mechanism of action in detail. Newly synthetized by us xanthone derivative 44 exhibited strong antifungal activity against reference and fluconazole resistant C. albicans strains. Our results indicate that the most active compounds 42 and 44 are not substrates for fungal ABC transporters (Cdr1p and Cdr2p) and Mdr1p, the main representative of the major facilitator superfamily efflux pumps, membrane proteins that are responsible for the development of resistance. Moreover, fungicidal mode of action reduces the probability of persistent or recurrent infections and resistance development. In this light, the demonstrated killing activity of the examined derivatives is their undoubted advantage. Novel synthesized compounds exhibited moderate cytotoxicity against human cell lines, although the selectivity index value for human pathogenic strains remained favourable. Our results also indicate that novel synthetized compounds 42 and 44 with antifungal activity target yeast topoisomerase II activity. In summary, further validation of xanthones applicability as antifungals is highly valuable.

C-Glucosyl Xanthone derivative Mangiferin downregulates the JNK3 mediated caspase activation in Almal induced neurotoxicity in differentiated SHSY-5Y neuroblastoma cells.

INTRODUCTION: C-Glucosyl Xanthone derivatives were assessed to inhibit the JNK3 mediated Caspase pathway in Almal (Aluminum Maltolate) induced neurotoxicity in SHSY-5Y cells. METHODS: Mangiferin was selected among 200 C-Glucosyl Xanthones based on molecular interaction, docking score (-10.22 kcal/mol), binding free energy (-71.12 kcal/mol), ADME/tox properties and by molecular dynamic studies. Further, it was noticed that glycone moiety of Mangiferin forms H-bond with ASN 194, SER 193, GLY 76, and OH group in the first position of the aglycone moiety shows interaction at Met 149 which is exceptionally crucial for JNK3 inhibitory activity. RESULTS AND DISCUSSION: Mangiferin (0.5, 1, 10, 20 and 30 µM) and standard SP600125 (20 µM) treatment increased the cell survival rate against Almal 200 µM, with EC50 of Mangiferin (8 µM) and standard SP600125 (4.9 µM) respectively. Mangiferin significantly impedes kinase activation, indicating suppression of JNK3 signaling with IC50 (98.26 nM). Mangiferin (10 and 15 µM) dose-dependently inhibits the caspase 3, 8, and 9 enzyme activation in comparison to Almal group. CONCLUSION: Mangiferin demonstrated neuroprotection in SHSY-5Y cells against apoptosis induced by Almal by adapting the architecture of the neurons and increasing their density. Among all Xanthone derivatives, Mangiferin could improve neuronal toxicity by inhibiting JNK3 and down-regulating the Caspase activation.

Biosynthesis of polyprenylated xanthones in Hypericum perforatum roots involves 4-prenyltransferase.

Polyprenylated xanthones are natural products with a multitude of biological and pharmacological activities. However, their biosynthetic pathway is not completely understood. In this study, metabolic profiling revealed the presence of 4-prenylated 1,3,5,6-tetrahydroxyxanthone derivatives in St. John's wort (Hypericum perforatum) root extracts. Transcriptomic data mining led to the detection of 5 variants of xanthone 4-prenyltransferase (HpPT4px) comprising 4 long variants (HpPT4px-v1 to HpPT4px-v4) and 1 short variant (HpPT4px-sh). The full-length sequences of all 5 variants were cloned and heterologously expressed in yeast (Saccharomyces cerevisiae). Microsomes containing HpPT4px-v2, HpPT4px-v4, and HpPT4px-sh catalyzed the addition of a prenyl group at the C-4 position of 1,3,5,6-tetrahydroxyxanthone; 1,3,5-trihydroxyxanthone; and 1,3,7-trihydroxyxanthone, whereas microsomes harboring HpPT4px-v1 and HpPT4px-v3 additionally accepted 1,3,6,7-tetrahydroxyxanthone. HpPT4px-v1 produced in Nicotiana benthamiana displayed the same activity as in yeast, while HpPT4px-sh was inactive. The kinetic parameters of HpPT4px-v1 and HpPT4px-sh chosen as representative variants indicated 1,3,5,6-tetrahydroxyxanthone as the preferred acceptor substrate, rationalizing that HpPT4px catalyzes the first prenylation step in the biosynthesis of polyprenylated xanthones in H. perforatum. Dimethylallyl pyrophosphate was the exclusive prenyl donor. Expression of the HpPT4px transcripts was highest in roots and leaves, raising the question of product translocation. C-terminal yellow fluorescent protein fusion of HpPT4px-v1 localized to the envelope of chloroplasts in N. benthamiana leaves, whereas short, truncated, and masked signal peptides led to the disruption of plastidial localization. These findings pave the way for a better understanding of the prenylation of xanthones in plants and the identification of additional xanthone-specific prenyltransferases.

Design, Synthesis, Biological Evaluation, and Preliminary Mechanistic Study of a Novel Mitochondrial-Targeted Xanthone.

α-Mangostin, a natural xanthone, was found to have anticancer effects, but these effects are not sufficient to be effective. To increase anticancer potential and selectivity, a triphenylphosphonium cation moiety (TPP) was introduced to α-mangostin to specifically target cancer cell mitochondria. Compared to the parent compound, the cytotoxicity of the synthesized compound 1b increased by one order of magnitude. Mechanistic analysis revealed that the anti-tumor effects were involved in the mitochondrial apoptotic pathway by prompting apoptosis and arresting the cell cycle at the G0/G1 phase, increasing the production of reactive oxygen species (ROS), and reducing mitochondrial membrane potential (Δψm). More notably, the antitumor activity of compound 1b was further confirmed by zebrafish models, which remarkably inhibited cancer cell proliferation and migration, as well as zebrafish angiogenesis. Taken together, our results for the first time indicated that TPP-linked 1b could lead to the development of new mitochondrion-targeting antitumor agents.

Flavonoid derivative DMXAA attenuates cisplatin-induced acute kidney injury independent of STING signaling.

Cisplatin-induced nephrotoxicity is the main adverse effect of cisplatin-based chemotherapy and highly limits its clinical use. DMXAA, a flavonoid derivative, is a promising vascular disrupting agent and known as an agonist of STING. Although cGAS-STING activation has been demonstrated to mediate cisplatin-induced acute kidney injury (AKI), the role of DMXAA in this condition is unclear. Here, we defined an unexpected and critical role of DMXAA in improving renal function, ameliorating renal tubular injury and cell apoptosis, and suppressing inflammation in cisplatin-induced AKI. Moreover, we confirmed that DMXAA combated AKI in a STING-independent manner, as evidenced by its protective effect in STING global knockout mice subjected to cisplatin. Furthermore, we compared the role of DMXAA with another STING agonist SR717 in cisplatin-treated mice and found that DMXAA but not SR717 protected animals against AKI. To better evaluate the role of DMXAA, we performed transcriptome analyses and observed that both inflammatory and metabolic pathways were altered by DMXAA treatment. Due to the established role of metabolic disorders in AKI, which contributes to kidney injury and recovery, we also performed metabolomics using kidney tissues from cisplatin-induced AKI mice with or without DMXAA treatment. Strikingly, our results revealed that DMXAA improved the metabolic disorders in kidneys of AKI mice, especially regulated the tryptophan metabolism. Collectively, therapeutic administration of DMXAA ameliorates cisplatin-induced AKI independent of STING, suggesting a promising potential for preventing nephrotoxicity induced by cisplatin-based chemotherapy.

The 'emodin family' of fungal natural products-amalgamating a century of research with recent genomics-based advances.

Covering: up to 2022A very large group of biosynthetically linked fungal secondary metabolites are formed via the key intermediate emodin and its corresponding anthrone. The group includes anthraquinones such as chrysophanol and cladofulvin, the grisandienes geodin and trypacidin, the diphenyl ether pestheic acid, benzophenones such as monodictyphenone and various xanthones including the prenylated shamixanthones, the agnestins and dimeric xanthones such as the ergochromes, cryptosporioptides and neosartorin. Such compounds exhibit a wide range of bioactivities and as such have been utilised in traditional medicine for centuries, as well as garnering more recent interest from the pharmaceutical sector. Additional interest comes from industries such as textiles and cosmetics due to their use as natural colourants. A variety of biosynthetic routes and mechanisms have been proposed for this family of compounds, being altered and updated as new biosynthetic methods develop and new results emerge. After nearly 100 years of such research, this review aims to provide a comprehensive overview of what is currently known about the biosynthesis of this important family, amalgamating the early chemical and biosynthetic studies with the more recent genetics-based advances and comparative bioinformatics.

Biosynthetic Pathways of Dimeric Natural Products Containing Bisanthraquinone and Related Xanthones.

Many dimeric natural products containing bisanthraquinone and related xanthones with diverse structures and versatile bioactivities have been isolated over the years. However, the complicated biosynthetic pathways of such natural products, which have remained elusive until recently, negatively impact their mass bioproduction and biosynthetic structural modification for drug discovery. In this concept, we summarize the recent progress in gene cluster mining and biosynthetic pathway elucidation of natural products containing bisanthraquinone and related xanthones. These pioneering works may pave the way for further biosynthetic pathway elucidation and structure modification of dimeric natural products through gene and protein engineering.

Chemistry and biosynthesis of bacterial polycyclic xanthone natural products.

Covering: up to the end of 2021Bacterial polycyclic xanthone natural products (BPXNPs) are a growing family of natural xanthones featuring a pentangular architecture with various modifications to the tricyclic xanthone chromophore. Their structural diversities and various activities have fueled biosynthetic and chemical synthetic studies. Moreover, their more potent activities than the clinically used drugs make them potential candidates for the treatment of diseases. Future unraveling of structure activity relationships (SARs) will provide new options for the (bio)-synthesis of drug analogues with higher activities. This review summarizes the isolation, structural elucidation and biological activities and more importantly, the recent strategies for the microbial biosynthesis and chemical synthesis of BPXNPs. Regarding their biosynthesis, we discuss the recent progress in enzymes that synthesize tricyclic xanthone, the protein candidates for structural moieties (methylene dioxygen bridge and nitrogen heterocycle), tailoring enzymes for methylation and halogenation. The chemical synthesis part summarizes the recent methodology for the division synthesis and coupling construction of achiral molecular skeletons. Ultimately, perspectives on the biosynthetic study of BPXNPs are discussed.

IL1ß/ TNFα/COX-2/VEGF axis responsible for effective healing potential of C-glucoside xanthone (mangiferin) based ointment in immunocompromised rats.

Present study was conducted to undermine the wound healing potential of mangiferin vis a vis its molecular dynamics in immunocompromised excisional rat model. 120 rats were randomly and equally divided into five groups viz. group I (Healthy control), group II (Immunocompromised control), group III (Immunocompromised group treated with silver sulphadiazine), group IV (Immunocompromised group treated with 2.5 %Mangiferin) and group V (Immunocompromised group treated with 5 %Mangiferin). Immuno compromised state was achieved following intramuscular injection of Hydrocortisone @ 80 mg/kg body weight. Study was conducted for a period of 28 days. Six animals from each group were humanely sacrificed at weekly interval till day 28th of study. Planimetric analysis, biochemical studies viz. hydroxyproline assay, total protein and DNA content, antioxidative potential through LPO assay was done along with molecular studies involving expression profiling of IL1ß, TNFα and COX-2 and Immunohistochemistry of angiogenic marker i.e. VEGF was performed to undermine the pharmacodynamics of mangiferin. Histopathological studies including H&E and Masson's Trichome was also performed to study histoarchitectural changes in wound healing and reparative process following application of mangiferin ointment. Study revealed significant (P ≤ 0.05) reduction in wound area measurement and significant (P ≤ 0.05) increase in wound contraction (%) following mangiferin administration in immunocompromised rats. Hydroxyproline, DNA and total protein showed significant (P ≤ 0.05) increase in skin tissues of mangiferin treated immunocompromised rats. LPO assay revealed significant (P ≤ 0.05) reduction in mangiferin treated animals. Histopathological studies of skin tissues revealed complete restoration advocating grade III of healing in 2.5% mangiferin treated group. Higher expression and strong signal intensity of VEGF was noticed in 2.5% mangiferin treatment group along with significant (P ≤ 0.05) upregulation IL1ß and TNFα on day 7 in 2.5% mangiferin treatment group with significant (P ≤ 0.05) down regulation of COX-2 in mangiferin treatment group as compared to other groups i.e. group II and III. It is concluded from our study that mangiferin facilitates wound healing through improved wound closure, organized deposition of collagen deposition and granulation matrix formation.

The protective effect of mangiferin on osteoarthritis: An in vitro and in vivo study.

Mangiferin is a kind of polyphenol chemical compound separated from these herbal medicines of Mangifera indica L., Anemarrhena asphodeloides Bge. and Belamcanda chinensis L., which has anti-inflammatory, anti-virus, and other physiological activities without toxic effects. Osteoarthritis (OA) is a chronic disease that is also a kind of arthritis disease in which articular cartilage or bones under the joint is damaged. In addition, artificial replacements are required in severe cases. At present, there are not too much researches on the potential biological activities of mangiferin that plays a protective role in the treatment of OA. In this study, we evaluated the protective effect of mangiferin on osteoarthritis (OA) in vitro and in vivo. First, the effect of different concentrations of mangiferin on rat chondrocytes was determined by MTT assay. Second, the effects of mangiferin on the expression levels of matrix metalloproteinase (MMP)-13, TNF alpha, Collagen II, Caspase-3, and cystatin-C in interleukin-1beta (IL-1beta)-induced rat chondrocytes were examined by the real-time polymerase chain reaction in vitro, meanwhile the effects of mangiferin on the nuclear factor kappa-B (NF-kappaB) signaling pathway were also investigated by Western Blot. Finally, the anti-osteoarthritic protective effect of mangiferin was evaluated in the rat model by anterior cruciate ligament transection (ACLT) combined with bilateral ovariectomy-induced OA in vivo. The results showed that the mangiferin was found to inhibit the expression of MMP-13, TNF-alpha, and Caspase-3 which also increased the expression of Collagen II and cystatin-C in IL 1beta induced rat chondrocytes. In addition, IL-1beta-induced activation of nuclear factor kappa-B (NF-kappaB) and the degradation of inhibitor of kappaB (IkappaB)-alpha were suppressed by mangiferin. For the in vivo study in a rat model of OA, 100 microl of mangiferin was administered by intra-articular injections for rats, the results showed that the cartilage degradation was suppressed by mangiferin through Micro CT and Histological Examination. According to both in vitro and in vivo results, mangiferin has a protective effect in the treatment of OA which may be a promising therapeutic agent for OA.

Speed dating for enzymes! Finding the perfect phosphopantetheinyl transferase partner for your polyketide synthase.

The biosynthetic pathways for the fungal polyketides bikaverin and bostrycoidin, from Fusarium verticillioides and Fusarium solani respectively, were reconstructed and heterologously expressed in S. cerevisiae alongside seven different phosphopantetheinyl transferases (PPTases) from a variety of origins spanning bacterial, yeast and fungal origins. In order to gauge the efficiency of the interaction between the ACP-domains of the polyketide synthases (PKS) and PPTases, each were co-expressed individually and the resulting production of target polyketides were determined after 48 h of growth. In co-expression with both biosynthetic pathways, the PPTase from Fusarium verticillioides (FvPPT1) proved most efficient at producing both bikaverin and bostrycoidin, at 1.4 mg/L and 5.9 mg/L respectively. Furthermore, the remaining PPTases showed the ability to interact with both PKS's, except for a single PKS-PPTase combination. The results indicate that it is possible to boost the production of a target polyketide, simply by utilizing a more optimal PPTase partner, instead of the commonly used PPTases; NpgA, Gsp and Sfp, from Aspergillus nidulans, Brevibacillus brevis and Bacillus subtilis respectively.

Marine and terrestrial endophytic fungi: a mine of bioactive xanthone compounds, recent progress, limitations, and novel applications.

Endophytic fungi are a kind of fungi that colonizes living plant tissues presenting a myriad of microbial adaptations that have been developed in such a hidden environment. Owing to its large diversity and particular habituation, they present a golden mine for research in the field of drug discovery. Endophytic fungal communities possess unique biocatalytic machinery that furnishes a myriad of complex natural product scaffolds. Xanthone compounds are examples of endophytic secondary metabolic products with pronounced biological activity to include: antioxidant, antimicrobial, anti-inflammatory, antithrombotic, antiulcer, choleretic, diuretic, and monoamine oxidase inhibiting activity.The current review compiles the recent progress made on the microbiological production of xanthones using fungal endophytes obtained from both marine and terrestrial origins, with comparisons being made among both natural resources. The biosynthesis of xanthones in endophytic fungi is outlined along with its decoding enzymes. Biotransformation reactions reported to be carried out using different endophytic microbial models are also outlined for xanthones structural modification purposes and the production of novel molecules.A promising application of novel computational tools is presented as a future direction for the goal of optimizing microbial xanthones production to include establishing metabolic pathway databases and the in silico analysis of microbial interactions. Metagenomics methods and related bioinformatics platforms are highlighted as unexplored tools for the biodiversity analysis of endophytic microbial communities that are difficult to be cultured.

Fusaroxazin, a novel cytotoxic and antimicrobial xanthone derivative from Fusarium oxysporum.

Oxazines and their derivatives are an uncommon natural heterocyclic compounds, which contain oxygen and nitrogen atoms and possess various bioactivities. A novel 1,4-oxazine-xanthone derivative, fusarioxazin (4) and three known sterols (1-3) were separated from Fusarium oxysporum EtOAc extract associated with Vicia faba L. (broad bean, Fabaceae) roots. Their structural assignment was accomplished using various spectroscopic tools and comparing with literature data. The cytotoxic and antimicrobial potentials of the novel metabolite (4) were evaluated. It possessed a significant antibacterial activity towards S. aureus (IZD 14.8 mm and MIC 5.3 µg/mL) and B. cereus (IZD 18.9 mm and MIC 3.7 µg/mL), in comparison to ciprofloxacin (IZDs 16.9 and 20.5 mm; MICs 3.9 and 2.3 µg/mL, respectively). Furthermore, it displayed a promising cytotoxic effect toward HCT-116 (IC50 2.1 µM), MCF-7 (IC50 1.8 µM), and A549 (IC50 3.2 µM) comparable to doxorubicin (IC50s 0.68, 0.54, and 0.39 µM, respectively).

Secondary metabolites of Hülle cells mediate protection of fungal reproductive and overwintering structures against fungivorous animals.

Fungal Hülle cells with nuclear storage and developmental backup functions are reminiscent of multipotent stem cells. In the soil, Hülle cells nurse the overwintering fruiting bodies of Aspergillus nidulans. The genome of A. nidulans harbors genes for the biosynthesis of xanthones. We show that enzymes and metabolites of this biosynthetic pathway accumulate in Hülle cells under the control of the regulatory velvet complex, which coordinates development and secondary metabolism. Deletion strains blocked in the conversion of anthraquinones to xanthones accumulate emodins and are delayed in maturation and growth of fruiting bodies. Emodin represses fruiting body and resting structure formation in other fungi. Xanthones are not required for sexual development but exert antifeedant effects on fungivorous animals such as springtails and woodlice. Our findings reveal a novel role of Hülle cells in establishing secure niches for A. nidulans by accumulating metabolites with antifeedant activity that protect reproductive structures from animal predators.

Molecularly engineered carrier-free co-delivery nanoassembly for self-sensitized photothermal cancer therapy.

BACKGROUND: Photothermal therapy (PTT) has been extensively investigated as a tumor-localizing therapeutic modality for neoplastic disorders. However, the hyperthermia effect of PTT is greatly restricted by the thermoresistance of tumor cells. Particularly, the compensatory expression of heat shock protein 90 (HSP90) has been found to significantly accelerate the thermal tolerance of tumor cells. Thus, a combination of HSP90 inhibitor and photothermal photosensitizer is expected to significantly enhance antitumor efficacy of PTT through hyperthermia sensitization. However, it remains challenging to precisely co-deliver two or more drugs into tumors. METHODS: A carrier-free co-delivery nanoassembly of gambogic acid (GA, a HSP90 inhibitor) and DiR is ingeniously fabricated based on a facile and precise molecular co-assembly technique. The assembly mechanisms, photothermal conversion efficiency, laser-triggered drug release, cellular uptake, synergistic cytotoxicity of the nanoassembly are investigated in vitro. Furthermore, the pharmacokinetics, biodistribution and self-enhanced PTT efficacy were explored in vivo. RESULTS: The nanoassembly presents multiple advantages throughout the whole drug delivery process, including carrier-free fabrication with good reproducibility, high drug co-loading efficiency with convenient dose adjustment, synchronous co-delivery of DiR and GA with long systemic circulation, as well as self-tracing tumor accumulation with efficient photothermal conversion. As expected, HSP90 inhibition-augmented PTT is observed in a 4T1 tumor BALB/c mice xenograft model. CONCLUSION: Our study provides a novel and facile dual-drug co-assembly strategy for self-sensitized cancer therapy.

Identification and characterization of glycosyltransferases catalyzing direct xanthone 4-C-glycosylation in Hypericum perforatum.

Xanthones are compounds with a diphenyl ether skeleton mainly found in plants and often glycosylated at carbon atoms. Although many C-glycosyltransferases (CGTs) participating in flavone C-glycosylation have been identified, MiCGT from Mangifera indica, adding sugar to an open-chain benzophenone skeleton, is the only identified xanthone biosynthesis-related CGT. Here, we identified two CGTs from Hypericum perforatum that add sugar to the closed-ring xanthone, but not benzophenone. These CGTs catalyze sugar transfer to the C-4 position of norathyriol (1,3,6,7-tetrahydroxyxanthone) to form isomangiferin (1,3,6,7-tetrahydroxyxanthone 4-C-glucoside), a major xanthone C-glucoside. This is the first study to report CGTs that mediate the direct C-glycosylation of xanthone.

Identification and characterization of xanthone biosynthetic genes contributing to the vivid red coloration of red-flowered gentian.

Cultivated Japanese gentians traditionally produce vivid blue flowers because of the accumulation of delphinidin-based polyacylated anthocyanins. However, recent breeding programs developed several red-flowered cultivars, but the underlying mechanism for this red coloration was unknown. Thus, we characterized the pigments responsible for the red coloration in these cultivars. A high-performance liquid chromatography with photodiode array analysis revealed the presence of phenolic compounds, including flavones and xanthones, as well as the accumulation of colored cyanidin-based anthocyanins. The chemical structures of two xanthone compounds contributing to the coloration of red-flowered gentian petals were determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The compounds were identified as norathyriol 6-O-glucoside (i.e., tripteroside designated as Xt1) and a previously unreported norathyriol-6-O-(6'-O-malonyl)-glucoside (designated Xt2). The copigmentation effects of these compounds on cyanidin 3-O-glucoside were detected in vitro. Additionally, an RNA sequencing analysis was performed to identify the cDNAs encoding the enzymes involved in the biosynthesis of these xanthones. Recombinant proteins encoded by the candidate genes were produced in a wheat germ cell-free protein expression system and assayed. We determined that a UDP-glucose-dependent glucosyltransferase (StrGT9) catalyzes the transfer of a glucose moiety to norathyriol, a xanthone aglycone, to produce Xt1, which is converted to Xt2 by a malonyltransferase (StrAT2). An analysis of the progeny lines suggested that the accumulation of Xt2 contributes to the vivid red coloration of gentian flowers. Our data indicate that StrGT9 and StrAT2 help mediate xanthone biosynthesis and contribute to the coloration of red-flowered gentians via copigmentation effects.

Biotransformation of Benzoate to 2,4,6-Trihydroxybenzophenone by Engineered Escherichia coli.

The synthesis of natural products by E. coli is a challenging alternative method of environmentally friendly minimization of hazardous waste. Here, we establish a recombinant E. coli capable of transforming sodium benzoate into 2,4,6-trihydroxybenzophenone (2,4,6-TriHB), the intermediate of benzophenones and xanthones derivatives, based on the coexpression of benzoate-CoA ligase from Rhodopseudomonas palustris (BadA) and benzophenone synthase from Garcinia mangostana (GmBPS). It was found that the engineered E. coli accepted benzoate as the leading substrate for the formation of benzoyl CoA by the function of BadA and subsequently condensed, with the endogenous malonyl CoA by the catalytic function of BPS, into 2,4,6-TriHB. This metabolite was excreted into the culture medium and was detected by the high-resolution LC-ESI-QTOF-MS/MS. The structure was elucidated by in silico tools: Sirius 4.5 combined with CSI FingerID web service. The results suggested the potential of the new artificial pathway in E. coli to successfully catalyze the transformation of sodium benzoate into 2,4,6-TriHB. This system will lead to further syntheses of other benzophenone derivatives via the addition of various genes to catalyze for functional groups.