RESUMO
Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world's supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.
Assuntos
Antineoplásicos , Reatores Biológicos , Vias Biossintéticas , Engenharia Metabólica , Saccharomyces cerevisiae , Vimblastina , Alcaloides de Vinca , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/provisão & distribuição , Catharanthus/química , Genes Fúngicos , Genes de Plantas , Engenharia Metabólica/métodos , Fosfatos de Poli-Isoprenil , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Triptofano , Vimblastina/biossíntese , Vimblastina/química , Vimblastina/provisão & distribuição , Alcaloides de Vinca/biossíntese , Alcaloides de Vinca/química , Alcaloides de Vinca/provisão & distribuiçãoRESUMO
Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications, including cancer, malaria and hypertension. Halogenated MIAs have shown improved pharmaceutical properties; however, synthesis of new-to-nature halogenated MIAs remains a challenge. Here we demonstrate a platform for de novo biosynthesis of two MIAs, serpentine and alstonine, in baker's yeast Saccharomyces cerevisiae and deploy it to systematically explore the biocatalytic potential of refactored MIA pathways for the production of halogenated MIAs. From this, we demonstrate conversion of individual haloindole derivatives to a total of 19 different new-to-nature haloserpentine and haloalstonine analogs. Furthermore, by process optimization and heterologous expression of a modified halogenase in the microbial MIA platform, we document de novo halogenation and biosynthesis of chloroalstonine. Together, this study highlights a microbial platform for enzymatic exploration and production of complex natural and new-to-nature MIAs with therapeutic potential.
Assuntos
Catharanthus , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Monoterpenos/metabolismo , Alcaloides Indólicos/metabolismo , Plantas/metabolismo , Preparações Farmacêuticas/metabolismo , Proteínas de Plantas/metabolismoRESUMO
PURPOSE: Benzoxazinoids (BXs) are a group of wholegrain phytochemicals with potential pharmacological properties; however, limited information exists on their absorption, metabolism, and excretion in humans. The aim of this study was to investigate the dose-dependent uptake and excretion of dietary BXs in a healthy population. METHODS: Blood and urine were collected from 19 healthy participants from a crossover study after a washout, a LOW BX diet or HIGH BX diet, and analysed for 12 BXs and 4 phenoxazinone derivatives. RESULTS: We found that the plasma BX level peaked approximately 3 h after food intake, whereas BXs in urine were present even at 36 h after consuming a meal. No phenoxazinone derivatives could be detected in either plasma or urine. The dominant BX metabolite in both plasma and urine was 2-ß-D-glucopyranosyloxy-1,4-benzoxazin-3-one (HBOA-Glc), even though 2-ß-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-Glc) was the major component in the diet. CONCLUSION: The dietary BX treatment correlated well with the plasma and urine levels, illustrating strong dose-dependent BX absorption, which also had a rapid washout, especially from the plasma compartment.
Assuntos
Benzoxazinas/farmacocinética , Dieta , Fibras na Dieta/administração & dosagem , Adolescente , Adulto , Idoso , Benzoxazinas/sangue , Benzoxazinas/urina , Índice de Massa Corporal , Estudos Cross-Over , Grão Comestível/química , Estudos de Avaliação como Assunto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Compostos Fitoquímicos/sangue , Compostos Fitoquímicos/farmacocinética , Compostos Fitoquímicos/urina , Adulto JovemRESUMO
Monoterpene indole alkaloids (MIAs) make up a highly bioactive class of metabolites produced by a range of tropical and subtropical plants. The corynanthe-type MIAs are a stereochemically complex subclass with therapeutic potential against a large number of indications including cancer, psychotic disorders, and erectile dysfunction. Here, we report yeast-based cell factories capable of de novo production of corynanthe-type MIAs rauwolscine, yohimbine, tetrahydroalstonine, and corynanthine. From this, we demonstrate regioselective biosynthesis of 4 fluorinated derivatives of these compounds and de novo biosynthesis of 7-chlororauwolscine by coexpression of a halogenase with the biosynthetic pathway. Finally, we capitalize on the ability of these cell factories to produce derivatives of these bioactive scaffolds to establish a proof-of-principle drug discovery pipeline in which the corynanthe-type MIAs are screened for bioactivity on human drug targets, expressed in yeast. In doing so, we identify antagonistic and agonistic behavior against the human adrenergic G protein-coupled receptors ADRA2A and ADRA2B, and the serotonergic receptor 5HT4b, respectively. This study thus demonstrates a proto-drug discovery pipeline for bioactive plant-inspired small molecules based on one-pot biocatalysis of natural and new-to-nature corynanthe-type MIAs in yeast.
Assuntos
Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Humanos , Vias Biossintéticas , Ioimbina/metabolismo , Ioimbina/farmacologia , Alcaloides de Triptamina e Secologanina/metabolismo , Alcaloides Indólicos/metabolismo , Descoberta de Drogas/métodosRESUMO
Benzoxazinoids are secondary metabolites with plant defense properties and possible health-promoting effects in humans. In this study, the transcriptional activity of ScBx genes (ScBx1-ScBx5; ScBx6-like), involved in benzoxazinoid biosynthesis, was analyzed during germination and early seedling development in rye. Our results showed that ScBx genes had highest levels of expression at 24-30 h after germination, followed by a decrease at later stages. For ScBx1-ScBx5 genes expression was higher in shoots compared with root tissues and vice versa for ScBx6-like gene transcripts. Moreover, methylated forms of benzoxazinoids accumulated in roots rather than in shoots during seedling development, in particular reaching high levels of HMBOA-glc in roots. Chemical profiles of benzoxazinoid accumulation in the developing seedling reflected the combined effects of de novo biosynthesis of the compounds as well as the turnover of compounds either pre-stored in the embryo or de novo biosynthesized. Bioinformatic analysis, together with the differential distribution of ScBx6-like transcripts in root and shoot tissues, suggested the presence of a ZmBx6 homolog encoding a 2-oxoglutarate dependent dehydrogenase in rye. The ScBx6-like cDNA was expressed in E. coli for functional characterization in vitro. LC-MS/MS analysis showed that the purified enzyme was responsible for the oxidation of DIBOA-glc into TRIBOA-glc, strongly suggesting the ScBX6-like enzyme in rye to be a functional ortholog of maize ZmBX6.
Assuntos
Benzoxazinas/química , Genes de Plantas , Germinação , Secale/química , Dioxigenases/genética , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/química , Secale/enzimologia , Secale/genética , Sementes/química , Zea mays/enzimologia , Zea mays/genéticaRESUMO
Lolines, fungal metabolites of the grass-endophyte association, were identified and quantified using newly developed LC-MS/MS methods in endophyte-infected grasses belonging to the Lolium and Festuca genera after extraction with three different solvents using two extraction methods. The shaking extraction method with isopropanol/water was superior to the other methods due to its high sensitivity, high accuracy (recovery within or close to the range of 80-120%), and high precision (coefficient of variation of <10%). Seven loline alkaloids were identified and quantified using our newly established LC-MS/MS methods, and N-formylloline was the most abundant (5 mg/g dry matter), followed by N-acetylloline. These LC-MS/MS methods used the shortest sample handling time and the fewest sample preparation steps and proved to be good alternatives to existing GC and GC-MS analytical methods without compromising analytical efficiency. In conclusion, we developed for the first time a highly sensitive quantitative LC-MS/MS analytical method for the accurate and reproducible quantification and a LightSight-assisted LC-QTRAP/MS qualitative method for the tentative identification of loline-type alkaloids in endophyte-infected grasses.
Assuntos
Alcaloides/química , Cromatografia Líquida/métodos , Endófitos/metabolismo , Festuca/microbiologia , Fungos/metabolismo , Lolium/microbiologia , Poaceae/microbiologia , Espectrometria de Massas em Tandem/métodos , Alcaloides/metabolismo , Festuca/química , Lolium/química , Poaceae/químicaRESUMO
Rye bread contains high amounts of benzoxazinoids, and in vitro studies have shown suppressive effects of selected benzoxazinoids on prostate cancer cells. Thus, research into benzoxazinoids as possible suppressors of prostate cancer is demanded. A pilot study was performed in which ten prostate cancer patients received a rye-enriched diet 1 week prior to prostatectomy. Plasma and urine samples were collected pre- and postintervention. Ten prostate biopsies were obtained from each patient and histologically evaluated. The biopsies exhibited concentrations above the detection limit of seven benzoxazinoids ranging from 0.15 to 10.59 ng/g tissue. An OPLS-DA analysis on histological and plasma concentrations of benzoxazinoids classified the subjects into two clusters. A tendency of higher benzoxazinoid concentrations toward the benign group encourages further investigations. Benzoxazinoids were quantified by an optimized LC-MS/MS method, and matrix effects were evaluated. At low concentrations in biopsy and plasma matrices the matrix effect was concentration-dependent and nonlinear. For the urine samples the general matrix effects were small but patient-dependent.
Assuntos
Benzoxazinas/análise , Neoplasias da Próstata/dietoterapia , Neoplasias da Próstata/patologia , Secale , Benzoxazinas/sangue , Benzoxazinas/urina , Biópsia com Agulha de Grande Calibre , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Limite de Detecção , Masculino , Reprodutibilidade dos TestesRESUMO
Benzoxazinoids (BXs) are a group of natural chemical compounds with putative pharmacological and health-protecting properties. BXs were formerly identified in and isolated from selected dicot medicinal plants and young cereal plants. Recently, BXs were found to be present in mature cereal grains and bakery products, such that knowledge about the pharmacological properties of BXs, which until now have unknowingly been consumed through the daily bread and breakfast cereals, has come into new focus. This review discusses published results from in vitro studies and a few human and animal model studies on the health effects and pharmacological responses of various BX compounds. Many of these studies have reported antimicrobial, anticancer, reproductive system stimulatory, central nervous system stimulatory, immunoregulatory, and appetite- and weight-reducing effects of BXs and/or BX derivatives. The health benefits of wholegrain intake may be associated with the solitary and/or overlapping biological effects of fibers, lignans, phenolic acids, alkylresorcinols, BXs, and other bioactive compounds. In the context of BXs as dietary ingredients, further comprehensive investigations are required to understand their biological functions, to elucidate the underlying mechanisms, to explore their potential contribution on the health effects associated with wholegrain consumption, and to examine their potential as functional food ingredients.
Assuntos
Anti-Infecciosos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Depressores do Apetite/farmacologia , Benzoxazinas/farmacologia , Grão Comestível/química , Animais , Antineoplásicos Fitogênicos/química , Benzoxazinas/efeitos adversos , Benzoxazinas/imunologia , Humanos , Sistema Imunitário/efeitos dos fármacos , Redução de PesoRESUMO
SCOPE: Benzoxazinoids, which are natural compounds recently identified in mature whole grain cereals and bakery products, have been suggested to have a range of pharmacological properties and health-protecting effects. There are no published reports concerned with the absorption and metabolism of bioactive benzoxazinoids in humans. METHODS AND RESULTS: The absorption, metabolism, and excretion of ten different dietary benzoxazinoids were examined by LC-MS/MS by analyzing plasma and urine from 20 healthy human volunteers after daily intake of 143 µmol of total benzoxazinoids from rye bread and rye buns. The results showed that 2-ß-D-glucopyranosyloxy-1,4-benzoxazin-3-one (HBOA-Glc) and its oxidized analog, 2-ß-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-Glc), were the major circulating benzoxazinoids. After consuming a benzoxazinoid diet for 1 week, morning urine contained eight benzoxazinoids with abundant HBOA-Glc (219 nmol × µmol⻹ of creatinine). The sulfate and glucuronide conjugates of 2-hydroxy-1,4-benzoxazin-3-one (HBOA) and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) were detected in plasma and urine, indicating substantial phase II metabolism. Direct absorption of lactam glycosides, the reduction of hydroxamic acid glycosides, glucuronidation, and sulfation were the main mechanisms of the absorption and metabolism of benzoxazinoids. CONCLUSION: These results indicate that following ingestion in healthy humans, a range of unmetabolized bioactive dietary benzoxazinoids and their sulfate and glucuronide conjugates appear in circulation and urine.
Assuntos
Benzoxazinas/farmacocinética , Dieta , Absorção , Adulto , Benzoxazinas/administração & dosagem , Benzoxazinas/sangue , Benzoxazinas/metabolismo , Benzoxazinas/urina , Índice de Massa Corporal , Pão , Cromatografia Líquida , Feminino , Glucuronídeos/metabolismo , Glicosídeos/metabolismo , Voluntários Saudáveis , Humanos , Modelos Lineares , Masculino , Reprodutibilidade dos Testes , Secale/química , Espectrometria de Massas em TandemRESUMO
Thorough knowledge of the absorption and metabolism of dietary benzoxazinoids is needed to understand their health-promoting effects. In this study, the fates of these bioactive compounds were examined by LC-MS/MS in plasma, urine, and feces after ingesting a daily dose of 4780 ± 68 nmol benzoxazinoids from rye bread using Wistar rats as a model. HBOA-glc (2-ß-D-glucopyranosyloxy-1,4-benzoxazin-3-one) was the predominant benzoxazinoid in the plasma (74 ± 27 nmol/L), followed by DIBOA-glc (2-ß-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one) and HBOA. The total level of benzoxazinoids in the urine was 1176 ± 66 nmol/d, which corresponds to approximately 25% of the total dietary intake. The urinary benzoxazinoid profile differed from that of plasma with HBOA-glc and DIBOA-glc (647 ± 31 and 466 ± 33 nmol/d, respectively) as the major urinary components. The glucuronide conjugates of HBOA and DIBOA were detected in both the plasma and urine. N-dehydroxylation was found to be a critical step in the absorption of hydroxamic acids. This unprecedented study will trigger future interest in the biological effects of benzoxazinoids in whole grain rye and wheat diets in humans and other animals.
Assuntos
Benzoxazinas/sangue , Benzoxazinas/urina , Pão , Glucuronídeos/metabolismo , Secale/metabolismo , Animais , Benzoxazinas/metabolismo , Humanos , Masculino , Espectrometria de Massas , Ratos , Ratos Wistar , Secale/química , Triticum/química , Triticum/metabolismoRESUMO
Recently, bioactive benzoxazinoids were discovered in cereal grains and bakery products. In this study, we studied the uptake, distribution, and metabolism of these secondary metabolites using a pig model. Twelve benzoxazinoid compounds and their 4 transformation products were quantified in the pigs' diets and biofluids using high-performance liquid chromatography coupled to electrospray ionization triple quadrupole mass spectrometry. The 2-ß-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-glc) was the most dominant benzoxazinoid (232 nmol/g DM) seconded by the double-hexose derivative of DIBOA (provisionally characterized here as DIBOA-glc-hex) in the rye-based diet. DIBOA-glc (derived from the diet and intestinal deglycosylation of DIBOA-glc-hex) was apparently reduced to 2-ß-D-glucopyranosyloxy-1,4-benzoxazin-3-one (HBOA-glc), the most dominant benzoxazinoid in the blood (829 nmol/L). The benzoxazinoid compounds were excreted in the urine, with HBOA-glc (18 µmol/L) as a major metabolite. In this study, we determined for the first time the bioavailability of dietary benzoxazinoids that have high digestibility, distribution, and metabolism in mammals. These findings could be a milestone for the exploitation of healthful and pharmacological properties of benzoxazinoids.