Metabolic engineering of yeast for de novo production of kratom monoterpene indole alkaloids
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1114704 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Monoterpene indole alkaloids (MIAs) from Mitragyna speciosa (“kratom”), such as mitragynine and speciogynine, are promising novel scaffolds for opioid receptor ligands for treatment of pain, addiction, and depression. While kratom leaves have been used for centuries in South-East Asia as stimulant and pain management substance, the biosynthetic pathway of these psychoactives have only recently been partially elucidated. Here, we demonstrate the de novo production of mitragynine and speciogynine in Saccharomyces cerevisiae through the reconstruction of a five-step synthetic pathway from common MIA precursor strictosidine comprising fungal tryptamine 4-monooxygenase to bypass an unknown kratom hydroxylase. Upon optimizing cultivation conditions, a titer of ~290 µg/L kratom MIAs from glucose was achieved. Untargeted metabolomics analysis of lead production strains led to the identification of numerous shunt products derived from the activity of strictosidine synthase (STR) and dihydrocorynantheine synthase (DCS), highlighting them as candidates for enzyme engineering to further improve kratom MIAs production in yeast. Finally, by feeding fluorinated tryptamine and expressing a human tailoring enzyme, we further demonstrate production of fluorinated and hydroxylated mitragynine derivatives with potential applications in drug discovery campaigns. Altogether, this study introduces a yeast cell factory platform for the biomanufacturing of complex natural and new-to-nature kratom MIAs derivatives with therapeutic potential.
2024-12-31 | GSE268113 | GEO
Project description:Catharanthus roseus produces a variety of indole alkaloids with significant biological activities. The indole alkaloids including catharanthine, vindolinine, ajmalicine and the precursor strictosidine were dramatically induced in the leaves following binary stress. To profile the modification of indole alkaloids in C. roseus seedlings under the binary stress of ultraviolet-B irradiation and dark incubation, gel-free proteomic analysis was carried out to uncover the underlying molecular mechanism.
2015-08-24 | PXD001937 | Pride
Project description:This dataset was used to test the extracted spectrometric patterns of monoterpene indole alkaloid skeletons
2024-10-19 | MSV000096137 | MassIVE
Project description:Tropane alkaloids (TAs) are a class of phytochemicals produced by plants of the nightshade family used for treating diverse neurological disorders. Here, we demonstrate de novo production of tropine, a key intermediate in the biosynthetic pathway of medicinal TAs such as scopolamine, from simple carbon and nitrogen sources in yeast (Saccharomyces cerevisiae). Our engineered strain incorporates 15 additional genes, including 11 derived from diverse plants and bacteria, and 7 disruptions to yeast regulatory or biosynthetic proteins to produce tropine at titers of 6 mg/L. We also demonstrate the utility of our engineered yeast platform for the discovery of TA derivatives by combining biosynthetic modules from distant plant lineages to achieve de novo production of cinnamoyltropine, a non-canonical TA. Our engineered strain constitutes a starting point for future optimization efforts towards realizing industrial fermentation of medicinal TAs and a platform for the synthesis of TA derivatives with enhanced bioactivities.
| S-EPMC6690885 | biostudies-other
Project description:MIADB: a cumulative collection of 172 tandem mass spectrometry (MS/MS) of a vast array of monoterpene indole alkaloids. Samples were analyzed using an Agilent LC-MS system composed of an Agilent 1260 Infinity HPLC coupled to an Agilent 6530 ESI-Q-TOF-MS operating in positive mode. A Sunfire analytical C18 column (150 × 2.1 mm; i.d. 3.5 μm, Waters) was used, with a flow rate of 250 μL/min and a linear gradient from 5% B (A: H2O + 0.1% formic acid, B: MeOH) to 100% B over 30 min. ESI conditions were set with the capillary temperature at 320 °C, source voltage at 3.5 kV, and a sheath gas flow rate of 10 L/min. The divert valve was set to waste for the first 3 min. There were four scan events: positive MS, window from m/z 100−1200, then three data-dependent MS/MS scans of the first, second, and third most intense ions from the first scan event. MS/MS settings were three fixed collision energies (30, 50, and 70 eV), default charge of 1, minimum intensity of 5000 counts, and isolation width of m/z 2. In the positive-ion mode, purine C5H4N4 [M + H]+ ion (m/z 121.050873) and the hexakis(1H,1H,3H-tetrafluoropropoxy)-phosphazene C18H18F24N3O6P3 [M + H]+ ion (m/z 922.009 798) were used as internal lock masses. Full scans were acquired at a resolution of 11 000 (at m/z 922). A permanent MS/MS exclusion list criterion was set to prevent oversampling of the internal calibrant.
2018-12-03 | MTBLS142 | MetaboLights
Project description:BackgroundAnthocyanins are polyphenolic pigments which provide pink to blue colours in fruits and flowers. There is an increasing demand for anthocyanins, as food colorants and as health-promoting substances. Plant production of anthocyanins is often seasonal and cannot always meet demand due to low productivity and the complexity of the plant extracts. Therefore, a system of on-demand supply is useful. While a number of other (simpler) plant polyphenols have been successfully produced in the yeast Saccharomyces cerevisiae, production of anthocyanins has not yet been reported.ResultsSaccharomyces cerevisiae was engineered to produce pelargonidin 3-O-glucoside starting from glucose. Specific anthocyanin biosynthetic genes from Arabidopsis thaliana and Gerbera hybrida were introduced in a S. cerevisiae strain producing naringenin, the flavonoid precursor of anthocyanins. Upon culturing, pelargonidin and its 3-O-glucoside were detected inside the yeast cells, albeit at low concentrations. A number of related intermediates and side-products were much more abundant and were secreted into the culture medium. To optimize titers of pelargonidin 3-O-glucoside further, biosynthetic genes were stably integrated into the yeast genome, and formation of a major side-product, phloretic acid, was prevented by engineering the yeast chassis. Further engineering, by removing two glucosidases which are known to degrade pelargonidin 3-O-glucoside, did not result in higher yields of glycosylated pelargonidin. In aerated, pH controlled batch reactors, intracellular pelargonidin accumulation reached 0.01 µmol/gCDW, while kaempferol and dihydrokaempferol were effectively exported to reach extracellular concentration of 20 µM [5 mg/L] and 150 µM [44 mg/L], respectively.ConclusionThe results reported in this study demonstrate the proof-of-concept that S. cerevisiae is capable of de novo production of the anthocyanin pelargonidin 3-O-glucoside. Furthermore, while current conversion efficiencies are low, a number of clear bottlenecks have already been identified which, when overcome, have huge potential to enhance anthocyanin production efficiency. These results bode very well for the development of fermentation-based production systems for specific and individual anthocyanin molecules. Such systems have both great scientific value for identifying and characterising anthocyanin decorating enzymes as well as significant commercial potential for the production of, on-demand, pure bioactive compounds to be used in the food, health and even pharma industries.
| S-EPMC6029064 | biostudies-literature
Project description:We propose a strain engineering approach for synthesizing sedoheptulose using glucose as sole feedstock. The gene pfkA encoding 6-phosphofructokinase in Corynebacterium glutamicum was inactivated to direct the carbon flux towards pentose phosphate pathway in the cellular metabolic network. This genetic modification successfully enabled the synthesis of sedoheptulose from glucose. Additionally, we identified key enzymes responsible for product formation through transcriptome analysis, and their corresponding genes were overexpressed, resulting in a further 20% increase in sedoheptulose production.
2024-05-31 | GSE254540 | GEO
Project description:Altered tryptophan catabolism has been identified in inflammatory diseases like rheumatoid arthritis (RA) and spondyloarthritis (SpA), but the causal mechanisms linking tryptophan metabolites to disease are unknown. Using the collagen-induced arthritis (CIA) model we identify alterations in tryptophan metabolism, and specifically indole, that correlate with disease. We demonstrate that both bacteria and dietary tryptophan are required for disease, and indole supplementation is sufficient to induce disease in their absence. When mice with CIA on a low-tryptophan diet were supplemented with indole, we observed significant increases in serum IL-6, TNF, and IL-1β; splenic RORγt+CD4+ T cells and ex vivo collagen-stimulated IL-17 production; and a pattern of anti-collagen antibody isotype switching and glycosylation that corresponded with increased complement fixation. IL-23 neutralization reduced disease severity in indole-induced CIA. Finally, exposure of human colon lymphocytes to indole increased expression of genes involved in IL-17 signaling and plasma cell activation. Altogether, we propose a mechanism by which intestinal dysbiosis during inflammatory arthritis results in altered tryptophan catabolism, leading to indole stimulation of arthritis development. Blockade of indole generation may present a novel therapeutic pathway for RA and SpA.
2023-11-13 | GSE241655 | GEO
Project description: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 vincristine. As MIAs are difficult to chemically synthesize, the world’s supply chain for vinblastine relies on low-yielding extractions of precursors vindoline and catharanthine from the plant Catharanthus roseus, followed by chemical coupling and reduction to form vinblastine. Here, we demonstrate de novo microbial biosynthesis of vindoline and catharanthine from renewable feedstocks such as simple sugar and amino acids using highly engineered yeast. The study showcases the longest biosynthetic pathway refactored into a microbial cell factory to date, including 29 enzymatic steps from the yeast native metabolite geranyl pyrophosphate to catharanthine and vindoline. We made 44 genetic edits to yeast that include expression of 35 heterologous genes from plants as well as deletions, knock-downs, and overexpression of 10 yeast genes or variants thereof to improve the precursor supply. Finally, we demonstrate one-step in vitro vinblastine production using chemical coupling and reduction of vindoline and catharanthine. Not only is the yeast a scalable platform for production of vinblastine, it is also a platform for production of more than 2,000 different natural and new-to-nature MIAs.
2022-08-21 | PXD025067 | Pride