Enhanced bioproduction of anticancer precursor vindoline by yeast cell factories.
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ABSTRACT:
SUBMITTER: Kulagina N
PROVIDER: S-EPMC8601169 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Plant specialized metabolites are widely used in the pharmaceutical industry, including the monoterpene indole alkaloids (MIAs) vinblastine and vincristine, which both display anticancer activity. Both compounds can be obtained through the chemical condensation of their precursors vindoline and catharanthine extracted from leaves of the Madagascar periwinkle. However, the extensive use of these molecules in chemotherapy increases precursor demand and results in recurrent shortages, explaining why the development of alternative production approaches, such microbial cell factories, is mandatory. In this context, the precursor-directed biosynthesis of vindoline from tabersonine in yeast-expressing heterologous biosynthetic genes is of particular interest but has not reached high production scales to date. To circumvent production bottlenecks, the metabolic flux was channeled towards the MIA of interest by modulating the copy number of the first two genes of the vindoline biosynthetic pathway, namely tabersonine 16-hydroxylase and tabersonine-16-O-methyltransferase. Increasing gene copies resulted in an optimized methoxylation of tabersonine and overcame the competition for tabersonine access with the third enzyme of the pathway, tabersonine 3-oxygenase, which exhibits a high substrate promiscuity. Through this approach, we successfully created a yeast strain that produces the fourth biosynthetic intermediate of vindoline without accumulation of other intermediates or undesired side-products. This optimization will probably pave the way towards the future development of yeast cell factories to produce vindoline at an industrial scale.
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Project description:Antitumor substances related to vinblastine and vincristine are exclusively found in the Catharanthus roseus (Madagascar periwinkle), a member of the Apocynaceae plant family, and continue to be extensively used in cancer chemotherapy. Although in high demand, these valuable compounds only accumulate in trace amounts in C. roseus leaves. Vinblastine and vincristine are condensed from the monoterpenoid indole alkaloid (MIA) precursors catharanthine and vindoline. Although catharanthine biosynthesis remains poorly characterized, the biosynthesis of vindoline from the MIA precursor tabersonine is well understood at the molecular and biochemical levels. This study uses virus-induced gene silencing (VIGS) to identify a cytochrome P450 [CYP71D1V2; tabersonine 3-oxygenase (T3O)] and an alcohol dehydrogenase [ADHL1; tabersonine 3-reductase (T3R)] as candidate genes involved in the conversion of tabersonine or 16-methoxytabersonine to 3-hydroxy-2,3-dihydrotabersonine or 3-hydroxy-16-methoxy-2,3-dihydrotabersonine, which are intermediates in the vindorosine and vindoline pathways, respectively. Biochemical assays with recombinant enzymes confirm that product formation is only possible by the coupled action of T3O and T3R, as the reaction product of T3O is an epoxide that is not used as a substrate by T3R. The T3O and T3R transcripts were identified in a C. roseus database representing genes preferentially expressed in leaf epidermis and suggest that the subsequent reaction products are transported from the leaf epidermis to specialized leaf mesophyll idioblast and laticifer cells to complete the biosynthesis of these MIAs. With these two genes, the complete seven-gene pathway was engineered in yeast to produce vindoline from tabersonine.
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Project description:In this present study, a newly isolated strain, Streptomyces sp. NEAE-H, capable of producing high amount of black extracellular melanin pigment on peptone-yeast extract iron agar and identified as Streptomyces glaucescens NEAE-H. Plackett-Burman statistical design was conducted for initial screening of 17 independent (assigned) variables for their significances on melanin pigment production by Streptomyces glaucescens NEAE-H. The most significant factors affecting melanin production are incubation period, protease-peptone and ferric ammonium citrate. The levels of these significant variables and their interaction effects were optimized by using face-centered central composite design. The maximum melanin production (31.650??g/0.1?ml) and tyrosinase activity (6089.10?U/ml) were achieved in the central point runs under the conditions of incubation period (6 days), protease-peptone (5?g/L) and ferric ammonium citrate (0.5?g/L). Melanin pigment was recovered by acid-treatment. Higher absorption of the purified melanin pigment was observed in the UV region at 250?nm. It appeared to have defined small spheres by scanning electron microscopy imaging. The maximum melanin yield was 350?mg dry wt/L of production medium. In vitro anticancer activity of melanin pigment was assayed against skin cancer cell line using MTT assay. The IC50 value was 16.34?±?1.31??g/ml for melanin and 8.8?±?0.5??g/ml for standard 5-fluorouracil.
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Project description:This review gives an overview of different yeast strains and enzyme classes involved in yeast whole-cell biotransformations. A focus was put on the synthesis of compounds for fine chemical and API (= active pharmaceutical ingredient) production employing single or only few-step enzymatic reactions. Accounting for recent success stories in metabolic engineering, the construction and use of synthetic pathways was also highlighted. Examples from academia and industry and advances in the field of designed yeast strain construction demonstrate the broad significance of yeast whole-cell applications. In addition to Saccharomyces cerevisiae, alternative yeast whole-cell biocatalysts are discussed such as Candida sp., Cryptococcus sp., Geotrichum sp., Issatchenkia sp., Kloeckera sp., Kluyveromyces sp., Pichia sp. (including Hansenula polymorpha = P. angusta), Rhodotorula sp., Rhodosporidium sp., alternative Saccharomyces sp., Schizosaccharomyces pombe, Torulopsis sp., Trichosporon sp., Trigonopsis variabilis, Yarrowia lipolytica and Zygosaccharomyces rouxii.
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Project description:Isoflavonoids comprise a class of plant natural products with great nutraceutical, pharmaceutical and agricultural significance. Their low abundance in nature and structural complexity however hampers access to these phytochemicals through traditional crop-based manufacturing or chemical synthesis. Microbial bioproduction therefore represents an attractive alternative. Here, we engineer the metabolism of Saccharomyces cerevisiae to become a platform for efficient production of daidzein, a core chemical scaffold for isoflavonoid biosynthesis, and demonstrate its application towards producing bioactive glucosides from glucose, following the screening-reconstruction-application engineering framework. First, we rebuild daidzein biosynthesis in yeast and its production is then improved by 94-fold through screening biosynthetic enzymes, identifying rate-limiting steps, implementing dynamic control, engineering substrate trafficking and fine-tuning competing metabolic processes. The optimized strain produces up to 85.4 mg L-1 of daidzein and introducing plant glycosyltransferases in this strain results in production of bioactive puerarin (72.8 mg L-1) and daidzin (73.2 mg L-1). Our work provides a promising step towards developing synthetic yeast cell factories for de novo biosynthesis of value-added isoflavonoids and the multi-phased framework may be extended to engineer pathways of complex natural products in other microbial hosts.
| S-EPMC8526750 | biostudies-literature
Project description:Sustainable production of oleochemicals requires establishment of cell factory platform strains. The yeast Saccharomyces cerevisiae is an attractive cell factory as new strains can be rapidly implemented into existing infrastructures such as bioethanol production plants. Here we show high-level production of free fatty acids (FFAs) in a yeast cell factory, and the production of alkanes and fatty alcohols from its descendants. The engineered strain produces up to 10.4 g l(-1) of FFAs, which is the highest reported titre to date. Furthermore, through screening of specific pathway enzymes, endogenous alcohol dehydrogenases and aldehyde reductases, we reconstruct efficient pathways for conversion of fatty acids to alkanes (0.8 mg l(-1)) and fatty alcohols (1.5 g l(-1)), to our knowledge the highest titres reported in S. cerevisiae. This should facilitate the construction of yeast cell factories for production of fatty acids derived products and even aldehyde-derived chemicals of high value.
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Project description:BACKGROUND:A microorganism engineered for non-native tasks may suffer stresses it never met before. Therefore, we examined whether a Kluyveromyces marxianus strain engineered with a carotenoid biosynthesis pathway can serve as an anti-stress chassis for building cell factories. RESULTS:Carotenoids, a family of antioxidants, are valuable natural products with high commercial potential. We showed that the free radical removal ability of carotenoids can confer the engineered host with a higher tolerance to ethanol, so that it can produce more bio-ethanol than the wild type. Moreover, we found that this engineered strain has improved tolerance to other toxic effects including furfurals, heavy metals such as arsenate (biomass contaminant) and isobutanol (end product). Furthermore, the enhanced ethanol tolerance of the host can be applied to bioconversion of a natural medicine that needs to use ethanol as the delivery solvent of hydrophobic precursors. The result suggested that the engineered yeast showed enhanced tolerance to ethanol-dissolved hydrophobic 10-deacetylbaccatin III, which is considered a sustainable precursor for paclitaxel (taxol) bioconversion. CONCLUSIONS:The stress tolerances of the engineered yeast strain showed tolerance to several toxins, so it may serve as a chassis for cell factories to produce target products, and the co-production of carotenoids may make the biorefinary more cost-effective.
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Project description:Fatty acid alkyl esters have broad applications in biofuels, lubricant formulas, paints, coatings, and cosmetics. Traditionally, these esters are mostly produced through unsustainable and energy-intensive processes. In contrast, microbial production of esters from renewable and sustainable feedstocks may provide a promising alternative and has attracted widespread attention in recent years. At present, yeasts are used as ideal hosts for producing such esters, due to their availability for high-density fermentation, resistance to phage infection, and tolerance against toxic inhibitors. Here, we summarize recent development on the biosynthesis of alkyl esters, including fatty acid ethyl esters (FAEEs), fatty acid short-branched chain alkyl esters (FASBEs), and wax esters (WEs) by various yeast cell factories. We focus mainly on the enzyme engineering strategies of critical wax ester synthases, and the pathway engineering strategies employed for the biosynthesis of various ester products. The bottlenecks that limit productivity and their potential solutions are also discussed in this review.
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