Production of 3-hydroxypropionic acid from glucose and xylose by metabolically engineered Saccharomyces cerevisiae.
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ABSTRACT:
SUBMITTER: Kildegaard KR
PROVIDER: S-EPMC8193239 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Yeasts do not possess an endogenous biochemical pathway for the synthesis of vitamin C. However, incubated with l-galactose, L-galactono-1,4-lactone, or L-gulono-1,4-lactone intermediates from the plant or animal pathway leading to l-ascorbic acid, Saccharomyces cerevisiae and Zygosaccharomyces bailii cells accumulate the vitamin intracellularly. Overexpression of the S. cerevisiae enzymes d-arabinose dehydrogenase and D-arabinono-1,4-lactone oxidase enhances this ability significantly. In fact, the respective recombinant yeast strains even gain the capability to accumulate the vitamin in the culture medium. An even better result is obtainable by expression of the plant enzyme L-galactose dehydrogenase from Arabidopsis thaliana. Budding yeast cells overexpressing the endogenous D-arabinono-1,4-lactone oxidase as well as L-galactose dehydrogenase are capable of producing about 100 mg of L-ascorbic acid liter(-1), converting 40% (wt/vol) of the starting compound L-galactose.
| S-EPMC522139 | biostudies-literature
Project description:BACKGROUND:Glycyrrhetinic acid (GA) is the most important ingredient in licorice due to its outstanding anti-inflammatory activity and wide application in the medicine and cosmetics industries. Contemporary industrial production of GA by acid hydrolysis of glycyrrhizin which was extracted from Glycyrrhiza plants, is not environment-friendly and devastates farmland since the Glycyrrhiza rhizomes grow up to 10 m underground. RESULTS:In this study, GA was produced through metabolically engineering Saccharomyces cerevisiae by introducing the entire heterogeneous biosynthetic pathway of GA. Codon optimized CYP88D6 and CYP72A154, combined with ?-AS (?-amyrin synthase encoding gene) and the NADPH-cytochrome P450 reductase gene of Arabidopsis thaliana were introduced into S. cerevisiae. The resulting strain (Y1) produced 2.5 mg/L of ?-amyrin and 14 ?g/L of GA. The cytochrome b5 from G. uralensis (GuCYB5) was identified and the introduction of this novel GuCYB5 increased the efficiency of GA production by eightfold. The joint utilization of the GuCYB5 gene along with 10 known MVA pathway genes from S. cerevisiae were overexpressed in a stable chromosome integration to achieve higher GA production. Using the combined strategy, GA concentration improved by 40-fold during batch fermentation. The production was further improved to 8.78 mg/L in fed-batch fermentation, which was increased by a factor of nearly 630. CONCLUSIONS:This study first investigated the influence of carbon flux in the upstream module and the introduction of a newly identified GuCYB5 on GA production. The newly identified GuCYB5 was highly effective in improving GA production. An integrated strategy including enzyme discovery, pathway optimization, and fusion protein construction was provided in improving GA production, achieving a 630 fold increase in GA production. The metabolically engineered yeast cell factories provide an alternative approach to glycyrrhetinic acid production, replacing the traditional method of plant extraction.
| S-EPMC6540369 | biostudies-literature
Project description:Vindoline is a plant derived monoterpene indole alkaloid (MIA) with potential therapeutic applications and more importantly serves as the precursor to vinblastine and vincristine. To obtain a yeast strain for high yield production of vindoline from tabersonine, multiple metabolic engineering strategies were employed via the CRISPR/Cas9 mediated multiplex genome integration technology in the present study. Through increasing and tuning the copy numbers of the pathway genes, pairing cytochrome P450 enzymes (CYPs) with appropriate cytochrome P450 reductases (CPRs), engineering the microenvironment for functional expression of CYPs, enhancing cofactor supply, and optimizing fermentation conditions, the production of vindoline was increased to a final titer as high as ∼16.5 mg/L, which is more than 3,800,000-fold higher than the parent strain and the highest tabersonine to vindoline conversion yield ever reported. This work represents a key step of the engineering efforts to establish de novo biosynthetic pathways for vindoline, vinblastine, and vincristine.
| S-EPMC8446080 | biostudies-literature
Project description:BackgroundSaccharomyces cerevisiae, engineered for L-lactic acid production from glucose and xylose, is a promising production host for lignocellulose-to-lactic acid processes. However, the two principal engineering strategies-pyruvate-to-lactic acid conversion with and without disruption of the competing pyruvate-to-ethanol pathway-have not yet resulted in strains that combine high lactic acid yields (YLA) and productivities (QLA) on both sugar substrates. Limitations seemingly arise from a dependency on the carbon source and the aeration conditions, but the underlying effects are poorly understood. We have recently presented two xylose-to-lactic acid converting strains, IBB14LA1 and IBB14LA1_5, which have the L-lactic acid dehydrogenase from Plasmodium falciparum (pfLDH) integrated at the pdc1 (pyruvate decarboxylase) locus. IBB14LA1_5 additionally has its pdc5 gene knocked out. In this study, the influence of carbon source and oxygen on YLA and QLA in IBB14LA1 and IBB14LA1_5 was investigated.ResultsIn anaerobic fermentation IBB14LA1 showed a higher YLA on xylose (0.27 g g Xyl-1 ) than on glucose (0.18 g g Glc-1 ). The ethanol yields (YEtOH, 0.15 g g Xyl-1 and 0.32 g g Glc-1 ) followed an opposite trend. In IBB14LA1_5, the effect of the carbon source on YLA was less pronounced (~?0.80 g g Xyl-1 , and 0.67 g g Glc-1 ). Supply of oxygen accelerated glucose conversions significantly in IBB14LA1 (QLA from 0.38 to 0.81 g L-1 h-1) and IBB14LA1_5 (QLA from 0.05 to 1.77 g L-1 h-1) at constant YLA (IBB14LA1 ~?0.18 g g Glc-1 ; IBB14LA1_5 ~?0.68 g g Glc-1 ). In aerobic xylose conversions, however, lactic acid production ceased completely in IBB14LA1 and decreased drastically in IBB14LA1_5 (YLA aerobic???0.25 g g Xyl-1 and anaerobic?~?0.80 g g Xyl-1 ) at similar QLA (~?0.04 g L-1 h-1). Switching from aerobic to microaerophilic conditions (pO2?~?2%) prevented lactic acid metabolization, observed for fully aerobic conditions, and increased QLA and YLA up to 0.11 g L-1 h-1 and 0.38 g g Xyl-1 , respectively. The pfLDH and PDC activities in IBB14LA1 were measured and shown to change drastically dependent on carbon source and oxygen.ConclusionEvidence from conversion time courses together with results of activity measurements for pfLDH and PDC show that in IBB14LA1 the distribution of fluxes at the pyruvate branching point is carbon source and oxygen dependent. Comparison of the performance of strain IBB14LA1 and IBB14LA1_5 in conversions under different aeration conditions (aerobic, anaerobic, and microaerophilic) further suggest that xylose, unlike glucose, does not repress the respiratory response in both strains. This study proposes new genetic engineering targets for rendering genetically engineering S. cerevisiae better suited for lactic acid biorefineries.
| S-EPMC5894196 | biostudies-literature
Project description:As the importance of reducing carbon emissions as a means to limit the serious effects of global climate change becomes apparent, synthetic biologists and metabolic engineers are looking to develop renewable sources for transportation fuels and petroleum-derived chemicals. In recent years, microbial production of high-energy fuels has emerged as an attractive alternative to the traditional production of transportation fuels. In particular, the Baker's yeast Saccharomyces cerevisiae, a highly versatile microbial chassis, has been engineered to produce a wide array of biofuels. Nevertheless, a key limitation of S. cerevisiae is its inability to utilize xylose, the second most abundant sugar in lignocellulosic biomass, for both growth and chemical production. Therefore, the development of a robust S. cerevisiae strain that is able to use xylose is of great importance. Here, we engineered S. cerevisiae to efficiently utilize xylose as a carbon source and produce the advanced biofuel isobutanol. Specifically, we screened xylose reductase (XR) and xylose dehydrogenase (XDH) variants from different xylose-metabolizing yeast strains to identify the XR-XDH combination with the highest activity. Overexpression of the selected XR-XDH variants, a xylose-specific sugar transporter, xylulokinase, and isobutanol pathway enzymes in conjunction with the deletions of PHO13 and GRE3 resulted in an engineered strain that is capable of producing isobutanol at a titer of 48.4?±?2.0 mg/L (yield of 7.0 mg/g D-xylose). This is a 36-fold increase from the previous report by Brat and Boles and, to our knowledge, is the highest isobutanol yield from D-xylose in a microbial system. We hope that our work will set the stage for an economic route for the production of advanced biofuel isobutanol and enable efficient utilization of lignocellulosic biomass.
| S-EPMC6787123 | biostudies-literature
Project description:Adipic acid is an important industrial chemical used in the synthesis of nylon-6,6. The commercial synthesis of adipic acid uses petroleum-derived benzene and releases significant quantities of greenhouse gases. Biocatalytic production of adipic acid from renewable feedstocks could potentially reduce the environmental damage and eliminate the need for fossil fuel precursors. Recently, we have demonstrated the first enzymatic hydrogenation of muconic acid to adipic acid using microbial enoate reductases (ERs) - complex iron-sulfur and flavin containing enzymes. In this work, we successfully expressed the Bacillus coagulans ER in a Saccharomyces cerevisiae strain producing muconic acid and developed a three-stage fermentation process enabling the synthesis of adipic acid from glucose. The ability to express active ERs and significant acid tolerance of S. cerevisiae highlight the applicability of the developed yeast strain for the biocatalytic production of adipic acid from renewable feedstocks.
| S-EPMC5814376 | biostudies-literature
Project description:BackgroundDiterpenoids are a large class of natural products with complex structures and broad commercial applications as food additives, important medicines, and fragrances. However, their low abundance in plants and high structural complexity limit their applications. Therefore, it is important to create an efficient diterpenoid-producing yeast cell factory of the production of various high-value diterpenoid compounds in a cost-effective manner RESULTS: In this study, 13R-manoyl oxide (13R-MO; 2.31 mg/L) was produced by expressing CfTPS2 and CfTPS3 from Coleus forskohlii in Saccharomyces cerevisiae. The 13R-MO titer was increased by 142-fold to 328.15 mg/L via the stepwise metabolic engineering of the original strain, including the overexpression of the rate-limiting genes (tHMG1 and ERG20) of the mevalonate pathway, transcription and protein level regulation of ERG9, Bts1p and Erg20F96Cp fusion, and the overexpression of tCfTPS2 and tCfTPS3 (excision of the N-terminal plastid transit peptide sequences of CfTPS2 and CfTPS3). The final titer of 13R-MO reached up to 3 g/L by fed-batch fermentation in a 5 L bioreactor.ConclusionsIn this study, an efficient 13R-MO yeast cell factory was constructed, which achieved the de novo production of 3 g/L of 13R-MO from glucose. To the best of our knowledge, this is the highest 13R-MO titer reported to date. Furthermore, the metabolic engineering strategies presented here could be used to produce other valuable diterpenoid compounds in yeast.
| S-EPMC6480505 | biostudies-literature
Project description:Background2,3-Butanediol (2,3-BDO) is a valuable chemical for industrial applications. Bacteria can produce 2,3-BDO with a high productivity, though most of their classification as pathogens makes them undesirable for the industrial-scale production. Though Saccharomyces cerevisiae (GRAS microorganism) was engineered to produce 2,3-BDO efficiently in the previous studies, their 2,3-BDO productivity, yield, and titer were still uncompetitive compared to those of bacteria production. Thus, we propose an industrial polyploid S. cerevisiae as a host for efficient production of 2,3-BDO with high growth rate, rapid sugar consumption rate, and resistance to harsh conditions. Genetic manipulation tools for polyploid yeast had been limited; therefore, we engineered an industrial polyploid S. cerevisiae strain based on the CRISPR-Cas9 genome-editing system to produce 2,3-BDO instead of ethanol.ResultsEndogenous genes coding for pyruvate decarboxylase and alcohol dehydrogenase were partially disrupted to prevent declined growth rate and C2-compound limitation. A bacterial 2,3-BDO-producing pathway was also introduced in engineered polyploid S. cerevisiae. A fatal redox imbalance was controlled through the heterologous NADH oxidase from Lactococcus lactis during the 2,3-BDO production. The resulting strain (YG01_SDBN) still retained the beneficial traits as polyploid strains for the large-scale fermentation. The combination of partially disrupted PDC (pyruvate decarboxylase) and ADH (alcohol dehydrogenase) did not cause the severe growth defects typically found in all pdc- or adh-deficient yeast. The YG01_SDBN strain produced 178 g/L of 2,3-BDO from glucose with an impressive productivity (2.64 g/L h). When a cassava hydrolysate was used as a sole carbon source, this strain produced 132 g/L of 2,3-BDO with a productivity of 1.92 g/L h.ConclusionsThe microbial production of 2,3-BDO has been limited to bacteria and haploid laboratorial S. cerevisiae strains. This study suggests that an industrial polyploid S. cerevisiae (YG01_SDBN) can produce high concentration of 2,3-BDO with various advantages. Integration of metabolic engineering of the industrial yeast at the gene level with optimization of fed-batch fermentation at the process scale resulted in a remarkable achievement of 2,3-BDO production at 178 g/L of 2,3-BDO concentration and 2.64 g/L h of productivity. Furthermore, this strain could make a bioconversion of a cassava hydrolysate to 2,3-BDO with economic and environmental benefits. The engineered industrial polyploid strain could be applicable to production of biofuels and biochemicals in large-scale fermentations particularly when using modified CRISPR-Cas9 tools.
| S-EPMC6714309 | biostudies-literature
Project description:Background2'-Fucosyllactose (2-FL), one of the most abundant oligosaccharides in human milk, has potential applications in foods due to its health benefits such as the selective promotion of bifidobacterial growth and the inhibition of pathogenic microbial binding to the human gut. Owing to the limited amounts of 2-FL in human milk, alternative microbial production of 2-FL is considered promising. To date, microbial production of 2-FL has been studied mostly in Escherichia coli. In this study, 2-FL was produced alternatively by using a yeast Saccharomyces cerevisiae, which may have advantages over E. coli.ResultsFucose and lactose were used as the substrates for the salvage pathway which was constructed with fkp coding for a bifunctional enzyme exhibiting L-fucokinase and guanosine 5'-diphosphate-L-fucose phosphorylase activities, fucT2 coding for α-1,2-fucosyltransferase, and LAC12 coding for lactose permease. Production of 2-FL by the resulting engineered yeast was verified by mass spectrometry. 2-FL titers of 92 and 503 mg/L were achieved from 48-h batch fermentation and 120-h fed-batch fermentation fed with ethanol as a carbon source, respectively.ConclusionsThis is the first report on 2-FL production by using yeast S. cerevisiae. These results suggest that S. cerevisiae can be considered as a host engineered for producing 2-FL via the salvage pathway.
| S-EPMC6020385 | biostudies-literature
Project description:BackgroundGinsenosides are Panax plant-derived triterpenoid with wide applications in cardiovascular protection and immunity-boosting. However, the saponins content of Panax plants is fairly low, making it time-consuming and unsustainable by direct extraction. Protopanaxadiol (PPD) is a common precursor of dammarane-type saponins, and its sufficient supply is necessary for the efficient synthesis of ginsenoside.ResultsIn this study, a combinational strategy was used for the construction of an efficient yeast cell factory for PPD production. Firstly, a PPD-producing strain was successfully constructed by modular engineering in Saccharomyces cerevisiae BY4742 at the multi-copy sites. Then, the INO2 gene, encoding a transcriptional activator of the phospholipid biosynthesis, was fine-tuned to promote the endoplasmic reticulum (ER) proliferation and improve the catalytic efficiency of ER-localized enzymes. To increase the metabolic flux of PPD, dynamic control, based on a carbon-source regulated promoter PHXT1, was introduced to repress the competition of sterols. Furthermore, the global transcription factor UPC2-1 was introduced to sterol homeostasis and up-regulate the MVA pathway, and the resulting strain BY-V achieved a PPD production of 78.13 ± 0.38 mg/g DCW (563.60 ± 1.65 mg/L). Finally, sugarcane molasses was used as an inexpensive substrate for the first time in PPD synthesis. The PPD titers reached 1.55 ± 0.02 and 15.88 ± 0.65 g/L in shake flasks and a 5-L bioreactor, respectively. To the best of our knowledge, these results were new records on PPD production.ConclusionThe high-level of PPD production in this study and the successful comprehensive utilization of low-cost carbon source -sugarcane molassesindicate that the constructed yeast cell factory is an excellent candidate strain for the production of high-value-added PPD and its derivativeswith great industrial potential.
| S-EPMC9636795 | biostudies-literature