Exploiting a heterologous construction of the 3-hydroxypropionic acid carbon fixation pathway with mesaconate as an indicator in Saccharomyces cerevisiae
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ABSTRACT:
SUBMITTER: Xu S
PROVIDER: S-EPMC10991142 | biostudies-literature | 2023 May
REPOSITORIES: biostudies-literature
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Project description:3-Hydroxypropionic acid (3-HP) is a commercially valuable platform compound. Klebsiella pneumoniae has been concerned as an appropriate host for 3-HP production because of its robust capacity to metabolize glycerol. Glycerol conversion to 3-HP in K. pneumoniae comprises two successive reactions: glycerol dehydratase catalyzes glycerol to 3-hydroxypropionaldehyde (3-HPA); aldehyde dehydrogenase catalyzes 3-HPA to 3-HP. Previous studies focusing on inducible expression of aldehyde dehydrogenase have shown defects of high cost of inducer and low catalytic activity due to inclusion body. Here we show a different strategy that a native promoter in the host K. pneumoniae was used to drive the heterologous expression of aldehyde dehydrogenase gene ald4 from Saccharomyces cerevisiae. The 3-HP yield of the recombinant reached a peak of 4.23 g/L at log phase, but it decreased during later period of fermentation. Except the validation of high activity of ald4, particularly, the 3-HP formation was uncovered to be closely coupled with cell division, and the lacking of NAD and ATP at latter fermentation phase became the bottleneck for cell growth and 3-HP accumulation. Furthermore, 3-HP is postulated to be converted to 3-HPA via feedback inhibition or other metabolite via unknown mechanism. Since glycerol dissimilation is a common mechanism in a variety of bacteria, the expression strategy using native promoter and implications may provide significant insight into the metabolic engineering for 3-HP production.
| S-EPMC3460132 | biostudies-literature
Project description:BackgroundThe microbial production of succinic acid (SA) from renewable carbon sources via the reverse TCA (rTCA) pathway is a process potentially accompanied by net-fixation of carbon dioxide (CO2). Among reduced carbon sources, glycerol is particularly attractive since it allows a nearly twofold higher CO2-fixation yield compared to sugars. Recently, we described an engineered Saccharomyces cerevisiae strain which allowed SA production in synthetic glycerol medium with a maximum yield of 0.23 Cmol Cmol-1. The results of that previous study suggested that the glyoxylate cycle considerably contributed to SA accumulation in the respective strain. The current study aimed at improving the flux into the rTCA pathway accompanied by a higher CO2-fixation and SA yield.ResultsBy changing the design of the expression cassettes for the rTCA pathway, overexpressing PYC2, and adding CaCO3 to the batch fermentations, an SA yield on glycerol of 0.63 Cmol Cmol-1 was achieved (i.e. 47.1% of the theoretical maximum). The modifications in this 2nd-generation SA producer improved the maximum biomass-specific glycerol consumption rate by a factor of nearly four compared to the isogenic baseline strain solely equipped with the dihydroxyacetone (DHA) pathway for glycerol catabolism. The data also suggest that the glyoxylate cycle did not contribute to the SA production in the new strain. Cultivation conditions which directly or indirectly increased the concentration of bicarbonate, led to an accumulation of malate in addition to the predominant product SA (ca. 0.1 Cmol Cmol-1 at the time point when SA yield was highest). Off-gas analysis in controlled bioreactors with CO2-enriched gas-phase indicated that CO2 was fixed during the SA production phase.ConclusionsThe data strongly suggest that a major part of dicarboxylic acids in our 2nd-generation SA-producer was formed via the rTCA pathway enabling a net fixation of CO2. The greatly increased capacity of the rTCA pathway obviously allowed successful competition with other pathways for the common precursor pyruvate. The overexpression of PYC2 and the increased availability of bicarbonate, the co-substrate for the PYC reaction, further strengthened this capacity. The achievements are encouraging to invest in future efforts establishing a process for SA production from (crude) glycerol and CO2.
| S-EPMC9148483 | biostudies-literature
Project description:BackgroundLevopimaric acid (LA), a type of diterpene resin acid produced by plants, is a significant industrial intermediate that is mainly produced via phytoextraction. This work aimed to apply synthetic biology to produce LA in yeast strains from a simple carbon source.ResultsLevopimaradiene (LP), the precursor of LA, was produced via LP synthase (LPS) expression in yeast. LPS was then modified by N-terminal truncating and site-directed mutagenesis. The strain containing t79LPSMM (79 N-terminal amino acid truncating and M593I/Y700F mutation) produced 6.92 mg/L of LP, which were 23-fold higher than the strain containing LPS. Next, t79LPSMM was expressed in a new metabolically engineered chassis, and the final LP production increased 164-folds to 49.21 mg/L. Three cytochrome P450 reductases (CPRs) were co-expressed with CYP720B1 (the enzyme responsible for LA production from LP) in yeast to evaluate their LA producing abilities, and the CPR from Taxus cuspidata (TcCPR) was found to be the best (achieved 23.13 mg/L of LA production). CYP720B1 and TcCPR genes overexpression in the multi-copy site of the S.cerevisiae genome led to a 1.9-fold increase in LA production to 45.24 mg/L in a shake-flask culture. Finally, LA production was improved to 400.31 mg/L via fed-batch fermentation in a 5-L bioreactor.ConclusionsThis is the first report to produce LA in a yeast cell factory and the highest titer of LA is achieved.
| S-EPMC6050663 | biostudies-literature
Project description:BackgroundDue to excellent performance in antitumor, antioxidation, antihypertension, antiatherosclerotic and antidepressant activities, crocetin, naturally exists in Crocus sativus L., has great potential applications in medical and food fields. Microbial production of crocetin has received increasing concern in recent years. However, only a patent from EVOVA Inc. and a report from Lou et al. have illustrated the feasibility of microbial biosynthesis of crocetin, but there was no specific titer data reported so far. Saccharomyces cerevisiae is generally regarded as food safety and productive host, and manipulation of key enzymes is critical to balance metabolic flux, consequently improve output. Therefore, to promote crocetin production in S. cerevisiae, all the key enzymes, such as CrtZ, CCD and ALD should be engineered combinatorially.ResultsBy introduction of heterologous CrtZ and CCD in existing β-carotene producing strain, crocetin biosynthesis was achieved successfully in S. cerevisiae. Compared to culturing at 30 °C, the crocetin production was improved to 223 μg/L at 20 °C. Moreover, an optimal CrtZ/CCD combination and a titer of 351 μg/L crocetin were obtained by combinatorial screening of CrtZs from nine species and four CCDs from Crocus. Then through screening of heterologous ALDs from Bixa orellana (Bix_ALD) and Synechocystis sp. PCC6803 (Syn_ALD) as well as endogenous ALD6, the crocetin titer was further enhanced by 1.8-folds after incorporating Syn_ALD. Finally a highest reported titer of 1219 μg/L at shake flask level was achieved by overexpression of CCD2 and Syn_ALD. Eventually, through fed-batch fermentation, the production of crocetin in 5-L bioreactor reached to 6278 μg/L, which is the highest crocetin titer reported in eukaryotic cell.ConclusionsSaccharomyces cerevisiae was engineered to achieve crocetin production in this study. Through combinatorial manipulation of three key enzymes CrtZ, CCD and ALD in terms of screening enzymes sources and regulating protein expression level (reaction temperature and copy number), crocetin titer was stepwise improved by 129.4-fold (from 9.42 to 1219 μg/L) as compared to the starting strain. The highest crocetin titer (6278 μg/L) reported in microbes was achieved in 5-L bioreactors. This study provides a good insight into key enzyme manipulation involved in serial reactions for microbial overproduction of desired compounds with complex structure.
| S-EPMC5371240 | biostudies-literature
Project description:Candida albicans is a major cause of opportunistic and life-threatening systemic fungal infections, especially in the immunocompromised. The plasma membrane proton-pumping ATPase (Pma1p) is an essential enzyme that generates the electrochemical gradient required for cell growth. We expressed C. albicans Pma1p (CaPma1p) in Saccharomyces cerevisiae to facilitate screening for inhibitors. Replacement of S. cerevisiae PMA1 with C. albicans PMA1 gave clones expressing CaPma1p that grew slowly at low pH. CaPma1p was expressed at significantly lower levels and had lower specific activity than the native Pma1p. It also conferred pH sensitivity, hygromycin B resistance, and low levels of glucose-dependent proton pumping. Recombination between CaPMA1 and the homologous nonessential ScPMA2 resulted in chimeric suppressor mutants that expressed functional CaPma1p with improved H(+) -ATPase activity and growth rates at low pH. Molecular models of suppressor mutants identified specific amino acids (between 531 and 595 in CaPma1p) that may affect regulation of the activity of Pma1p oligomers in S. cerevisiae. A modified CaPma1p chimeric construct containing only 5 amino acids from ScPma2p enabled the expression of a fully functional enzyme for drug screens and structural resolution.
| S-EPMC4428687 | biostudies-literature
Project description:Biomass, the most abundant carbon source on the planet, may in the future become the primary feedstock for production of fuels and chemicals, replacing fossil feedstocks. This will, however, require development of cell factories that can convert both C6 and C5 sugars present in lignocellulosic biomass into the products of interest. We engineered Saccharomyces cerevisiae for production of 3-hydroxypropionic acid (3HP), a potential building block for acrylates, from glucose and xylose. We introduced the 3HP biosynthetic pathways via malonyl-CoA or β-alanine intermediates into a xylose-consuming yeast. Using controlled fed-batch cultivation, we obtained 7.37±0.17 g 3HP L-1 in 120 hours with an overall yield of 29±1% Cmol 3HP Cmol-1 xylose. This study is the first demonstration of the potential of using S. cerevisiae for production of 3HP from the biomass sugar xylose.
| S-EPMC8193239 | biostudies-literature
Project description:Medicarpin is an important bioactive compound with multiple medicinal activities, including anti-tumor, anti-osteoporosis, and anti-bacterial effects. Medicarpin is associated with pterocarpans derived from medicinal plants, such as Sophora japonica, Glycyrrhiza uralensis Fisch., and Glycyrrhiza glabra L. However, these medicinal plants contain only low amounts of medicarpin. Moreover, the planting area for medicarpin-producing plants is limited; consequently, the current medicarpin supply cannot meet the high demands of medicinal markets. In this study, eight key genes involved in medicarpin biosynthesis were identified using comparative transcriptome and bioinformatic analyses. In vitro and in vivo enzymatic reaction confirmed the catalytic functions of candidate enzymes responsible for the biosynthesis of medicarpin and medicarpin intermediates. Further engineering of these genes in Saccharomyces cerevisiae achieved the heterologous biosynthesis of medicarpin using liquiritigenin as a substrate, with a final medicarpin yield of 0.82 ± 0.18 mg/L. By increasing the gene copy numbers of vestitone reductase (VR) and pterocarpan synthase (PTS), the final medicarpin yield was increased to 2.05 ± 0.72 mg/L. This study provides a solid foundation for the economic and sustainable production of medicarpin through a synthetic biology strategy.
| S-EPMC10711132 | biostudies-literature
Project description:Trichodermol, a fungal sesquiterpene derived from the farnesyl diphosphate pathway, is the biosynthetic precursor for trichodermin, a member of the trichothecene class of fungal toxins produced mainly by the genera of Trichoderma and Fusarium. Trichodermin is a promising candidate for the development of fungicides and antitumor agents due to its significant antifungal and cytotoxic effects. It can also serve as a scaffold to generate new congeners for structure-activity relationship (SAR) study. We reconstructed the biosynthetic pathway of trichodermol in Saccharomyces cerevisiae BY4741, and investigated the effect of produced trichodermol on the host by de novo RNA sequencing (RNA-Seq) and quantitative Real-time PCR analyses. Co-expression of pESC::FgTRI5 using plasmid pLLeu-tHMGR-UPC2.1 led to trichodiene production of 683 μg L-1, while integration of only the codon-optimized FgTRI5 into the chromosome of yeast improved the production to 6,535 μg L-1. Subsequent expression of the codon-optimized cytochrome P450 monooxygenase encoding genes, TaTRI4 and TaTRI11, resulted in trichodermol, with an estimated titer of 252 μg L-1 at shake flask level. RNA-Seq and qPCR analyses revealed that the produced trichodermol downregulated the expression of the genes involved in ergosterol biosynthesis, but significantly upregulated the expression of PDR5 related to membrane transport pathway in S. cerevisiae. Collectively, we achieved the first heterologous biosynthesis of trichodermol by reconstructing its biosynthetic pathway in yeast, and the reconstructed pathway will serve as a platform to generate trichodermin analogs as potential candidates for agrochemicals and anticancer agents through further optimizations.
| S-EPMC6087768 | biostudies-literature
Project description:Phosphoketolases catalyze an energy- and redox-independent cleavage of certain sugar phosphates. Hereby, the two-carbon (C2) compound acetyl-phosphate is formed, which enzymatically can be converted into acetyl-CoA-a key precursor in central carbon metabolism. Saccharomyces cerevisiae does not demonstrate efficient phosphoketolase activity naturally. In this study, we aimed to compare and identify efficient heterologous phosphoketolase enzyme candidates that in yeast have the potential to reduce carbon loss compared to the native acetyl-CoA producing pathway by redirecting carbon flux directly from C5 and C6 sugars towards C2-synthesis. Nine phosphoketolase candidates were expressed in S. cerevisiae of which seven produced significant amounts of acetyl-phosphate after provision of sugar phosphate substrates in vitro. The candidates showed differing substrate specificities, and some demonstrated activity levels significantly exceeding those of candidates previously expressed in yeast. The conducted studies also revealed that S. cerevisiae contains endogenous enzymes capable of breaking down acetyl-phosphate, likely into acetate, and that removal of the phosphatases Gpp1 and Gpp2 could largely prevent this breakdown. An evaluation of in vivo function of a subset of phosphoketolases was conducted by monitoring acetate levels during growth, confirming that candidates showing high activity in vitro indeed showed increased acetate accumulation, but expression also decreased cellular fitness. The study shows that expression of several bacterial phosphoketolase candidates in S. cerevisiae can efficiently divert intracellular carbon flux toward C2-synthesis, thus showing potential to be used in metabolic engineering strategies aimed to increase yields of acetyl-CoA derived compounds.
| S-EPMC5110461 | biostudies-literature
Project description:Chitin in the cyst wall of Entamoeba histolytica is made by two chitin synthases (Chs), one of which is unique (EhCHS-1) and one of which resembles those of insects and nematodes (EhCHS-2). EhCHS-1 is deposited chitin in the lateral wall of transformed Saccharomyces cerevisiae Chs mutants, independent of accessory proteins (Chs4p to Chs7p) required by yeast Chs3p.
| S-EPMC1360259 | biostudies-literature