Green Synthesis of the Flavor Esters with a Marine Candida parapsilosis Esterase Expressed in Saccharomyces cerevisiae.
Ontology highlight
ABSTRACT:
SUBMITTER: Xue D
PROVIDER: S-EPMC7105524 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The fungal cytochrome P450 lanosterol 14α-demethylase (CYP51) is required for the biosynthesis of fungal-specific ergosterol and is the target of azole antifungal drugs. Despite proven success as a clinical target for azole antifungals, there is an urgent need to develop next-generation antifungals that target CYP51 to overcome the resistance of pathogenic fungi to existing azole drugs, toxic adverse reactions and drug interactions due to human drug-metabolizing CYPs. Candida parapsilosis is a readily transmitted opportunistic fungal pathogen that causes candidiasis in health care environments. In this study, we have characterised wild type C. parapsilosis CYP51 and its clinically significant, resistance-causing point mutation Y132F by expressing these enzymes in a Saccharomyces cerevisiae host system. In some cases, the enzymes were co-expressed with their cognate NADPH-cytochrome P450 reductase (CPR). Constitutive expression of CpCYP51 Y132F conferred a 10- to 12-fold resistance to fluconazole and voriconazole, reduced to ~6-fold resistance for the tetrazoles VT-1161 and VT-1129, but did not confer resistance to the long-tailed triazoles. Susceptibilities were unchanged in the case of CpCPR co-expression. Type II binding spectra showed tight triazole and tetrazole binding by affinity-purified recombinant CpCYP51. We report the X-ray crystal structure of ScCYP51 in complex with VT-1129 obtained at a resolution of 2.1 Å. Structural analysis of azole-enzyme interactions and functional studies of recombinant CYP51 from C. parapsilosis have improved understanding of their susceptibility to azole drugs and will help advance structure-directed antifungal discovery.
| S-EPMC8781857 | biostudies-literature
Project description:BackgroundMarine mud is an abundant and largely unexplored source of enzymes with unique properties that may be useful for industrial and biotechnological purposes. However, since most microbes cannot be cultured in the laboratory, a cultivation-independent metagenomic approach would be advantageous for the identification of novel enzymes. Therefore, with the objective of screening novel lipolytic enzymes, a metagenomic library was constructed using the total genomic DNA extracted from marine mud.ResultsBased on functional heterologous expression, 34 clones that showed lipolytic activity were isolated. The five clones with the largest halos were identified, and the corresponding genes were successfully overexpressed in Escherichia coli. Molecular analysis revealed that these encoded proteins showed 48-79 % similarity with other proteins in the GenBank database. Multiple sequence alignment and phylogenetic tree analysis classified these five protein sequences as new members of known families of bacterial lipolytic enzymes. Among them, EST4, which has 316 amino acids with a predicted molecular weight of 33.8 kDa, was further studied in detail due to its strong hydrolytic activity. Characterization of EST4 indicated that it is an alkaline esterase that exhibits highest hydrolytic activity towards p-nitrophenyl butyrate (specific activity: 1389 U mg(-1)) at 45 °C and pH 8.0. The half-life of EST4 is 55 and 46 h at 40 and 45 °C, respectively, indicating a relatively high thermostability. EST4 also showed remarkable stability in organic solvents, retaining 90 % of its initial activity when incubated for 12 h in the presence of hydrophobic alkanes. Furthermore, EST4 was used as an efficient whole-cell biocatalyst for the synthesis of short-chain flavor esters, showing high conversion rate and good tolerance for high substrate concentrations (up to 3.0 M). These results demonstrate a promising potential for industrial scaling-up to produce short-chain flavor esters at high substrate concentrations in non-aqueous media.ConclusionsThis manuscript reports unprecedented alcohol tolerance and conversion of an esterase biocatalyst identified from a marine mud metagenomic library. The high organic solvent tolerance and thermostability of EST4 suggest that it has great potential as a biocatalyst.
| S-EPMC4758151 | biostudies-literature
Project description:Fusarium oxysporum laccase was functionally expressed in Saccharomyces cerevisiae and engineered towards higher expression levels and higher reactivity towards 2,6-dimethoxyphenol, that could be used as a mediator for lignin modification. A combination of classical culture optimization and protein engineering led to around 30 times higher activity in the culture supernatant. The winner mutant exhibited three times lower Km, four times higher kcat and ten times higher catalytic efficiency than the parental enzyme. The strategy for laccase engineering was composed of a combination of random methods with a rational approach based on QM/MM MD studies of the enzyme complex with 2,6-dimethoxyphenol. Laccase mediator system with 2,6-dimethoxyphenol caused fulvic acids release from biosolubilized coal.
| S-EPMC7039978 | 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:A whole-cell-based assay using Saccharomyces cerevisiae strains that overexpress Candida albicans CDR1 and MDR1 efflux pumps has been employed to screen natural product extracts for reversal of fluconazole resistance. The tropical green alga Penicillus capitatus was selected for bioassay-guided isolation, leading to the identification of capisterones A and B (1 and 2), which were recently isolated from this alga and shown to possess antifungal activity against the marine pathogen Lindra thallasiae. Current work has assigned their absolute configurations using electronic circular dichroism and determined their preferred conformations in solution based on detailed NOE analysis. Compounds 1 and 2 significantly enhanced fluconazole activity in S. cerevisiae, but did not show inherent antifungal activity when tested against several opportunistic pathogens or cytotoxicity to several human cancer and noncancerous cell lines (up to 35 microM). These compounds may have a potential for combination therapy of fungal infections caused by clinically relevant azole-resistant strains.
| S-EPMC2631560 | biostudies-literature
Project description:Despite the fact that beer is produced on a large scale, the effects of pesticide residues on beer have been rarely investigated. In this study, we used micro-brewing settings to determine the effect of triadimefon on the growth of Saccharomyces cerevisiae and beer flavor. The yeast growth in medium was significantly inhibited (45%) at concentrations higher than 5 mg L(-1), reaching 80% and 100% inhibition at 10 mg L(-1) and 50 mg L(-1), respectively. There were significant differences in sensory quality between beer samples fermented with and without triadimefon based on data obtained with an electronic tongue and nose. Such an effect was most likely underlain by changes in yeast fermentation activity, including decreased utilization of maltotriose and most amino acids, reduced production of isobutyl and isoamyl alcohols, and increased ethyl acetate content in the fungicide treated samples. Furthermore, yeast metabolic profiling by phenotype microarray and UPLC/TOF-MS showed that triadimefon caused significant changes in the metabolism of glutathione, phenylalanine and sphingolipids, and in sterol biosynthesis. Thus, triadimefon negatively affects beer sensory qualities by influencing the metabolic activity of S. cerevisiae during fermentation, emphasizing the necessity of stricter control over fungicide residues in brewing by the food industry.
| S-EPMC5024320 | biostudies-literature
Project description:ObjectiveSaccharomyces cerevisiae is used worldwide for the production of ale-type beers. This yeast is responsible for the production of the characteristic fruity aroma compounds. Esters constitute an important group of aroma active secondary metabolites produced by S. cerevisiae. Previous work suggests that esterase activity, which results in ester degradation, may be the key factor determining the abundance of fruity aroma compounds. Here, we test this hypothesis by deletion of two S. cerevisiae esterases, IAH1 and TIP1, using CRISPR-Cas9 genome editing and by studying the effect of these deletions on esterase activity and extracellular ester pools.ResultsSaccharomyces cerevisiae mutants were constructed lacking esterase IAH1 and/or TIP1 using CRISPR-Cas9 genome editing. Esterase activity using 5-(6)-carboxyfluorescein diacetate (cFDA) as substrate was found to be significantly lower for ?IAH1 and ?IAH1?TIP1 mutants compared to wild type (WT) activity (P?<?0.05 and P?<?0.001, respectively). As expected, we observed an increase in relative abundance of acetate and ethyl esters and an increase in ethyl esters in ?IAH1 and ?TIP1, respectively. Interestingly, the double gene disruption mutant ?IAH1?TIP1 showed an aroma profile comparable to WT levels, suggesting the existence and activation of a complex regulatory mechanism to compensate multiple genomic alterations in aroma metabolism.
| S-EPMC6161353 | biostudies-literature
Project description:BackgroundSaccharomyces cerevisiae AN120 osw2∆ spores were used as a host with good resistance to unfavorable environment. This work was undertaken to develop a new yeast spore-encapsulation of Candida parapsilosis Glu228Ser/(S)-carbonyl reductase II and Bacillus sp. YX-1 glucose dehydrogenase for efficient chiral synthesis in organic solvents.ResultsThe spore microencapsulation of E228S/SCR II and GDH in S. cerevisiae AN120 osw2∆ catalyzed (R)-phenylethanol in a good yield with an excellent enantioselectivity (up to 99%) within 4 h. It presented good resistance and catalytic functions under extreme temperature and pH conditions. The encapsulation produced several chiral products with over 70% yield and over 99% enantioselectivity in ethyl acetate after being recycled for 4-6 times. It increased substrate concentration over threefold and reduced the reaction time two to threefolds compared to the recombinant Escherichia coli containing E228S and glucose dehydrogenase.ConclusionsThis work first described sustainable enantioselective synthesis without exogenous cofactors in organic solvents using yeast spore-microencapsulation of coupled alcohol dehydrogenases.
| S-EPMC6526602 | biostudies-literature
Project description:Conyza blinii H.Lév. is a widely used medicinal herb in southwestern China. The main pharmacological components of C. blinii are a class of oleanane-type pentacyclic triterpene glycosides known as conyzasaponins, which are thought to be synthesized from ?-amyrin. However, no genes involved in the conyzasaponin pathway have previously been identified. Here, we identify an oxidosqualene cyclase (OSC), a ?-amyrin synthase, which mediates cyclization of 2,3-oxidosqualene to yield ?-amyrin. Ten OSC sequences were isolated from C. blinii transcript tags. Phylogenetic analysis was used to select the tag Cb18076 as the putative ?-amyrin synthase, named Cb?AS. The open reading frame of Cb?AS is 2286 bp and encodes 761 amino acids. Its mature protein contains the highly conserved motifs (QXXXGXW/DCTAE) of OSCs and (MWCYCR) of ?-amyrin synthases. Transcription of Cb?AS was upregulated 4-24 h after treatment of the seedlings of the C. blinii cultivar with methyl jasmonate. Furthermore, expression of Cb?AS in Saccharomyces cerevisiae successfully yielded ?-amyrin. The chemical structures and concentrations of ?-amyrin were confirmed by GC-MS/MS. The target yeast ultimately produced 4.432 mg·L-1 ?-amyrin. Thus, Cb?AS is an OSC involved in conyzasaponin biosynthesis.
| S-EPMC5623702 | biostudies-literature
Project description:BackgroundThe only hitherto known biological role of yeast Saccharomyces cerevisiae Tum1 protein is in the tRNA thiolation pathway. The mammalian homologue of the yeast TUM1 gene, the thiosulfate sulfurtransferase (a.k.a. rhodanese) Tst, has been proposed as an obesity-resistance and antidiabetic gene. To assess the role of Tum1 in cell metabolism and the putative functional connection between lipid metabolism and tRNA modification, we analysed evolutionary conservation of the rhodanese protein superfamily, investigated the role of Tum1 in lipid metabolism, and examined the phenotype of yeast strains expressing the mouse homologue of Tum1, TST.ResultsWe analysed evolutionary relationships in the rhodanese superfamily and established that its members are widespread in bacteria, archaea and in all major eukaryotic groups. We found that the amount of sterol esters was significantly higher in the deletion strain tum1Δ than in the wild-type strain. Expression of the mouse TST protein in the deletion strain did not rescue this phenotype. Moreover, although Tum1 deficiency in the thiolation pathway was complemented by re-introducing TUM1, it was not complemented by the introduction of the mouse homologue Tst. We further showed that the tRNA thiolation pathway is not involved in the regulation of sterol ester content in S. cerevisiae, as overexpression of the tEUUC, tKUUU and tQUUG tRNAs did not rescue the lipid phenotype in the tum1Δ deletion strain, and, additionally, deletion of the key gene for the tRNA thiolation pathway, UBA4, did not affect sterol ester content.ConclusionsThe rhodanese superfamily of proteins is widespread in all organisms, and yeast TUM1 is a bona fide orthologue of mammalian Tst thiosulfate sulfurtransferase gene. However, the mouse TST protein cannot functionally replace yeast Tum1 protein, neither in its lipid metabolism-related function, nor in the tRNA thiolation pathway. We show here that Tum1 protein is involved in lipid metabolism by decreasing the sterol ester content in yeast cells, and that this function of Tum1 is not exerted through the tRNA thiolation pathway, but through another, currently unknown pathway.
| S-EPMC5567475 | biostudies-literature