D.C. Carqueija Cardoso
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3 records found
1
Endocytosis of the Saccharomyces cerevisiae Jen1 monocarboxylate-proton symporter under mildly alkaline conditions
A mechanism to prevent metabolite loss?
The Saccharomyces cerevisiae Jen1 transporter mediates electroneutral proton symport of lactate and pyruvate. In lactate-grown batch cultures, growth-coupled medium alkalinization was previously shown to coincide with endocytosis of Jen1.
Results
To investigate the physiological relevance of pH-dependent Jen1 endocytosis, S. cerevisiae was grown in carbon-limited continuous cultures on a mixed ethanol-lactate feed. When applying a linearly increasing pH (6.75–7.25) to these cultures, lactate and pyruvate concentrations in the external medium progressively increased. Up to a culture pH of 7.0, these extracellular concentrations aligned with a predicted thermodynamic equilibrium of reversible, Jen1-mediated electroneutral carboxylate-proton symport. Consistent with earlier reports, pronounced Jen1 internalization occurred above pH 7.0. At these mildly alkaline pH values, a more pronounced increase of residual lactate concentrations and transcriptional upregulation of genes involved in oxidative phosphorylation were consistent with increased cellular energy demands.
Conclusion
This study reveals how pH-dependent regulation of carboxylate transporters shapes cellular adaptation to changing environmental conditions. Insights into these regulatory mechanisms can inform strategies to optimize microbial cell factories operating under variable pH regimes in industrial settings. The integrated analysis of transport, Jen1 localization, and transcriptional responses in growing continuous cultures uncovered physiological challenges associated with electroneutral carboxylate/proton symport under mildly alkaline conditions. The data support the hypothesis that Jen1 internalization evolved to prevent intracellular metabolite loss under unfavorable pH conditions. ...
The Saccharomyces cerevisiae Jen1 transporter mediates electroneutral proton symport of lactate and pyruvate. In lactate-grown batch cultures, growth-coupled medium alkalinization was previously shown to coincide with endocytosis of Jen1.
Results
To investigate the physiological relevance of pH-dependent Jen1 endocytosis, S. cerevisiae was grown in carbon-limited continuous cultures on a mixed ethanol-lactate feed. When applying a linearly increasing pH (6.75–7.25) to these cultures, lactate and pyruvate concentrations in the external medium progressively increased. Up to a culture pH of 7.0, these extracellular concentrations aligned with a predicted thermodynamic equilibrium of reversible, Jen1-mediated electroneutral carboxylate-proton symport. Consistent with earlier reports, pronounced Jen1 internalization occurred above pH 7.0. At these mildly alkaline pH values, a more pronounced increase of residual lactate concentrations and transcriptional upregulation of genes involved in oxidative phosphorylation were consistent with increased cellular energy demands.
Conclusion
This study reveals how pH-dependent regulation of carboxylate transporters shapes cellular adaptation to changing environmental conditions. Insights into these regulatory mechanisms can inform strategies to optimize microbial cell factories operating under variable pH regimes in industrial settings. The integrated analysis of transport, Jen1 localization, and transcriptional responses in growing continuous cultures uncovered physiological challenges associated with electroneutral carboxylate/proton symport under mildly alkaline conditions. The data support the hypothesis that Jen1 internalization evolved to prevent intracellular metabolite loss under unfavorable pH conditions.
Flocculation in Saccharomyces cerevisiae is a critical phenotype with ecological and industrial significance. This study aimed to functionally dissect the contributions of individual FLO genes (FLO1, FLO5, FLO9, FLO10, FLO11) to flocculation by employing an optogenetic circuit (OptoQ-AMP5) for precise, light-inducible control of gene expression. A FLO-null platform yeast strain was engineered allowing the expression of individual FLO genes without native background interference. Each FLO gene was reintroduced into the FLO-null background under the control of OptoQ-AMP5. Upon light induction, strains expressing FLO1, FLO5, or FLO10 demonstrated strong flocculation, with FLO1 and FLO5 forming large and structurally distinct aggregates. FLO9 induced a weaker phenotype. Sugar inhibition assays revealed distinct sensitivities among flocculins, notably FLO9’s novel sensitivity to fructose and maltotriose. Additionally, FLO-induced changes in cell surface hydrophobicity were quantified, revealing that FLO10 and FLO1 conferred the greatest hydrophobicity, correlating with their aggregation strength. This work establishes a robust platform for investigating flocculation mechanisms in yeast with temporal precision. It highlights the phenotypic diversity encoded within the FLO gene family and their differential responses to environmental cues. The optogenetic system provides a valuable tool for both fundamental studies and the rational engineering of yeast strains for industrial fermentation processes requiring controlled flocculation.
Chemically defined media for cultivation of Saccharomyces cerevisiae strains are commonly supplemented with a mixture of multiple Class-B vitamins, whose omission leads to strongly reduced growth rates. Fast growth without vitamin supplementation is interesting for industrial applications, as it reduces costs and complexity of medium preparation and may decrease susceptibility to contamination by auxotrophic microbes. In this study, suboptimal growth rates of S. cerevisiae CEN.PK113-7D in the absence of pantothenic acid, para-aminobenzoic acid (pABA), pyridoxine, inositol and/or biotin were corrected by single or combined overexpression of ScFMS1, ScABZ1/ScABZ2, ScSNZ1/ScSNO1, ScINO1 and Cyberlindnera fabianii BIO1, respectively. Several strategies were explored to improve growth of S. cerevisiae CEN.PK113-7D in thiamine-free medium. Overexpression of ScTHI4 and/or ScTHI5 enabled thiamine-independent growth at 83% of the maximum specific growth rate of the reference strain in vitamin-supplemented medium. Combined overexpression of seven native S. cerevisiae genes and CfBIO1 enabled a maximum specific growth rate of 0.33 ± 0.01 h−1 in vitamin-free synthetic medium. This growth rate was only 17 % lower than that of a congenic reference strain in vitamin-supplemented medium. Physiological parameters of the engineered vitamin-independent strain in aerobic glucose-limited chemostat cultures (dilution rate 0.10 h−1) grown on vitamin-free synthetic medium were similar to those of similar cultures of the parental strain grown on vitamin-supplemented medium. Transcriptome analysis revealed only few differences in gene expression between these cultures, which primarily involved genes with roles in Class-B vitamin metabolism. These results pave the way for development of fast-growing vitamin-independent industrial strains of S. cerevisiae.