Increase of anion and proton permeability of <i>Saccharomyces carlsbergensis</i> plasmalemma by n‐alcohols as a possible cause of its de‐energization

Петров, В. А.; Okorokov, Lev A.

doi:10.1002/yea.320060404

Cited by 42 publications

(34 citation statements)

References 24 publications

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“…The improved secretion of laccase in the presence of ethanol was in accordance with previous reports of laccase production in other organisms (35,37). In addition to increasing cytoplasmic membrane permeability (38,48), ethanol may generate a stress response in the cell, inducing the expression of the chaperones that are involved in the protein folding/secretion process (62). Copper uptake is essential for laccase expression (28), establishing a compromise between protein secretion and copper toxicity when choosing the final copper concentration (in our case, 2 mM CuSO 4 ).…”

Section: Discussionsupporting

confidence: 91%

Engineering Platforms for Directed Evolution of Laccase from Pycnoporus cinnabarinus

Camarero

Pardo

Cañas

et al. 2012

Appl Environ Microbiol

123

173

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While the Pycnoporus cinnabarinus laccase (PcL) is one of the most promising high-redox-potential enzymes for environmental biocatalysis, its practical use has to date remained limited due to the lack of directed evolution platforms with which to improve its features. Here, we describe the construction of a PcL fusion gene and the optimization of conditions to induce its functional expression in Saccharomyces cerevisiae, facilitating its directed evolution and semirational engineering. The native PcL signal peptide was replaced by the ␣-factor preproleader, and this construct was subjected to six rounds of evolution coupled to a multiscreening assay based on the oxidation of natural and synthetic redox mediators at more neutral pHs. The laccase total activity was enhanced 8,000-fold: the evolved ␣-factor preproleader improved secretion levels 40-fold, and several mutations in mature laccase provided a 13.7-fold increase in k cat . While the pH activity profile was shifted to more neutral values, the thermostability and the broad substrate specificity of PcL were retained. Evolved variants were highly secreted by Aspergillus niger (ϳ23 mg/ liter), which addresses the potential use of this combined-expression system for protein engineering. The mapping of mutations onto the PcL crystal structure shed new light on the oxidation of phenolic and nonphenolic substrates. Furthermore, some mutations arising in the evolved preproleader highlighted its potential for heterologous expression of fungal laccases in yeast (S. cerevisiae).

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Section: Discussionsupporting

confidence: 91%

Engineering Platforms for Directed Evolution of Laccase from Pycnoporus cinnabarinus

Camarero

Pardo

Cañas

et al. 2012

Appl Environ Microbiol

123

173

View full text Add to dashboard Cite

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“…This result showed that the initial pH had a significant influence on the PER value. One possible mechanism for the effect of initial pH on PER could be explained by the fact that the H + concentration gradient from outside to inside the cells increases, with the extra-cellular pH falling, which could lead to better proton extrusion 20 . However, the proton efflux of cells would be inhibited when the pH value was low enough.…”

Section: Optimization Of the Proton Efflux Rate (Per) Testmentioning

confidence: 99%

The Dynamic Changes of Proton Efflux Rate in Saccharomyces pastorianus Strains During High Gravity or Very High Gravity Brewing

Zhang

Zhao

Wan

et al. 2011

Journal of the Institute of Brewing

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The changes in the proton efflux rate (PER) during fermentation of normal gravity (NG), high gravity (HG) and very high gravity (VHG) wort by a lager yeast (Saccharomyces pastorianus) were monitored using an optimized PER test method. The values of the proton efflux rate in S. pastorianus decreased with increasing initial wort gravity. Moreover, the difference in the proton efflux rate values at the beginning of the fermentation was lower than at the end of fermentation from normal gravity to very high gravity brewing. These results demonstrated that the proton efflux rate in S. pastorianus was inhibited in the later stages of high gravity and very high gravity brewing. Furthermore, the changes of the proton efflux rate in S. pastorianus under the high ethanol concentration conditions appeared to depend on the concentration of ethanol in the fermentation liquid. A better negative correlation (P < 0.001, r = -0.95) between the ethanol concentration at >4% (w/v) and the proton efflux rate was found. The changes of the proton efflux rate in the cells treated with exogenous ethanol confirmed that higher concentrations of ethanol could significantly inhibit proton efflux in S. pastorianus. This study offers a possible way to monitor and explain the performance of yeast in the complex environment of high gravity and very high gravity brewing.

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“…Most observations on the effect of ethanol suggest an increased membrane permeability, leading to leakage of amino acids (Salgueiro 1988), enhancement of the uncoupling effect of acetate (Pampulha & Loureiro 1989), dissipation of the proton-motive force (pmf) across the plasma membrane (Cartwright et al 1986) and inhibition of some enzymes, notably the plasma membrane ATPase (Cartwright et al 1987). The proposed inhibition of the plasma membrane ATPase (Cartwright et al 1987) has been questioned by Petrov & Okarokov (1990). Experiments with plasma membrane vesicles of S. carlbergensis indicated that the pmf across the plasma membrane decreased in the presence of ethanol but that this was not due to inhibition of the ATPase.…”

Section: Alcoholsmentioning

confidence: 99%

Physiology of yeasts in relation to biomass yields

Verduyn¹

1992

Quantitative Aspects of Growth and Metabolism of Microorganisms

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The stoichiometric limit to the biomass yield (maximal assimilation of the carbon source) is determined by the amount of CO 2 lost in anabolism and the amount of carbon source required for generation of NADPH. This stoichiometric limit may be reached when yeasts utilize formate as an additional energy source. Factors affecting the biomass yield on single substrates are discussed under the following headings: -Energy requirement for biomass formation (YATP)' YATP depends strongly on the nature of the carbon source. Cell composition. The macroscopic composition of the biomass, and in particular the protein content, has a considerable effect on the ATP requirement for biomass formation. Hence, determination of for instance the protein content of biomass is relevant in studies on bioenergetics. Transport of the carbon source. Active (i.e. energy-requiring) transport, which occurs for a number of sugars and polyols, may contribute significantly to the calculated theoretical ATP requirement for biomass formation. Pia-ratio. The efficiency of mitochondrial energy generation has a strong effect on the cell yield. The Pia-ratio is determined to a major extent by the number of proton-translocating sites in the mitochondrial respiratory chain. Maintenance and environmental factors. Factors such as osmotic stress, heavy metals, oxygen and carbon dioxide pressures, temperature and pH affect the yield of yeasts. Various mechanisms may be involved, often affecting the maintenance energy requirement. Metabolites such as ethanol and weak acids. Ethanol increases the permeability ofthe plasma membrane, whereas weak acids can act as proton conductors. Energy content of the growth substrate. It has often been attempted in the literature to predict the biomass yield by correlating the energy content of the carbon source (represented by the degree of reduction) to the biomass yield or the percentage assimilation of the carbon source. An analysis of biomass yields of Candida uti/is on a large number of carbon sources indicates that the biomass yield is mainly determined by the biochemical pathways leading to biomass formation, rather than by the energy content of the substrate.

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Increase of anion and proton permeability of Saccharomyces carlsbergensis plasmalemma by n‐alcohols as a possible cause of its de‐energization

Cited by 42 publications

References 24 publications

Engineering Platforms for Directed Evolution of Laccase from Pycnoporus cinnabarinus

Engineering Platforms for Directed Evolution of Laccase from Pycnoporus cinnabarinus

The Dynamic Changes of Proton Efflux Rate in Saccharomyces pastorianus Strains During High Gravity or Very High Gravity Brewing

Physiology of yeasts in relation to biomass yields

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