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Expression of Mitochondrial Cytochrome C Oxidase Chaperone Gene (COX20) Improves Tolerance to Weak Acid and Oxidative Stress during Yeast Fermentation.

Kumar V, Hart AJ, Keerthiraju ER, Waldron PR, Tucker GA, Greetham D - PLoS ONE (2015)

Bottom Line: Saccharomyces cerevisiae is the micro-organism of choice for the conversion of fermentable sugars released by the pre-treatment of lignocellulosic material into bioethanol.Pre-treatment of lignocellulosic material releases acetic acid and previous work identified a cytochrome oxidase chaperone gene (COX20) which was significantly up-regulated in yeast cells in the presence of acetic acid.This is a study which has utilised tetracycline-regulated protein expression in a fermentation system, which was characterised by improved (or enhanced) tolerance to acetic acid and oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: University of Nottingham, School of Biosciences, Sutton Bonington Campus, Loughborough, LE12 5RD, United Kingdom.

ABSTRACT

Introduction: Saccharomyces cerevisiae is the micro-organism of choice for the conversion of fermentable sugars released by the pre-treatment of lignocellulosic material into bioethanol. Pre-treatment of lignocellulosic material releases acetic acid and previous work identified a cytochrome oxidase chaperone gene (COX20) which was significantly up-regulated in yeast cells in the presence of acetic acid.

Results: A Δcox20 strain was sensitive to the presence of acetic acid compared with the background strain. Overexpressing COX20 using a tetracycline-regulatable expression vector system in a Δcox20 strain, resulted in tolerance to the presence of acetic acid and tolerance could be ablated with addition of tetracycline. Assays also revealed that overexpression improved tolerance to the presence of hydrogen peroxide-induced oxidative stress.

Conclusion: This is a study which has utilised tetracycline-regulated protein expression in a fermentation system, which was characterised by improved (or enhanced) tolerance to acetic acid and oxidative stress.

No MeSH data available.


Related in: MedlinePlus

Metabolic activity of WT and Δcox20 under 0–100 mM acetic acid, (A) WT and (B) Δcox20.Results presented are a representative of triplicate values (Mean +/- SD n = 3).
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pone.0139129.g002: Metabolic activity of WT and Δcox20 under 0–100 mM acetic acid, (A) WT and (B) Δcox20.Results presented are a representative of triplicate values (Mean +/- SD n = 3).

Mentions: The metabolic activity of wild type (BY4741) and the Δcox20 strain during incubation in the presence of acetic acid (0–100 mM) was determined by use of a phenotypic microarray (Fig 2A and 2B). The two strains demonstrated similar metabolic activity under control (no acetic acid) conditions but the Δcox20 strain was characterised by an increase in acetic acid sensitivity when compared with wild-type in particular at higher concentrations. The impact of the overexpression of COX20 on tolerance to acetic acid was then assessed, Δcox20 strains which had been transformed with plasmids (pCM161 or pCM173) containing COX20 under a tetracycline-regulatable promoter were assessed for acetic acid tolerance in comparison with their empty vector controls (Fig 3A). Presence of empty vectors (pCM161 or pCM173) had no discernible effect on tolerance to 75 mM acetic acid when compared to the Δcox20 background (Fig 3A). However, Δcox20 strains containing the overexpression vectors pCM161(COX20) or pCM173(COX20) displayed increased tolerance to 75 mM acetic acid as compared to their corresponding empty vector controls (Fig 3A). The effect of pCM173(COX20) being particularly effective at increasing metabolic activity, compatible with the fact that this construct employs a stronger promoter. Protein expression using a pCM173 vector has been shown to be higher when compared with expression level using a pCM161 vector [16]. The metabolic activity of the wild type yeast under these conditions was intermediate between the Δcox20 and pCM173 (COX20) strains (Fig 3A). Assays with Δcox20 strains expressing COX20 in a pCM161 plasmid appeared to recover the phenotype when compared with wild-type for the first 30 hours of the assay; after this time point there was no further increase metabolic output when compared with wild-type. For this reason all further studies were undertaken using the pCM173(COX20) strain. Tolerance to acetic acid was further confirmed in assays using 100 mM acetic acid in which a strain with an empty vector was sensitive whereas a strain overexpressing COX20 was resistant (S1A Fig).


Expression of Mitochondrial Cytochrome C Oxidase Chaperone Gene (COX20) Improves Tolerance to Weak Acid and Oxidative Stress during Yeast Fermentation.

Kumar V, Hart AJ, Keerthiraju ER, Waldron PR, Tucker GA, Greetham D - PLoS ONE (2015)

Metabolic activity of WT and Δcox20 under 0–100 mM acetic acid, (A) WT and (B) Δcox20.Results presented are a representative of triplicate values (Mean +/- SD n = 3).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591339&req=5

pone.0139129.g002: Metabolic activity of WT and Δcox20 under 0–100 mM acetic acid, (A) WT and (B) Δcox20.Results presented are a representative of triplicate values (Mean +/- SD n = 3).
Mentions: The metabolic activity of wild type (BY4741) and the Δcox20 strain during incubation in the presence of acetic acid (0–100 mM) was determined by use of a phenotypic microarray (Fig 2A and 2B). The two strains demonstrated similar metabolic activity under control (no acetic acid) conditions but the Δcox20 strain was characterised by an increase in acetic acid sensitivity when compared with wild-type in particular at higher concentrations. The impact of the overexpression of COX20 on tolerance to acetic acid was then assessed, Δcox20 strains which had been transformed with plasmids (pCM161 or pCM173) containing COX20 under a tetracycline-regulatable promoter were assessed for acetic acid tolerance in comparison with their empty vector controls (Fig 3A). Presence of empty vectors (pCM161 or pCM173) had no discernible effect on tolerance to 75 mM acetic acid when compared to the Δcox20 background (Fig 3A). However, Δcox20 strains containing the overexpression vectors pCM161(COX20) or pCM173(COX20) displayed increased tolerance to 75 mM acetic acid as compared to their corresponding empty vector controls (Fig 3A). The effect of pCM173(COX20) being particularly effective at increasing metabolic activity, compatible with the fact that this construct employs a stronger promoter. Protein expression using a pCM173 vector has been shown to be higher when compared with expression level using a pCM161 vector [16]. The metabolic activity of the wild type yeast under these conditions was intermediate between the Δcox20 and pCM173 (COX20) strains (Fig 3A). Assays with Δcox20 strains expressing COX20 in a pCM161 plasmid appeared to recover the phenotype when compared with wild-type for the first 30 hours of the assay; after this time point there was no further increase metabolic output when compared with wild-type. For this reason all further studies were undertaken using the pCM173(COX20) strain. Tolerance to acetic acid was further confirmed in assays using 100 mM acetic acid in which a strain with an empty vector was sensitive whereas a strain overexpressing COX20 was resistant (S1A Fig).

Bottom Line: Saccharomyces cerevisiae is the micro-organism of choice for the conversion of fermentable sugars released by the pre-treatment of lignocellulosic material into bioethanol.Pre-treatment of lignocellulosic material releases acetic acid and previous work identified a cytochrome oxidase chaperone gene (COX20) which was significantly up-regulated in yeast cells in the presence of acetic acid.This is a study which has utilised tetracycline-regulated protein expression in a fermentation system, which was characterised by improved (or enhanced) tolerance to acetic acid and oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: University of Nottingham, School of Biosciences, Sutton Bonington Campus, Loughborough, LE12 5RD, United Kingdom.

ABSTRACT

Introduction: Saccharomyces cerevisiae is the micro-organism of choice for the conversion of fermentable sugars released by the pre-treatment of lignocellulosic material into bioethanol. Pre-treatment of lignocellulosic material releases acetic acid and previous work identified a cytochrome oxidase chaperone gene (COX20) which was significantly up-regulated in yeast cells in the presence of acetic acid.

Results: A Δcox20 strain was sensitive to the presence of acetic acid compared with the background strain. Overexpressing COX20 using a tetracycline-regulatable expression vector system in a Δcox20 strain, resulted in tolerance to the presence of acetic acid and tolerance could be ablated with addition of tetracycline. Assays also revealed that overexpression improved tolerance to the presence of hydrogen peroxide-induced oxidative stress.

Conclusion: This is a study which has utilised tetracycline-regulated protein expression in a fermentation system, which was characterised by improved (or enhanced) tolerance to acetic acid and oxidative stress.

No MeSH data available.


Related in: MedlinePlus