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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

(A) 10% SDS-PAGE gel of protein samples from wild-type (BY4741), Δcox20, Δcox20 pCM161, Δcox20 pCM173, Δcox20 pCM161(COX20), Δcox20 pCM173(COX20). (B) β-galactosidase activity (U/ mL) for Δcox20 (pCM161), Δcox20 (pCM161) + 1 μg/mL tetracycline, Δcox20 (pCM161(COX20)), Δcox20 (pCM161(COX20)) + 1 μg/mL tetracycline.Results presented are a representative of triplicate values (Mean +/- SD n = 3).
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pone.0139129.g001: (A) 10% SDS-PAGE gel of protein samples from wild-type (BY4741), Δcox20, Δcox20 pCM161, Δcox20 pCM173, Δcox20 pCM161(COX20), Δcox20 pCM173(COX20). (B) β-galactosidase activity (U/ mL) for Δcox20 (pCM161), Δcox20 (pCM161) + 1 μg/mL tetracycline, Δcox20 (pCM161(COX20)), Δcox20 (pCM161(COX20)) + 1 μg/mL tetracycline.Results presented are a representative of triplicate values (Mean +/- SD n = 3).

Mentions: Centromeric plasmids containing COX20 under the regulation of tetO2 (pCM161) or tetO7 (pCM173) promoters were constructed; these were then used to transform a Δcox20 yeast strain. The Δcox20 strain was also transformed with empty vector as a further control. The wild type and all the transformed strains were assessed for expression of COX20 on a SDS-PAGE gel (Fig 1A). COX20 has a predicted molecular weight of 23 KDa [15]. There was no detectable expression in the wild type, Δcox20 or either of the empty vector controls (Fig 1A), a clear band was evident in both over-expression lines, protein concentrations being higher in the strain with the tetO7 regulated promoter (pCM173(COX20)) than the strain with a tet02 regulated promoter (pCM161(COX20)) (Fig 1A). Expression from these two promoters is suppressed by the addition of tetracycline (1 μg/mL) [16], and this was demonstrated in this instance as the addition of tetracycline revealed that there was no detectable protein expression (data not shown). Expression of β-galactosidase is an additional method to test the efficiency of tetracycline repression and activity was detected for a Δcox20 strain carrying either a pCM161 vector or a pCM161 vector containing COX20. Addition of tetracycline suppressed β-galactosidase activity in each case (Fig 1B).


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)

(A) 10% SDS-PAGE gel of protein samples from wild-type (BY4741), Δcox20, Δcox20 pCM161, Δcox20 pCM173, Δcox20 pCM161(COX20), Δcox20 pCM173(COX20). (B) β-galactosidase activity (U/ mL) for Δcox20 (pCM161), Δcox20 (pCM161) + 1 μg/mL tetracycline, Δcox20 (pCM161(COX20)), Δcox20 (pCM161(COX20)) + 1 μg/mL tetracycline.Results presented are a representative of triplicate values (Mean +/- SD n = 3).
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Related In: Results  -  Collection

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pone.0139129.g001: (A) 10% SDS-PAGE gel of protein samples from wild-type (BY4741), Δcox20, Δcox20 pCM161, Δcox20 pCM173, Δcox20 pCM161(COX20), Δcox20 pCM173(COX20). (B) β-galactosidase activity (U/ mL) for Δcox20 (pCM161), Δcox20 (pCM161) + 1 μg/mL tetracycline, Δcox20 (pCM161(COX20)), Δcox20 (pCM161(COX20)) + 1 μg/mL tetracycline.Results presented are a representative of triplicate values (Mean +/- SD n = 3).
Mentions: Centromeric plasmids containing COX20 under the regulation of tetO2 (pCM161) or tetO7 (pCM173) promoters were constructed; these were then used to transform a Δcox20 yeast strain. The Δcox20 strain was also transformed with empty vector as a further control. The wild type and all the transformed strains were assessed for expression of COX20 on a SDS-PAGE gel (Fig 1A). COX20 has a predicted molecular weight of 23 KDa [15]. There was no detectable expression in the wild type, Δcox20 or either of the empty vector controls (Fig 1A), a clear band was evident in both over-expression lines, protein concentrations being higher in the strain with the tetO7 regulated promoter (pCM173(COX20)) than the strain with a tet02 regulated promoter (pCM161(COX20)) (Fig 1A). Expression from these two promoters is suppressed by the addition of tetracycline (1 μg/mL) [16], and this was demonstrated in this instance as the addition of tetracycline revealed that there was no detectable protein expression (data not shown). Expression of β-galactosidase is an additional method to test the efficiency of tetracycline repression and activity was detected for a Δcox20 strain carrying either a pCM161 vector or a pCM161 vector containing COX20. Addition of tetracycline suppressed β-galactosidase activity in each case (Fig 1B).

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