Metabolic activity of WT and Δ<i>cox20</i> under 0–100 mM acetic acid, (A) WT and (B) Δ<i>cox20</i>.

<p>Results presented are a representative of triplicate values (Mean <i>+</i>/- SD n = 3).</p

Growth rates for Δ<i>cox20</i> pCM173 and Δ<i>cox20</i> pCM173(COX20) under control conditions (4% glucose) and 25–75 mM acetic acid (A) Δ<i>cox20</i> pCM173 and (B) Δ<i>cox20</i> pCM173(COX20).

<p>Results presented are a representative of triplicate values (Mean <i>+</i>/- SD n = 3).</p

Primers used in this study for the knockout of <i>TRP1</i> and insertion of pCM vectors into yeast strains.

<p>Primers used in this study for the knockout of <i>TRP1</i> and insertion of pCM vectors into yeast strains.</p

Strains used in this study, all strains are derived from BY4741 (MATa <i>his3</i>Δ0 <i>leu2</i>Δ0 <i>met15</i>Δ0 <i>ura3</i>Δ0), with the additional <i>TRP1</i> gene knocked out to allow for selection with pCM plasmids.

<p>* Integrative plasmids were constructed as indicated in material and methods.</p

(A) Metabolic activity of WT, Δ<i>cox20</i>, Δ<i>cox20</i> pCM161, Δ<i>cox20</i> pCM173, Δ<i>cox20</i> pCM161(<i>COX20</i>), Δ<i>cox20</i> pCM173(<i>COX20</i>) grown under 75 mM acetic acid, (B) Metabolic activity of Δ<i>cox20 and</i> Δ<i>cox20</i> pCM173(<i>COX20</i>) with and without tetracycline, under 75 mM acetic acid (C) Spot plate analysis of Δ<i>cox20</i> pCM161, Δ<i>cox20</i> pCM173, Δ<i>cox20</i> pCM161(<i>COX20</i>), Δ<i>cox20</i> pCM173(<i>COX20</i>) in the presence of control and 75 mM acetic acid.

<p>Results presented are a representative of triplicate values (Mean <i>+</i>/- SD n = 3).</p

Metabolic activity (redox signal intensity) of WT, Δ<i>cox20</i>, Δ<i>cox20</i> pCM173, and pCM173(<i>COX20</i>) on hydrolysates derived from an acid pre-treatment of wheat.

<p>Results presented are a representative of triplicate values (Mean <i>+</i>/- SD n = 3).</p

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

<div><p>Introduction</p><p><i>Saccharomyces cerevisiae</i> 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 (<i>COX20</i>) which was significantly up-regulated in yeast cells in the presence of acetic acid.</p><p>Results</p><p>A Δ<i>cox20</i> strain was sensitive to the presence of acetic acid compared with the background strain. Overexpressing <i>COX20</i> using a tetracycline-regulatable expression vector system in a Δ<i>cox20</i> 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.</p><p>Conclusion</p><p>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.</p></div

(A) Metabolic activity (redox signal intensity) of Δ<i>cox20</i> pCM173 or Δ<i>cox20</i> pCM173(<i>COX20</i>) in the presence of 1 mM hydrogen peroxide (B) Growth rates (OD600) for Δ<i>cox20</i> pCM173 or Δ<i>cox20</i> pCM173(<i>COX20</i>) in the presence of 1 mM hydrogen peroxide (C) Viability of Δ<i>cox20</i> pCM173 or Δ<i>cox20</i> pCM173(<i>COX20</i>) in the presence of 1 mM hydrogen peroxide measured over 120 mins.

<p>Results presented are a representative of triplicate values (Mean <i>+</i>/- SD n = 3).</p