Ethanol production using nuclear petite yeast mutants

Two respiratory-deficient nuclear petites, FY23Δpet191 and FY23Δcox5a, of the yeast Saccharomyces cerevisiae were generated using polymerase-chain-reaction-mediated gene disruption, and their respective ethanol tolerance and productivity assessed and compared to those of the parental grande, FY23WT, and a mitochondrial petite, FY23ρ⁰. Batch culture studies demonstrated that the parental strain was the most tolerant to exogenously added ethanol with an inhibition constant. K _i, of 2.3% (w/v) and a specific rate of ethanol production, q _p, of 0.90 g ethanol g dry cells⁻¹ h⁻¹. FY23ρ⁰ was the most sensitive to ethanol, exhibiting a K _i of 1.71% (w/v) and q _p of 0.87 g ethanol g dry cells⁻¹ h⁻¹. Analyses of the ethanol tolerance of the nuclear petites demonstrate that functional mitochondria are essential for maintaining tolerance to the toxin with the 100% respiratory-deficient nuclear petite, FY23Δpet191, having a K _i of 2.14% (w/v) and the 85% respiratory-deficient FY23Δcox5a, having a K _i of 1.94% (w/v). The retention of ethanol tolerance in the nuclear petites as compared to that of FY23ρ⁰ is mirrored by the ethanol productivities of these nuclear mutants, being respectively 43% and 30% higher than that of the respiratory-sufficient parent strain. This demonstrates that, because of their respiratory deficiency, the nuclear petites are not subject to the Pasteur effect and so exhibit higher rates of fermentation. Received: 22 September 1997 / Accepted: 7 December 1997