Improvement of ethanol production by an industrial yeast strain via multiple gene deletions

The genetic engineering of yeasts used in commercial processes can be both time-consuming and laborious. This is because industrial yeasts possess largely uncharacterised genomes, which frequently carry at least two copies of any gene. Such strains are usually devoid of auxotrophic or other genetic markers and this requires the incorporation of positively selectable (and often heterologous) genes into plasmids or other transforming DNA molecules. In this paper, we demonstrate that multiple gene deletions may be readily performed in industrial yeasts. Using a specially designed 1oxPkanMX4 gene replacement cassette, we deleted the two PET191 alleles essential to respiration in the diploid, high alcohol-producing, wine yeast, K1. The two integrated deletion cassettes, which rendered the respiratory-deficient mutant, K1Δpet191ab, resistant to the antibiotic geneticin were then excised from the genome following the expression of a ere recombinase gene harboured on the multi-copy plasmid YEP351-cre-cyh. This plasmid was maintained in the mutant under the selective pressure of the antibiotic cycloheximide and then removed when both genes had been successfully deleted. Batch fermentations were performed in homebrew style for strains K1 and K1Δpet191ab and revealed a 40% higher volumetric ethanol production rate and a 9% higher ethanol ceiling for the mutant. This demonstrates that, because of their respiratory deficiency, nuclear petites are not subject to the Pasteur effect and so exhibit higher rates of fermentation. Furthermore, nuclear petites cannot metabolise the product of fermentation, ethanol, allowing higher ethanol titres to be achieved. We believe that the method of strain manipulation demonstrated here will be of interest to scientists in the alcoholic beverages industry, who wish to delete genes in production yeast strains, while simultaneously ensuring the removal of all foreign coding sequences.