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 lar gely uncharacterised genomes, which frequently carry at feast 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 o ften heterologous) genes into plasmids or other transforming DNA molecules. In this paper we demonstrate that multiple gene deletions may be readily p erformed in industrial yeasts. Using a specially designed loxPkanMX4 gene r eplacement cassette, we deleted the two PET191 alleles essential to respira tion in the diploid high alcohol-producing, wine yeast, K1. The two integra ted deletion cassettes, which rendered the respiratory-deficient mutant, K1 Delta pet191ab, resistant to the antibiotic geneticin were then excised fr om the genome following the expression of a cre recombinase gene harboured on the multi-copy plasmid YEP351-cre-cyh. This plasmid teas maintained in t he mutant under the selective pressure of the antibiotic cycloheximide and then removed when both genes had been successfully deleted. Batch fermentat ions were performed in homebrew style for strains K1 and K1 Delta pet191ab and revealed a 40% higher volumetric ethanol production rate and a 9% highe r ethanol ceiling for the mutant. This demonstrates that, because of their respiratory deficiency, nuclear petites are not subject to the Pasteur effe ct and so exhibit higher rates of fermentation. Furtherrnore, nuclear petit es cannot metabolise the product of fermentation ethanol, allowing higher e thanol titres to be achieved. We believe that the method of strain manipula tion demonstrated here will be of interest to scientists in the alcoholic b everages industry, who wish to delete genes in production yeast strains, wh ile simultaneously ensuring the removal of all foreign coding sequences.