Improvement of Ethanol Production by an Industrial Yeast Strain via Multiple Gene Deletions

Abstract

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 tzvo 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 loxPkanMX4 gene replacement cassette, we deleted the tzvo PET191 alleles essential to respiration in the diploid, high alcohol-producing, wine yeast, Kl. The tivo integrated deletion cassettes, which rendered the respiratory-deficient mutant, K2Apetl91ab, resistant to the antibiotic geneticin were then excised from the genome folloiving the expression of a ere recombinase gene harboured on the multi-copy plasmid YEP351-cre-cyh. This plasmid ivas 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 Kl and JCZApetl91ab and revealed a 40% higher volumetric ethanol production rate and a 9% higher ethanol ceiling for the mutant. Tltis demonstrates that, because of their respiratory deficiency, nuclear petites are not siibject 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 ivill 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.

Flux Balance Analysis of a Genome‐Scale Yeast Model Constrained by Exometabolomic Data Allows Metabolic System Identification of Genetically Different Strains

“…Reduction in the copy number of the PET191 gene implies less respiratory capacity, and getting closer to “anaerobic” conditions in this manner results in elevated ethanol production rates, as could be predicted. The complete elimination of Pasteur effect (suppression of fermentation by oxygen) by the chromosomal lesion of respiratory chain enables higher rates of fermentation and subsequently higher ethanol flux through glycolysis (15, 25, 28). The use of minimal oxygen consumption as the objective function in FBA tends to mimic this cellular behavior, leading to predicted results that are very close to the experimental values (Figure 1).…”

“…We exemplify this approach using data on fermentation by wild‐type and mutant industrial strains of the yeast Saccharomyces cerevisiae . For this purpose, a genome‐scale model of S. cerevisiae (14) was used to simulate metabolic characteristics of the industrial nuclear petite mutants K1Δ pet191a and K1Δ pet191ab , which were generated by deleting copies of the PET191 gene from a high‐alcohol‐producing yeast, K1 (15). The deletions result in respiration deficiency in the mutants.…”

Repeated-batch fermentations of xylose and glucose–xylose mixtures using a respiration-deficient Saccharomyces cerevisiae engineered for xylose metabolism

“…Fermenting glucose, a respiration-deficient mutant of S. cerevisiae has already known for showing higher ethanol productivity and/or yield than that of the respiration-sufficient parental strain (Dikicioglu et al, 2008;Hutter and Oliver, 1998;Oner et al, 2005;Panoutsopoulou et al, 2001). A respirationdeficient mutant of engineered stain also produced more ethanol (0.25 g ethanol/g xylose) from xylose as compared to the parental strain (0.15 g g −1 ) (Jin et al, 2004).…”

Increased ethanol production by deletion of HAP4 in recombinant xylose-assimilating Saccharomyces cerevisiae

“…Completely respiration-deficient nuclear petites may facilitate such applications [11]. Indeed, respiration-deficient mutants of S. cerevisiae strains displayed 30-43 % higher ethanol productivity than the respiratory-sufficient parent strain when glucose was used as a substrate [11,12]. The use of respiration-deficient S. stipitis and S. cerevisiae strains has been shown to improve ethanol yield [13,14].…”