Determining the Roles of the Aldehyde Dehydrogenase Enzymes 5 and 6 during Icewine and Table Wine Fermentation in a Commercial Wine Yeast Using CRISPR-Cas9.

Abstract

Canada is known for Icewine which is fermented from grapes naturally frozen on the vine. Grapes are harvested below –8°C and pressed while frozen, trapping ice crystals inside the berries and releasing juice highly concentrated in sugars and acids. The high sugar juice environment (40°Brix) causes hyperosmotic stress for yeast, resulting in water loss, triggering glycerol production to act as an internal osmolyte to draw water back into the yeast cell, resulting in an NAD+ imbalance. Icewine has significantly higher acetic acid versus table wine, where acetic acid production may be used to reduce NAD(P)+ to NAD(P)H via NAD(P)+-dependent aldehyde dehydrogenase proteins (Aldp). Previous research linked the NAD+ redox imbalance during Icewine fermentation with ALD gene expression patterns indicating a role of Ald3p encoded by ALD3 in elevating the acetic acid whereas in table wine, ALD6 expression dominated. In the overall research program from our laboratory, the goal is to remove all five of the ALD genes, individually and in combination, from a commercial yeast genome using CRISPR-Cas9 gene editing. The objective of this project is to remove three (ALD6, ALD5, and ALD4) of the five ALD genes individually from the genome of a commercial wine yeast K1 V1116 using CRISPR-Cas9 gene editing to assess the roles of the aldehyde dehydrogenase enzymes in acetic acid production in both Icewine and table wine using these commercial yeast deletion strains. All copies of ALD6 and ALD5 were successfully knocked out of K1-V1116. The ald6Δ yeast had a significant impact on the acetic acid production with an 86% decrease in acetic acid production during Icewine fermentation and an 85% decrease during table wine fermentation in comparison to the wild-type control yeast. The ald5Δ had a significant but minimal impact on acetic acid production during table wine fermentation and no impact on acetic acid during Icewine fermentation. Under table wine conditions, ald5Δ had a 16% decrease in acetic acid production. One allele of the ALD4 gene was successfully knocked out of K1-V1116, creating a heterozygous ALD4 mutant. This heterozygous mutant was not evaluated in fermentations.