The CRISPR/Cas9 system as a molecular strategy to decrease urea production in wine yeasts

Genome editing throughout the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) represents an emerging engineering approach that could be very useful in industrial yeasts. Indeed, although several molecular tools are available for genetic analysis in laboratory haploid strains of Saccharomyces cerevisiae, the genetics of metabolism in commercially-relevant polyploid yeast strains is still poorly understood. The present study applied the CRISPR/Cas9 system in order to generate two commercial ‘starter’ strains of S. cerevisiae (EC1118, AWRI796) with reduced urea production. In particular the CAN1 gene, encoding for an arginine permease, was eliminated by introducing a stop codon in the nucleotide sequence; the resulting can1 mutants showed a urea decrease of 18.5 and 35.5% for EC1118 and AWRI796 strains, respectively. In a wine-model environment based on two grape musts obtained from Chardonnay and Cabernet Sauvignon cultivars, both S. cerevisiae starter strains and can1 mutants completed the must fermentation in 8-12 days. However, recombinant strains carrying the CAN1 mutation failed to produce urea suggesting that the genetic modification successfully impaired the arginine metabolism. In conclusion, the reduction of urea production in a wine-model environment confirms that the CRISPR/Cas9 system has been successfully established in S. cerevisiae wine yeasts.