Impact of CRISPRi-Mediated Titration of <i>GPD</i> Genes on the Fermentative Performance of <i>S. cerevisiae</i>

Glycerol is one of the main byproducts in ethanol fermentation due to its importance in redox balance and response to osmotic stress in <i>Saccharomyces cerevisiae</i>. Since its production diverts carbon from alcohol production, traditional gene-editing methods have been applied to the glycerol synthesis pathway. However, such approaches generate undesirable phenotypes for industrial applications. In the present study, we employed the CRISPR-dCas9 system to moderately downregulate the expression of <i>GPD1</i> and <i>GPD2</i>, the two main genes involved in this metabolism. <i>GPD2</i> gene expression downregulation and a graded reduction in glycerol production after repression of four different target sites in each paralogue were achieved. Employment of the CRISPRi approach for <i>GPD</i> gene modulation resulted in higher specific ethanol productivity (SEP) than that of single knockout cells. Targeted modulation in a region -140 basepairs upstream of the transcription start site (TSS) of <i>GPD1</i> resulted in a 3% increase in ethanol production compared to the wild type and gpd Δ strains. Such regulation, combined with <i>GPD2</i> deletion, revealed the higher SEP among all tested strains. Furthermore, a <i>GPD1</i>-modulated strain maintained tolerance to high osmolarity in very high-gravity (VHG) fermentation while maintaining its ethanol production levels above those observed in the control strain.