# Impact of CRISPRi-Mediated Titration of GPD Genes on the Fermentative Performance of S. cerevisiae

## TL;DR

This study uses CRISPRi to reduce glycerol production in yeast, improving ethanol output without harming yeast performance.

## Contribution

A CRISPRi-based approach is introduced to modulate GPD genes, achieving higher ethanol productivity than traditional knockout methods.

## Key findings

- CRISPRi downregulation of GPD1 and GPD2 reduced glycerol production.
- Modulated GPD1 strains increased ethanol production by 3% compared to wild type.
- Combining GPD1 modulation with GPD2 deletion achieved the highest ethanol productivity.

## Abstract

Glycerol is one of the main byproducts in ethanol fermentation
due to its importance in redox balance and response to osmotic stress
in Saccharomyces cerevisiae. 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 GPD1 and GPD2, the two main genes involved in this metabolism. GPD2 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 GPD 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 GPD1 resulted in a 3% increase
in ethanol production compared to the wild type and gpd Δ strains.
Such regulation, combined with GPD2 deletion, revealed
the higher SEP among all tested strains. Furthermore, a GPD1-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.