Growth-coupled CRISPRi screening reveals YBR013C as a novel genetic target for secretion of an ovalbumin-based fusion protein in Saccharomyces cerevisiae.

UNLABELLED: Ovalbumin (OVA) is an important egg white protein that is widely used in food applications owing to its functional properties. Although precision fermentation offers a sustainable alternative for OVA production, limited extracellular secretion remains a significant challenge. To address this gap, this study aimed to develop a growth-coupled screening strategy for identifying a novel gene that enhances OVA secretion in Saccharomyces cerevisiae. To achieve this, an OVA-β-glucosidase (GH1) fusion protein was used to link secretion efficiency to cellobiose-dependent growth. A genome-wide CRISPR interference (CRISPRi) library was introduced into an OVA-GH1-expressing strain and screened under isolated conditions. Repression of YBR013C, a gene of unknown function, consistently resulted in superior growth. Reintroduction of the YBR013C-targeting CRISPRi plasmid reproduced this phenotype and increased extracellular OVA-GH1 levels. This study identifies YBR013C as a novel factor associated with protein secretion and presents a generalizable screening platform for improving protein secretion in yeast. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-026-02231-8.