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

초록

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.

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