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<p>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 <i>Saccharomyces cerevisiae</i>. 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 <i>YBR013C</i>, a gene of unknown function, consistently resulted in superior growth. Reintroduction of the <i>YBR013C</i>-targeting CRISPRi plasmid reproduced this phenotype and increased extracellular OVA–GH1 levels. This study identifies <i>YBR013C</i> as a novel factor associated with protein secretion and presents a generalizable screening platform for improving protein secretion in yeast.</p>

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

  • 期刊论文

  • 发表日期：

    2026年6月30日

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  • Woohyuk Lee,
  • Da In Yun,
  • Sun-Ki Kim

摘要

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.