CRISPR-Cas9-Mediated Disruption of the Ethanol Fermentation Pathway in Komagataella phaffii through ADH2 Gene Deletion

Abstract

Komagataella phaffii is a non-conventional yeast that is widely used in industrial biotechnology due to its ability to grow to high cell densities and its effectiveness as a host for genetic engineering applications. A promising strategy for metabolic engineering in K. phaffii involves disrupting genes associated with ethanol metabolism, such as alcohol dehydrogenase 2 (KpADH2). This study aimed to construct and apply a CRISPR-Cas9 system for the disruption of the ADH2 gene in K. phaffii. A homologous repair template was created using overlap-extension PCR, while an ADH2-specific CRISPR-Cas9 plasmid was constructed using the GoldenPiCS cloning system. Both constructs were introduced into K. phaffii cells by electroporation and selected on kanamycin-containing media. Successful transformants were subsequently screened using colony PCR. The repair template and CRISPR-Cas9 plasmid were successfully constructed and validated. Transformation resulted in the formation of several putative colonies, indicating successful uptake of the CRISPR/Cas components. Colony PCR analysis showed that one out of three screened colonies produced the expected 2000 bp amplicon, which is 1000 bp shorter compared to the native KpADH2, suggesting successful removal of the ADH2 coding region. This study demonstrates the feasibility of using CRISPR-Cas9 to target and disrupt the KpADH2 gene, providing a basis for future research on ethanol metabolism and the development of metabolically engineered K. phaffii strains for industrial applications.