Metabolic Engineering of Komagataella phaffii for Conversion of Alcohol Fermentation to Lactic Acid Fermentation using CRISPR-Cas9

Abstract

Lactic acid is a valuable platform chemical used in the food, biomedical, and bioplastics industries, particularly as a precursor for biodegradable polylactic acid. Komagataella phaffii is a promising industrial yeast due to its robust growth, ability to utilize methanol as a carbon source, and suitability for genetic engineering. The native adh2 gene encodes an alcohol dehydrogenase involved in ethanol metabolism. This study aimed to establish a CRISPR/Cas9-based genome editing strategy for the integration of the Pediococcus acidilactici lactate dehydrogenase-l (Paldh) gene into the K. phaffii adh2 locus. A donor DNA construct consisting of the K. phaffii adh2 upstream flanking region promoter, Paldh coding sequence, and K. phaffii adh2 downstream flanking region terminator was assembled using overlap extension PCR. A single-guide RNA was designed using CHOPCHOP tools and cloned into the BB3cK CRISPR plasmid to direct Cas9-mediated cleavage at the adh2 locus. A successfully constructed donor DNA and sgRNA have 2972 bp and 251 bp length respectively. The recombinant BB3cK_sgRNA plasmid was validated using PCR, which showed the expected 251 bp amplicons, indicating the presence of sgRNA construct inside the BB3cK CRISPR plasmid. The donor DNA and sgRNA_BB3cK plasmid were co-transformed into K. phaffii. Transformants were subsequently screened by colony PCR using Paldh-specific primers to identify colonies carrying the integrated gene. Out of five single colonies screened, one colony showed a DNA band of a apporimately 1,027 bp DNA fragment, indicating successful integration of Paldh into the engineered K. phaffii strain.