Construction of Donor DNA Cassette Containing Lactiplantibacillus plantarum LDH for Gene Swap in Komagataella phaffii Within Its ADH2 Locus

Abstract

Lactic acid is a valuable monomer used that utilizes lactate dehydrogenase for its production. Lactate dehydrogenase found in many lactic acid bacteria including Lactiplantibacillus plantarum. Utilizing a heterofermentative lactic acid bacteria, such as L. plantarum, for lactic acid production is not efficient due to generation of other organic acid byproducts. The yeast Komagataella phaffii has emerged as a promising candidate for genetic and metabolic engineering, owing to its well-established engineering tools and strong promoter such as PAOX1 . This study explores the feasibility of metabolic engineering in K. phaffii by substituting its Alcohol Dehydrogenase 2 gene (KpADH2) with L. plantarum lactate dehydrogenase (LpLDH) gene using CRISPR/Cas9 technology. Primer pairs for constructing the donor DNA cassette were designed to consist of the LpLDH coding sequence which was regulated by the KpADH2 promoter and terminator. Additionally, sgRNA targeting KpADH2 was constructed and assembled into a Plasmid BB3cK_pGAP_23*_pLAT1_Cas9 plasmid. An in silico characterization of the LpLDH was performed. The gene fragment corresponding to the upstream of KpADH2, the LpLDH coding sequence, and the downstream of KpADH2 was successfully isolated, measuring approximately 985, 1019 and 985 base pairs in length. However, construction of these fragments into a donor DNA cassette requires further optimization. The sgRNA was successfully constructed and inserted into the CRISPi plasmid indicated by colony PCR. Moreover, In silico analysis of the isolated LpLDH coding sequence showed 100% similarity with the reference genome, its physicochemical properties suggest the protein to be structurally stable. It showed similar β-sheet and α-helix structures as LDH through protein primary and secondary structure analysis, and its predicted three-dimensional structure conformation. Lastly, the predicted structure showed similar protein motifs and domains. This preliminary research showed the feasibility of the integration of LpLDH within the KpADH2 locus using CRISPR/Cas9 approach.