Heterologous Single-Strand DNA-Annealing and Binding Protein Enhance CRISPR-Based Genome Editing Efficiency in Komagataella phaffii

Abstract

The industrial yeast Komagataella phaffii is a highly effective platform for heterologous protein production, owing to its high protein expression and secretion capacity. Heterologous genes and proteins are involved in multiple processes, including transcription, translation, protein folding, modification, transportation, and degradation; however, engineering these proteins and genes is challenging due to inefficient genome editing techniques. We employed Pseudomonas aeruginosa phage singlestranded DNA-annealing protein (SSAP) PapRecT and P. aeruginosa single-stranded DNA-binding protein (SSB) PaSSB to introduce SSAP-SSB-based homology recombination, which facilitated K. phaffii CRISPR-based genome engineering. Specifically, a host-independent method was developed by expressing sgRNA with PapRecT-PaSSB in a single plasmid, with which only a 50 bp short homologous arm (HA) reached a 100% positive rate for CRISPR-based gene insertion, reaching 18 colony-forming units (CFU) per μg of donor DNA. Single deletion using 1000 bp HA attained 100%, reaching 68 CFUs per μg of donor DNA. Using this efficient CRISPR-based genome editing tool, we integrated three genes (INO4, GAL4-like, and PAB1) at three different loci for overexpression to realize the collaborative regulation of human-lactalbumin (α-LA) production. Specifically, we strengthened phospholipid biosynthesis to facilitate endoplasmic reticulum membrane formation and enhanced recombinant protein transcription and translation by overexpressing transcription and translation factors. The final production of α-LA in the 3 L fermentation reached 113.4 mg L −1 , two times higher than that of the strain without multiple site gene editing, which is the highest reported titer in K. phaffii. The CRISPR-based genome editing method developed in this study is suitable for the synergistic multiple-site engineering of protein and biochemical biosynthesis pathways to improve the biomanufacturing efficiency.

Applications of the Methylotrophic Yeast Komagataella phaffii in the Context of Modern Biotechnology

“…Furthermore, its off-target potential was not evaluated. Recently, Liu et al [ 126 ] described a highly programmable expression platform based on CRISPR-Cas systems (SynPic-X), and Deng and collaborators achieved the highest reported titer in K. phaffii , using CRISPR-Cas to regulate human lactalbumin (α-LA) production [ 146 ].…”

Precision Fermentation as an Alternative to Animal Protein, a Review

“…Metabolic engineering should address these issues by boosting precursor availability, maintaining redox and energy balance, or co-expressing helper genes involved in the unfolded protein response (UPR) pathway, leading to the development of a more robust strain [44,91]. In addition, the target protein yields can be increased by adjusting secretion sequences and promoters, the number of gene copies, using helper proteins such as chaperones, and exploring changes in membrane lipid composition, as demonstrated by different academic groups and private companies [87,[92][93][94][95].…”