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

Oct 26, 2023ACS synthetic biology

Foreign Single-Strand DNA Binding Proteins May Improve CRISPR Genome Editing in Komagataella phaffii

AI simplified

Abstract

A CRISPR-based gene editing method achieved a 100% insertion rate for a 50 bp homologous arm, producing 18 colony-forming units (CFU) per μg of donor DNA.

The integration of three genes at different locations enhanced the production of human-lactalbumin (α-LA).
Phospholipid biosynthesis was strengthened to aid in the formation of the endoplasmic reticulum membrane.
Overexpression of transcription and translation factors improved the transcription and translation of recombinant proteins.
Final α-LA production reached 113.4 mg/L in 3 L fermentation, which is two times higher than the strain without multiple site gene editing.
The method may facilitate more efficient biomanufacturing by enabling synergistic multi-site engineering of protein and biochemical pathways.

AI simplified

The industrial yeastis 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 employedphage single-stranded DNA-annealing protein (SSAP) PapRecT andsingle-stranded DNA-binding protein (SSB) PaSSB to introduce SSAP-SSB-based homology recombination, which facilitatedCRISPR-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 (,, and) 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, two times higher than that of the strain without multiple site gene editing, which is the highest reported titer in. 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. Komagataella phaffii Pseudomonas aeruginosa P. aeruginosa K. phaffii INO4GAL4-like PAB1K. phaffii -1

Full Text

Full text is available at the source.

View full text (PDF) ↗View full text ↗

Abstract

Full Text

Related papers

what lands in your inbox each week: