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

Mengting Deng; Yaokang Wu; Xueqin Lv; Long Liu; Jianghua Li; Guocheng Du; Jian Chen; Yanfeng Liu

doi:10.1021/acssynbio.3c00494

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

ACS Synth Biol. 2023 Nov 17;12(11):3443-3453. doi: 10.1021/acssynbio.3c00494. Epub 2023 Oct 26.

Authors

Mengting Deng^{1

2

3

4}, Yaokang Wu^{1

2

3

4}, Xueqin Lv^{1

2

3

4}, Long Liu^{1

2

3

4}, Jianghua Li^{1

2

3

4}, Guocheng Du^{1

2

3

4}, Jian Chen^{2

3

4

5

6}, Yanfeng Liu^{1

2

3

4

6}

Affiliations

¹ Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
² Science Center for Future Foods, Jiangnan University, Wuxi 214122, China.
³ Engineering Research Center of Ministry of Education on Food Synthetic Biotechnology, Jiangnan University, Wuxi 214122, China.
⁴ Jiangsu Province Engineering Research Center of Food Synthetic Biotechnology, Jiangnan University, Wuxi 214122, China.
⁵ National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China.
⁶ Qingdao Special Food Research Institute, Qingdao 266109, China.

PMID: 37881961
DOI: 10.1021/acssynbio.3c00494

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 single-stranded 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.

Keywords: CRISPR-based genome editing; Komagataella phaffii; alpha-lactalbumin; single-strand DNA-annealing protein; single-strand DNA-binding protein; α-LA.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems* / genetics
Carrier Proteins / genetics
Gene Editing* / methods
Humans
RNA, Guide, CRISPR-Cas Systems

Substances

Carrier Proteins
RNA, Guide, CRISPR-Cas Systems

Supplementary concepts

Komagataella phaffii