Establishment and optimization of a genetic manipulation system for <i>Staphylococcus pasteuri</i>.

Tinghao ZHANG; Ziqi WANG; Yuxin SONG; Jinjin WANG; Feng GUO; Yongjun ZHANG; Fuping LU; Ming LI

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Establishment and optimization of a genetic manipulation system for Staphylococcus pasteuri.

Author: Tinghao ZHANG ¹ ; Ziqi WANG ¹ ; Yuxin SONG ¹ ; Jinjin WANG ¹ ; Feng GUO ¹ ; Yongjun ZHANG ² ; Fuping LU ¹ ; Ming LI ¹
Author Information

1. Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin 300457, China.
2. Henan Academy of Sericultural Science, Nanyang 450003, Henan, China.
Publication Type:Journal Article
Keywords: 1,4-diaminobutane; Staphylococcus pasteuri; electroporation; gene editing; genetic engineering; tolerance
MeSH: Staphylococcus/drug effects*; Gene Editing/methods*; Electroporation/methods*; Plasmids/genetics*; CRISPR-Cas Systems; Genetic Engineering/methods*
From: Chinese Journal of Biotechnology 2025;41(9):3604-3616
CountryChina
Language:Chinese
Abstract: One of the technical bottlenecks limiting the high yield of 1,4-butanediamine is the insufficient tolerance of strains to 1,4-butanediamine. Enhancing the tolerance of strains to 1,4-butanediamine is therefore a primary challenge that needs to be addressed for the construction of strains with high yields of 1,4-butanediamine. Staphylococcus pasteuri 326180 exhibits exceptional tolerance to high-concentration 1,4-butanediamine, serving as both an ideal model for studying the mechanism underlying the 1,4-butanediamine tolerance and a novel host for constructing strains capable of efficiently producing 1,4-butanediamine. However, for both the research on the tolerance mechanism and the modification of chassis strains, gene editing of S. pasteuri needs to be carried out at the molecular level. The research objective of this paper is to establish a genetic manipulation system for S. pasteuri, laying foundation for subsequent studies on tolerance mechanism and the modification of chassis strains. This study systematically optimized the electroporation conditions, including key parameters such as the growth phase of cells, electric field strength, electroporation buffer, and recovery medium, successfully establishing an electroporation method for S. pasteuri. Additionally, we constructed the gene editing plasmid pCpfOA by replacing the resistance expression cassette, optimized the selection markers for gene editing, and finally established a CRISPR/Cpf1-based gene editing technology for S. pasteuri, achieving an editing efficiency of 90%. The genetic manipulation system of S. pasteuri established in this study provides technical support for research into the tolerance mechanism of this bacterium and the genetic modification of chassis strains.