Impact of a random TN5 mutation on endoglucanase secretion in ruminal cellulolytic Escherichia coli

• Published in: Gene 2025-01, Vol.933, p.148936, Article 148936
• Main Authors: Pang, Jian, Zhang, Long, Qi, Qingsheng, Liu, Zhanying
• Format: Article
• Language: English
• Subjects: BcsZ; Cellulase - genetics; Cellulase - metabolism; DNA Transposable Elements - genetics; endo-1,4-beta-glucanase; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; genes; Genome, Bacterial; Mutant library; mutants; Mutation; Protease La - genetics; Protease La - metabolism; protein secretion; Secretion mechanism; Transposon; transposons
• ISSN: 0378-1119; 1879-0038; 1879-0038
• DOI: 10.1016/j.gene.2024.148936

•A mutant library was constructed using TN5 transposons.•Using the transparent circle method to screen mutants.•Find three genes that affect the secretion of E. coli BcsZ. Most protein secretion systems are found in gram-negative bacteria, but the mechanism of endoglucanase (BcsZ) secretion in Escherichia coli (E. coli) remains unclear. In this study, we used JBZ-DH5α (which overexpresses BcsZ on the E. coli DH5α genome) as the initial strain. A mutant library was created by randomly inserting the TN5 transposon into the genome, and mutants with reduced transparent circles were identified on Congo red plates. The insertion sites of transposons in the genome were determined through whole-genome sequencing. The results revealed that the genes rnc, lon, and suhB, which encode RNC-ribonuclease III (RNC), LON-protease (LON), and SuhB-inositol phosphatase (SuhB), respectively, were disrupted. BcsZ secretion decreased in E. coli DH5α when the lon, rnc, or suhB genes were deleted, but the overexpression of these genes restored their secretion levels. These findings suggest that the lon, rnc, and suhB genes play a role in BcsZ secretion in E. coli, potentially enhancing our knowledge of BcsZ secretion and offering a strategy to increase protein secretion in E. coli as a cell factory.