整合酶
基因组
酪氨酸
遗传学
生物
化学
计算生物学
基因
生物化学
作者
Xin Song,Yongliang Li,Yongjun Xia,Guangqiang Wang,Zhiqiang Xiong,Yijin Yang,Lianzhong Ai
标识
DOI:10.1016/j.fbio.2024.103694
摘要
Lacticaseibacillus casei is a common bacterium, due to its small genome and relatively simple metabolism, it will process the potential to become a cell factory. Although CRISPR has been successfully applied to the gene editing of L. casei, the technology is limited by the size of the knock-in gene and cannot achieve efficient integration of large fragments, which has impeded the application of L. casei in synthetic biology. Here, we develop a genome integration tool applicable to L. casei. Three possible phage integrases were screened and introduced into L. casei AR1088. A highly active integrase in L. casei was screened. Based on this, a single plasmid-based efficient gene integration tool for L. casei was established. With the assistance of CRISPR-Cas9 editing system, we realized multi-site integration of genome. Then, homologous modeling of integrase φA2 was conducted to explore its core amino acid site and minimum functional region. Two core sites of H334 and T366 were identified, and it was confirmed that the full-length protein of integrase was essential for integration. As a proof of concept, the bile salt hydrolase gene from Lactiplantibacillus plantarum was integrated into AR1088 genome at multiple sites, which improved the acid and bile salt tolerance of the engineered strain. The system developed in this study overcomes the deficiencies of currently available genetic tools in the multi-site chromosomal integration of large DNA fragment in L. casei, and may be extended to other lactobacillus.
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