State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
College of Life Science, University of Chinese Academy of Sciences, Beijing, China.
Appl Environ Microbiol. 2023 Feb 28;89(2):e0173822. doi: 10.1128/aem.01738-22. Epub 2023 Jan 31.
Site-specific recombinases (integrases) can mediate the horizontal transfer of genomic islands. The ability to integrate large DNA sequences into target sites is very important for genetic engineering in prokaryotic and eukaryotic cells. Here, we characterized an unprecedented catalogue of 530 tyrosine-type integrases by examining genes potentially encoding tyrosine integrases in bacterial genomic islands. The phylogeny of putative tyrosine integrases revealed that these integrases form an evolutionary clade that is distinct from those already known and are affiliated with novel integrase groups. We systematically searched for candidate integrase genes, and their integration activities were validated in a bacterial model. We verified the integration functions of six representative novel integrases by using a two-plasmid integration system consisting of a donor plasmid carrying the integrase gene and site and a recipient plasmid harboring an site in -deficient Escherichia coli. Further quantitative reverse transcription-PCR (qRT-PCR) assays validated that the six selected integrases can be expressed with their native promoters in E. coli. The region reductions showed that the extent of sites of integrases is approximately 200 bp for integration capacity. In addition, mutational analysis showed that the conserved tyrosine at the C terminus is essential for catalysis, confirming that these candidate proteins belong to the tyrosine-type recombinase superfamily, i.e., tyrosine integrases. This study revealed that the novel integrases from bacterial genomic islands have site-specific recombination functions, which is of physiological significance for their genomic islands in bacterial chromosomes. More importantly, our discovery expands the toolbox for genetic engineering, especially for efficient integration activity. Site-specific recombinases or integrases have high specificity for DNA large fragment integration, which is urgently needed for gene editing. However, known integrases are not sufficient for meeting multiple integrations. In this work, we discovered an array of integrases through bioinformatics analysis in bacterial genomes. Phylogeny and functional assays revealed that these new integrases belong to tyrosine-type integrases and have the ability to conduct site-specific recombination. Moreover, region extent and catalysis site analysis were characterized. Our study provides the methodology for discovery of novel integrases and increases the capacity of weapon pool for genetic engineering in bacteria.
位点特异性重组酶（整合酶）可介导基因组岛的水平转移。将大段 DNA 序列整合到靶位点的能力对于原核和真核细胞的基因工程非常重要。在这里，我们通过检查细菌基因组岛中潜在编码酪氨酸整合酶的基因，对 530 种前所未有的酪氨酸整合酶进行了特征描述。假定酪氨酸整合酶的系统发育揭示了这些整合酶形成了一个与已知整合酶明显不同的进化分支，并且与新的整合酶群有关。我们系统地搜索候选整合酶基因，并在细菌模型中验证了它们的整合活性。我们使用由携带整合酶基因和 位点的供体质粒和带有 位点的受体质粒组成的双质粒整合系统，验证了六个代表性新型整合酶的整合功能，该系统在 -缺陷型大肠杆菌中。进一步的定量逆转录-PCR（qRT-PCR）测定验证了这六种选定的整合酶可以在大肠杆菌中用其天然启动子表达。 区域减少表明整合酶的 位点大约为 200bp，整合能力为 200bp。此外，突变分析表明，C 末端保守的酪氨酸对于催化至关重要，这证实了这些候选蛋白属于酪氨酸型重组酶超家族，即酪氨酸整合酶。这项研究表明，来自细菌基因组岛的新型整合酶具有位点特异性重组功能，这对于它们在细菌染色体上的基因组岛具有生理意义。更重要的是，我们的发现扩展了遗传工程的工具包，特别是对于高效的整合活性。位点特异性重组酶或整合酶对 DNA 大片段的整合具有高度特异性，这是基因编辑所急需的。然而，已知的整合酶不足以满足多种整合需求。在这项工作中，我们通过细菌基因组中的生物信息学分析发现了一系列整合酶。系统发育和功能分析表明，这些新的整合酶属于酪氨酸整合酶，具有进行位点特异性重组的能力。此外，还对 区域范围和催化位点进行了分析。我们的研究为发现新型整合酶提供了方法，并为细菌的遗传工程增加了武器库的容量。
