PDF(Pubmed)
Abstract：
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 attP site and a recipient plasmid harboring an attB site in recA-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 attP region reductions showed that the extent of attP 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. IMPORTANCE 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, attP 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.
摘要：
位点特异性重组酶(整合酶)可以介导基因组岛的水平转移。将大DNA序列整合到靶位点的能力对于原核和真核细胞中的基因工程非常重要。这里,我们通过检查细菌基因组岛中可能编码酪氨酸整合酶的基因,鉴定了530个酪氨酸型整合酶的前所未有的目录.推定的酪氨酸整合酶的系统发育表明，这些整合酶形成了与已知的进化枝不同的进化枝，并与新的整合酶基团相关。我们系统地搜索了候选整合酶基因，并在细菌模型中验证了它们的整合活性。我们通过使用双质粒整合系统验证了六种具有代表性的新型整合酶的整合功能，该系统由携带整合酶基因和attP位点的供体质粒和在recA缺陷型大肠杆菌中带有attB位点的受体质粒组成。进一步的定量逆转录-PCR(qRT-PCR)测定验证了六种选择的整合酶可以用它们的天然启动子在大肠杆菌中表达。attP区的减少表明，整合酶的attP位点的程度约为整合能力的200bp。此外,突变分析表明，在C端保守的酪氨酸是必不可少的催化，确认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，这对它们在细菌染色体中的基因组岛具有生理意义。更重要的是,我们的发现扩展了基因工程的工具箱，特别是对于有效的集成活动。重要性位点特异性重组酶或整合酶对DNA大片段整合具有高特异性，这是基因编辑迫切需要的。然而,已知的整合酶不足以满足多重整合。在这项工作中,通过对细菌基因组的生物信息学分析，我们发现了一系列整合酶。系统发育和功能测定表明，这些新的整合酶属于酪氨酸型整合酶，并具有进行位点特异性重组的能力。此外，ATTP区域范围和催化位点分析进行了表征。我们的研究提供了发现新型整合酶的方法，并增加了细菌基因工程武器库的能力。
