Cui Guzhen, Hua Dengxiong, Zhao Xingxing, Zhou Jia, Yang Ying, Huang Tingyu, Wang Xinxin, Zhao Yan, Zhang Ting, Liao Jian, Guan Zhizhong, Luo Peng, Chen Zhenghong, Qi Xiaolan, Hong Wei
Key Laboratory of Endemic and Ethnic Diseases, Ministry of Education & Key Laboratory of Medical Molecular Biology of Guizhou Province & Key Laboratory of Microbiology and Parasitology of Education Department of Guizhou, Guizhou Medical University, Guiyang, Guizhou, China.
Joint Laboratory of Helicobacter Pylori and Intestinal Microecology of Affiliated Hospital of Guizhou Medical University.
Microbiol Spectr. 2023 Feb 21;11(2):e0315922. doi: 10.1128/spectrum.03159-22.
Thermophilic group II intron is one type of retrotransposon composed of intron RNA and intron-encoded protein (IEP), which can be utilized in gene targeting by harnessing their novel ribozyme-based DNA integration mechanism termed "retrohoming." It is mediated by a ribonucleoprotein (RNP) complex that contains the excised intron lariat RNA and an IEP with reverse transcriptase (RT) activity. The RNP recognizes targeting sites by exon-binding sequences 2 (EBS2)/intron-binding sequences 2 (IBS2), EBS1/IBS1, and EBS3/IBS3 bases pairing. Previously, we developed the TeI3c/4c intron as a thermophilic gene targeting system-Thermotargetron (TMT). However, we found that the targeting efficiency of TMT varies significantly at different targeting sites, which leads to a relatively low success rate. To further improve the success rate and gene-targeting efficiency of TMT, we constructed a Random Gene-targeting Plasmids Pool (RGPP) to analyze the sequence recognition preference of TMT. A new base pairing, located at the -8 site between EBS2/IBS2 and EBS1/IBS1 (named EBS2b-IBS2b), increased the success rate (2.45- to 5.07-fold) and significantly improved gene-targeting efficiency of TMT. A computer algorithm (TMT 1.0), based on the newly discovered sequence recognition roles, was also developed to facilitate the design of TMT gene-targeting primers. The present work could essentially expand the practicalities of TMT in the genome engineering of heat-tolerance mesophilic and thermophilic bacteria. The randomized base pairing in the interval of IBS2 and IBS1 of Tel3c/4c intron (-8 and -7 sites) in Thermotargetron (TMT) results in a low success rate and gene-targeting efficiency in bacteria. In the present work, we constructed a randomized gene-targeting plasmids pool (RGPP) to study whether there is a base preference in target sequences. Among all the successful "retrohoming" targets, we found that a new EBS2b-IBS2b base paring (A/T) significantly increased TMT's gene-targeting efficiency, and the concept is also applicable to other gene targets in redesigned gene-targeting plasmids pool in E. coli. The improved TMT is a promising tool for the genetic engineering of bacteria and could promote metabolic engineering and synthetic biology research in valuable microbes that recalcitrance for genetic manipulation.
嗜热II类内含子是一种由内含子RNA和内含子编码蛋白（IEP）组成的反转录转座子，它可以利用其基于核酶的新型DNA整合机制“反转归巢”来用于基因靶向。这一过程由核糖核蛋白（RNP）复合体介导，该复合体包含切除的内含子套索RNA和具有逆转录酶（RT）活性的IEP。RNP通过外显子结合序列2（EBS2）/内含子结合序列2（IBS2）、EBS1/IBS1和EBS3/IBS3碱基配对来识别靶向位点。此前，我们开发了TeI3c/4c内含子作为嗜热基因靶向系统——嗜热靶向子（TMT）。然而，我们发现TMT在不同靶向位点的靶向效率差异显著，这导致成功率相对较低。为了进一步提高TMT的成功率和基因靶向效率，我们构建了一个随机基因靶向质粒库（RGPP）来分析TMT的序列识别偏好。位于EBS2/IBS2和EBS1/IBS1之间-8位点的一种新的碱基配对（命名为EBS2b-IBS2b）提高了成功率（2.45至5.07倍），并显著提高了TMT的基因靶向效率。还开发了一种基于新发现的序列识别作用的计算机算法（TMT 1.0），以促进TMT基因靶向引物的设计。目前的工作基本上可以扩展TMT在耐热嗜温菌和嗜热菌基因组工程中的实用性。嗜热靶向子（TMT）中Tel3c/4c内含子的IBS2和IBS1区间（-8和-7位点）的随机碱基配对导致细菌中的成功率和基因靶向效率较低。在目前的工作中，我们构建了一个随机基因靶向质粒库（RGPP）来研究靶序列中是否存在碱基偏好。在所有成功的“反转归巢”靶点中，我们发现一种新的EBS2b-IBS2b碱基配对（A/T）显著提高了TMT的基因靶向效率，并且这一概念也适用于大肠杆菌中重新设计的基因靶向质粒库中的其他基因靶点。改进后的TMT是一种有前途的细菌基因工程工具，可以促进对遗传操作具有抗性的有价值微生物的代谢工程和合成生物学研究。
