Sakai Miyabi, Shimosaka Taichi, Katsumata Kosuke, Yohda Masafumi, Narumi Issay
Department of Biotechnology and Life Science, Faculty of Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan.
Department of Life Sciences, Faculty of Life Sciences, Toyo University, Asaka, Japan.
Front Microbiol. 2024 May 28;15:1387296. doi: 10.3389/fmicb.2024.1387296. eCollection 2024.
spp. are known for their radiation resistance, toxic compound removal, and production of valuable substances. Therefore, developing gene expression systems for spp. is crucial in advancing genetic engineering applications. To date, plasmid vectors that express foreign genes in and have been limited to plasmid pI3 and its derivatives. In contrast, plasmid vectors that express foreign genes in include plasmid pZT23 and its derivatives. In this study, we developed a new system for the stable introduction and retention of expression plasmids for . Two cryptic plasmids were removed from the wild-type strain to generate the TY3 strain. We then constructed a shuttle vector plasmid, pGRC5, containing the replication initiation region of the smallest cryptic plasmid, pDEGR-3, replication initiation region of the vector, pACYC184, and an antibiotic resistance gene. We introduced pGRC5, pZT23-derived plasmid pZT29H, and pI3-derived plasmid pRADN8 into strain TY3, and found their coexistence in cells. The quantitative PCR assay results found that pGRC5, pZT29H, and pRADN8 had relative copy numbers of 11, 26, and 5 per genome, respectively. Furthermore, we developed a new plasmid in which the luciferase gene was controlled by the promoter region, which contained radiation-desiccation response operator sequences for DdrO, a stress response regulon repressor in , hence inducing gene expression via ultraviolet-C light irradiation. These plasmids are expected to facilitate the removal and production of toxic and valuable substances, in , respectively, particularly of those involving multiple genes.
某些物种以其抗辐射性、有毒化合物去除能力以及产生有价值物质的能力而闻名。因此，开发用于这些物种的基因表达系统对于推进基因工程应用至关重要。迄今为止，在某些物种中表达外源基因的质粒载体仅限于质粒pI3及其衍生物。相比之下，在某些物种中表达外源基因的质粒载体包括质粒pZT23及其衍生物。在本研究中，我们开发了一种新系统，用于稳定导入和保留用于某些物种的表达质粒。从野生型菌株中去除了两个隐蔽质粒以产生TY3菌株。然后，我们构建了一个穿梭载体质粒pGRC5，它包含最小隐蔽质粒pDEGR - 3的复制起始区域、载体pACYC184的复制起始区域以及一个抗生素抗性基因。我们将pGRC5、pZT23衍生的质粒pZT29H和pI3衍生的质粒pRADN8导入TY3菌株，并发现它们在某些细胞中共存。定量PCR分析结果发现，pGRC5、pZT29H和pRADN8在每个基因组中的相对拷贝数分别为11、26和5。此外，我们开发了一种新质粒，其中荧光素酶基因由启动子区域控制，该启动子区域包含某些物种的辐射干燥反应操纵序列DdrO，DdrO是某些物种中的一种应激反应调节子阻遏物，因此通过紫外线C光照射诱导基因表达。这些质粒有望分别促进某些物种中有毒物质的去除和有价值物质的产生，特别是那些涉及多个基因的物质。
