pheSAG Based Rapid and Efficient Markerless Mutagenesis in Methylotuvimicrobium
pheSAG Based Rapid and Efficient Markerless Mutagenesis in Methylotuvimicrobium
复制标题
基于 pheS(AG) 的甲基微菌快速高效无标记诱变
DOI：
10.3389/fmicb.2020.00441
复制
发表时间：
2020-03-31
影响因子：
5.2
通讯作者：
Yan, Xin
中科院分区：
文献类型：
--
作者：
Liu, Yongchuang;He, Xiangrong;Yan, Xin
近年来，一些耐盐碱的甲烷氧化菌由于其生长速度快、生命力强，不仅成为甲烷转化的生物催化剂，而且成为研究甲烷氧化菌基础知识的有利材料。为了实现甲基化菌群基因组的无标记修饰，基于pheS构建了一个反选择标记（counterselectable marker， CSM），该标记编码苯丙酰trna合成酶的α亚基。将两个点突变（T252A和A306G）引入到methylotuvimicroium buryatense 5GB1C的PheS中，生成PheS(AG)，该PheS可以识别对氯苯丙氨酸（p-Cl-Phe）作为底物。从理论上讲，PheS（AG）在细胞中的表达会导致p-Cl-Phe与蛋白质结合，导致细胞死亡。利用mmoX的P-tac启动子和核糖体结合位点区域控制pheS(AG)，生成pheS(AG)-3 CSM。携带pheS(AG)-3的M. buryatense 5GB1C对0.5 mM p-Cl-Phe极为敏感。然后，将pheS(AG)-3与zeocin抗性基因结合，构建了长度仅为1.5 kb的正反选择盒PZ。利用PZ和pcr技术，在M. buryatense 5GB1C和Methylotuvimicrobium alcaliphilum 20Z中构建glgA1或整个smmo操纵子的无标记缺失。阳性检出率达92%以上，最快8天即可完成。
Due to their fast growth rate and robustness, some haloalkalitolerant methanotrophs from the genus Methylotuvimicrobium have recently become not only promising biocatalysts for methane conversion but also favorable materials for obtaining fundamental knowledge on methanotrophs. Here, to realize unmarked genome modification in Methylotuvimicrobium bacteria, a counterselectable marker (CSM) was developed based on pheS, which encodes the alpha-subunit of phenylalanyl-tRNA synthetase. Two-point mutations (T252A and A306G) were introduced into PheS in Methylotuvimicrobium buryatense 5GB1C, generating PheS(AG), which can recognize p-chloro-phenylalanine (p-Cl-Phe) as a substrate. Theoretically, the expression of PheS(AG) in a cell will result in the incorporation of p-Cl-Phe into proteins, leading to cell death. The P-tac promoter and the ribosome-binding site region of mmoX were employed to control pheS(AG), producing the pheS(AG)-3 CSM. M. buryatense 5GB1C harboring pheS(AG)-3 was extremely sensitive to 0.5 mM p-Cl-Phe. Then, a positive and counterselection cassette, PZ (only 1.5 kb in length), was constructed by combining pheS(AG)-3 and the zeocin resistance gene. A PZ- and PCR-based strategy was used to create the unmarked deletion of glgA1 or the whole smmo operon in M. buryatense 5GB1C and Methylotuvimicrobium alcaliphilum 20Z. The positive rates were over 92%, and the process could be accomplished in as few as eight days.