Development of a counterselectable seamless mutagenesis system in lactic acid bacteria.
Development of a counterselectable seamless mutagenesis system in lactic acid bacteria.
复制标题
乳酸菌可反选择无缝诱变系统的开发
DOI：
10.1186/s12934-017-0731-8
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发表时间：
2017-07-05
影响因子：
6.4
通讯作者：
Kong J
中科院分区：
文献类型：
--
作者：
Xin Y;Guo T;Mu Y;Kong J
乳酸菌（LAB）作为生产高价值代谢产物的细胞工厂受到越来越多的关注。然而，用于遗传修饰这些菌株的分子工具主要是基于载体的双交换策略，这是费力且低效的。为了解决这个问题，已经开发了几种反选择性标记，而其中很少有可以在没有预处理的情况下用于野生型宿主细胞。在乳酸乳球菌NZ 9000基因组中鉴定了编码苯丙氨酰-tRNA合成酶α亚基的pheS基因。当突变型pheS基因（pheS*）在Lc.乳酸菌NZ 9000的L-乳酸脱氢酶启动子（Pldh）在大肠杆菌中表达，获得了A312 G取代的PheS*，使细胞对苯丙氨酸类似物对氯苯丙氨酸（p-Cl-Phe）敏感。这一结果表明pheS* 适合作为Lc的反选择标记。乳酸。然而，来自染色体拷贝的pheS* 的表达水平太低而不能赋予p-Cl-Phe敏感性。因此，尝试了级联启动子的策略来增强pheS* 的表达水平。预期地，具有五个串联重复启动子Pldh的盒5 Pldh-pheS * 导致对15 mM p-Cl-Phe的敏感性。随后，在Lc中开发了基于含有5 Pldh-pheS * 盒的温度敏感性质粒pG+ host 9的反向选择性无缝诱变系统PheS*/pG+ host 9。乳酸。我们还证明了应用pheS* 作为干酪乳杆菌BL 23中的反向选择标记的可能性。如E.在大肠杆菌中，pheS* 作为反向选择标记已被证明在Lc中的靶基因缺失中是功能性的。lactis和L. casei。此外，PheS*/pG+ host 9反向选择性突变系统不需预处理，可用于野生型宿主细胞。
Lactic acid bacteria (LAB) are receiving more attention to act as cell factories for the production of high-value metabolites. However, the molecular tools for genetic modifying these strains are mainly vector-based double-crossover strategies, which are laborious and inefficient. To address this problem, several counterselectable markers have been developed, while few of them could be used in the wild-type host cells without pretreatment. The pheS gene encoding phenylalanyl-tRNA synthetase alpha subunit was identified in Lactococcus lactis NZ9000 genome. When mutant pheS gene (pheS*) under the control of the Lc. lactis NZ9000 l-lactate dehydrogenase promoter (Pldh) was expressed from a plasmid, the resulted PheS* with an A312G substitution rendered cells sensitive to the phenylalanine analog p-chloro-phenylalanine (p-Cl-Phe). This result suggested pheS* was suitable to be used as a counterselectable marker in Lc. lactis. However, the expression level of pheS* from a chromosomal copy was too low to confer p-Cl-Phe sensitivity. Therefore, a strategy of cascading promoters was attempted for strengthening the expression level of pheS*. Expectedly, a cassette 5Pldh-pheS* with five tandem repetitive promoters Pldh resulted in a sensitivity to 15 mM p-Cl-Phe. Subsequently, a counterselectable seamless mutagenesis system PheS*/pG+host9 based on a temperature-sensitive plasmid pG+host9 harboring a 5Pldh-pheS* cassette was developed in Lc. lactis. We also demonstrated the possibility of applying pheS* to be a counterselectable marker in Lactobacillus casei BL23. As reported in E. coli, pheS* as a counterselectable marker has been demonstrated to be functional in targeted gene(s) deletion in Lc. lactis as well as in L. casei. Moreover, the efficiency and timesaving counterselectable seamless mutagenesis system PheS*/pG+host9 could be used in the wild-type host cells without pretreatment.