High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors
High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors
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DOI：
10.1099/mic.0.28084-0
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发表时间：
2005-07-01
期刊：
影响因子：
2.8
通讯作者：
Axelsson, L
中科院分区：
文献类型：
--
作者：
Sorvig, E;Mathiesen, G;Axelsson, L
已经开发了用于在清酒乳杆菌和植物乳杆菌中诱导基因表达的载体，其中感兴趣的基因的表达由来自在乳杆菌中发现的细菌素操纵子的强的、受调节的启动子驱动。清酒品系。这些启动子的活性通过双组分信号转导系统控制，该系统响应于外部添加的肽信息素。载体具有模块化设计，允许容易地交换所有必需元件：诱导型启动子、同源调控系统、感兴趣的基因、抗生素抗性标记和复制子。构建并测试了这些所谓的“pSIP”载体的各种变体，这些变体在衍生调节元件的细菌素调节子（sakacin A或sakacin P）、选自这些调节子的调节启动子和复制子（衍生自p256或pSH 71）方面不同。使用β-葡萄糖醛酸酶（GusA）和氨肽酶N（PepN）作为报告，它表明，最好的载体允许诱导，信息素剂量依赖性基因表达在非常高的水平，而显示中等的基础活动时，不诱导。最有效的设置是使用含有pSH 71复制子、来自sakacin P调节子的orfX启动子和同源调节基因的载体在L.清酒主人。用这种设置获得的GusA水平比用原型pSIP版本获得的水平高约10倍，而PepN水平几乎占总细胞蛋白的50%。
Vectors have been developed for inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum in which expression of the gene of interest is driven by strong, regulated promoters from bacteriocin operons found in L. sakei strains. The activity of these promoters is controlled via a two-component signal transduction system, which responds to an externally added peptide pheromone. The vectors have a modular design, permitting easy exchange of all essential elements: the inducible promoter, the cognate regulatory system, the gene of interest, the antibiotic resistance marker and the replicon. Various variants of these so-called 'pSIP' vectors were constructed and tested, differing in terms of the bacteriocin regulon from which the regulatory elements were derived (sakacin A or sakacin P), the regulated promoter selected from these regulons, and the replicon (derived from p256 or pSH71). Using beta-glucuronidase (GusA) and aminopeptidase N (PepN) as reporters, it was shown that the best vectors permitted inducible, pheromone-dose-dependent gene expression at very high levels, while displaying moderate basal activities when not induced. The most effective set-up was obtained using a vector containing the pSH71 replicon, the orfX promoter from the sakacin P regulon, and the cognate regulatory genes, in a L. sakei host. GusA levels obtained with this set-up were approximately ten times higher than the levels obtained with prototype pSIP versions, whereas PepN levels amounted to almost 50% of total cellular protein.