Engineering of core promoter regions enables the construction of constitutive and inducible promoters in Halomonas sp.
Engineering of core promoter regions enables the construction of constitutive and inducible promoters in Halomonas sp.
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DOI：
10.1002/biot.201400828
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发表时间：
2016-02-01
影响因子：
4.7
通讯作者：
Ouyang, Qi
中科院分区：
文献类型：
--
作者：
Li, Tingting;Li, Teng;Ouyang, Qi
盐单胞菌TD 01是一株新发现的嗜盐菌，是一种有前途的低成本化工生产宿主菌。然而，在盐单胞菌属物种的遗传操作仍然是困难的，由于缺乏良好的表征和可调的表达系统。本研究利用一种系统、高效的方法构建了组成型启动子文库和诱导型启动子。孔蛋白是盐单胞菌TD 01中高度表达的一种蛋白质，是首次从盐单胞菌TD 01蛋白质组中发现的。随后对孔蛋白上游基因间区的研究鉴定了核心启动子区，包括-10和-35元件。通过随机化-35和-10元件之间的序列，获得了转录活性具有310倍变化的组成型启动子文库;通过将lac操纵子整合到核心启动子区域中，构建了具有>200倍诱导的诱导型启动子。组成型启动子和诱导型启动子作为两个互补的表达系统，分别用于盐单胞菌TD 01中聚3-羟基丁酸酯（poly-3-hydroxybutyrate，PHB）的生物合成调控，证明了两个表达系统的有效性，为盐单胞菌TD菌株的代谢工程研究提供了新的思路，同时也为其他缺乏特征的菌株的研究提供了新的思路。
Halomonas strain TD01, a newly identified halophilic bacterium, has proven to be a promising low-cost host for the production of chemicals. However, genetic manipulation in Halomonas sp. is still difficult due to the lack of well-characterized and tunable expression systems. In this study, a systematic, efficient method was exploited to construct both a constitutive promoter library and inducible promoters. Porin, a highly expressed protein in Halomonas TD01, was first identified from the Halomonas TD01 proteome. Subsequent study of the intergenic region upstream of porin led to the identification of a core promoter region, including -10 and -35 elements. By randomizing the sequence between the -35 and -10 elements, a constitutive promoter library was obtained with 310-fold variation in transcriptional activity; an inducible promoter with a >200-fold induction was built by integrating a lac operator into the core promoter region. As two complementary expression systems, the constitutive and inducible promoters were then employed to regulate the biosynthetic pathway of poly-3-hydroxybutyrate (PHB) in Halomonas TD01, demonstrating the usefulness of the expression systems, furthermore, they could be applied in future metabolic engineering of Halomonas TD strains, and the systematic method used in this study can be generalized to other less-characterized bacterial strains.