Abstract：
Lactic acid bacteria (LAB) are important for many biotechnological applications such as bioproduction and engineered probiotics for therapy. Inducible promoters are key gene expression control elements, yet those available in LAB are mainly based on bacteriocin systems and have many drawbacks, including large gene clusters, costly inducer peptides, and little portability to in vivo settings. Using Lactobacillus gasseri, a model commensal bacteria from the human gut, we report the engineering of synthetic LactoSpanks promoters (Pls), a collection of variable strength inducible promoters controlled by the LacI repressor from E. coli and induced by isopropyl β-d-1-thiogalactopyranoside (IPTG). We first show that the Phyper-spank promoter from Bacillus subtilis is functional in L. gasseri, albeit with substantial leakage. We then construct and screen a semirational library of Phyper-spank variants to select a set of four IPTG-inducible promoters that span a range of expression levels and exhibit reduced leakages and operational dynamic ranges (from ca. 9 to 28 fold-change). With their low genetic footprint and simplicity of use, LactoSpanks will support many applications in L. gasseri, and potentially other lactic acid and Gram-positive bacteria.
摘要：
乳酸菌（LAB）对于许多生物技术应用（例如生物生产和用于治疗的工程益生菌）很重要。诱导型启动子是关键的基因表达控制元件,然而，LAB中可用的细菌素系统主要基于细菌素系统，并且有许多缺点，包括大型基因簇，昂贵的诱导肽，和很少的便携性在体内设置。使用gasseri乳杆菌，来自人类肠道的共生细菌模型，我们报道了合成LactoSpanks启动子(Pls)的工程，由来自大肠杆菌的LacI阻遏物控制并由异丙基β-d-1-硫代吡喃半乳糖苷(IPTG)诱导的可变强度诱导型启动子的集合。我们首先表明，来自枯草芽孢杆菌的Phyper-spank启动子在L.gasseri中具有功能，尽管有大量泄漏。然后，我们构建并筛选了Phyper-spank变体的半理性文库，以选择一组四个IPTG诱导型启动子，这些启动子跨越一系列表达水平并表现出减少的泄漏和操作动态范围（从大约9至28倍变化)。由于其遗传足迹低和使用简单，LactoSpanks将支持L.gasseri的许多应用，以及潜在的其他乳酸和革兰氏阳性菌。
