Department of Biology, Laboratory for Microbiology, Philipps University Marburg, Marburg, Germany.
LOEWE Center for Synthetic Microbiology, Philipps University Marburg, Marburg, Germany.
Appl Environ Microbiol. 2018 Jan 2;84(2). doi: 10.1128/AEM.01772-17. Print 2018 Jan 15.
Ectoine and hydroxyectoine are widely synthesized by members of the and a few members of the as potent osmostress protectants. We have studied the salient features of the osmostress-responsive promoter directing the transcription of the ectoine/hydroxyectoine biosynthetic gene cluster from the plant-root-associated bacterium by transferring it into , an enterobacterium that does not produce ectoines naturally. Using reporter fusions, we found that the heterologous promoter reacted with exquisite sensitivity in its transcriptional profile to graded increases in sustained high salinity, responded to a true osmotic signal, and required the buildup of an osmotically effective gradient across the cytoplasmic membrane for its induction. The involvement of the -10, -35, and spacer regions of the sigma-70-type promoter in setting promoter strength and response to osmotic stress was assessed through site-directed mutagenesis. Moderate changes in the promoter sequence that increase its resemblance to housekeeping sigma-70-type promoters of afforded substantially enhanced expression, both in the absence and in the presence of osmotic stress. Building on this set of promoter mutants, we engineered an chassis strain for the heterologous production of ectoines. This synthetic cell factory lacks the genes for the osmostress-responsive synthesis of trehalose and the compatible solute importers ProP and ProU, and it continuously excretes ectoines into the growth medium. By combining appropriate host strains and different plasmid variants, excretion of ectoine, hydroxyectoine, or a mixture of both compounds was achieved under mild osmotic stress conditions. Ectoines are compatible solutes, organic osmolytes that are used by microorganisms to fend off the negative consequences of high environmental osmolarity on cellular physiology. An understanding of the salient features of osmostress-responsive promoters directing the expression of the ectoine/hydroxyectoine biosynthetic gene clusters is lacking. We exploited the promoter from an ectoine/hydroxyectoine-producing soil bacterium for such a study by transferring it into a surrogate bacterial host. Despite the fact that does not synthesize ectoines naturally, the promoter retained its exquisitely sensitive osmotic control, indicating that osmoregulation of transcription is an inherent feature of the promoter and its flanking sequences. These sequences were narrowed to a 116-bp DNA fragment. Ectoines have interesting commercial applications. Building on data from a site-directed mutagenesis study of the promoter, we designed a synthetic cell factory that secretes ectoine, hydroxyectoine, or a mixture of both compounds into the growth medium.
海藻糖和羟基海藻糖是广泛由 和少数 的成员合成的，作为有效的渗透压保护剂。我们研究了从植物根相关细菌 中指导海藻糖/羟基海藻糖生物合成基因簇转录的渗透压响应启动子的显著特征，通过将其转移到 中，一种天然不产生海藻糖的肠杆菌。使用 报告基因融合，我们发现异源 启动子在其转录谱中对持续高盐度的细微变化表现出高度敏感的反应，对真正的渗透信号作出反应，并需要在细胞质膜上建立渗透有效的梯度才能诱导。通过定点突变评估了 -10、-35 和间隔区在设定启动子强度和对渗透压胁迫的反应中的作用。适度改变类似于 的管家型 -70 型启动子的 启动子序列，可大大增强其表达，无论是在没有渗透压胁迫的情况下还是在有渗透压胁迫的情况下。基于这组 启动子突变体，我们设计了一种 底盘菌株，用于海藻糖的异源生产。这种合成细胞工厂缺乏渗透压响应型合成海藻糖的基因和相容性溶质转运蛋白 ProP 和 ProU，并且不断将海藻糖分泌到生长培养基中。通过结合适当的宿主菌株和不同的质粒变体，在温和的渗透压胁迫条件下，可实现海藻糖、羟基海藻糖或两者混合物的分泌。海藻糖是相容性溶质，是微生物用来抵御高环境渗透压对细胞生理负面影响的有机渗透物。缺乏指导海藻糖/羟基海藻糖生物合成基因簇表达的渗透压响应启动子的显著特征。我们利用一种从产海藻糖/羟基海藻糖的土壤细菌中获得的 启动子进行了这样的研究，将其转移到一个替代的细菌宿主中。尽管 天然不合成海藻糖，但 启动子保留了其极其敏感的渗透压控制，表明 转录的渗透压调节是启动子及其侧翼序列的固有特征。这些序列被缩小到 116 个碱基对的 DNA 片段。海藻糖具有有趣的商业应用。基于 启动子的定点突变研究数据，我们设计了一种合成细胞工厂，将海藻糖、羟基海藻糖或两者混合物分泌到生长培养基中。
