Tinkering with Osmotically Controlled Transcription Allows Enhanced Production and Excretion of Ectoine and Hydroxyectoine from a Microbial Cell Factory
Tinkering with Osmotically Controlled Transcription Allows Enhanced Production and Excretion of Ectoine and Hydroxyectoine from a Microbial Cell Factory
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DOI：
10.1128/aem.01772-17
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发表时间：
2018-01-01
影响因子：
4.4
通讯作者：
Bremer, Erhard
中科院分区：
文献类型：
--
作者：
Czech, Laura;Poehl, Sebastian;Bremer, Erhard
Ectoine和hydroxytocoine广泛地由细菌的成员和一些细菌属的成员合成，作为有效的植物胁迫保护剂。我们已经研究了显着的特点，植物根相关的细菌假单胞菌stutzeri的ectoine/hydroxyectoine生物合成基因簇的转录，通过将其转移到大肠杆菌，肠杆菌，不产生ectoines自然的逆境响应启动子指导。使用ectlacZ报告融合，我们发现，异源的ect启动子的反应与精致的敏感性，在其转录谱中的梯度增加持续高盐度，响应于一个真正的渗透信号，并需要建立一个有效的梯度，其诱导跨细胞质膜。通过定点诱变评估了sigma-70型ect启动子的-10、-35和间隔区在设定启动子强度和对渗透胁迫的响应中的参与。ECT启动子序列的适度变化增加了其与E.在渗透胁迫存在和不存在的情况下，大肠杆菌的表达均得到显著增强。基于这组ect启动子突变体，我们设计了一个E。大肠杆菌底盘菌株用于异源生产四氢嘧啶。这种合成细胞工厂缺乏海藻糖的抗应激反应合成基因和相容的溶质进口商ProP和ProU，并且它不断地将四氢嘧啶排泄到生长培养基中。通过结合适当的宿主菌株和不同的质粒变体，排泄的四氢嘧啶，羟基四氢嘧啶，或两种化合物的混合物在温和的渗透压stress conditions.Importance Ectoines是相容的溶质，有机渗透压被微生物用来抵御高环境渗透压对细胞生理的负面影响。缺乏对指导四氢嘧啶/羟基四氢嘧啶生物合成基因簇表达的胁迫响应启动子的显著特征的理解。我们利用ect启动子从四氢嘧啶/羟基四氢嘧啶生产土壤细菌这样的研究，通过将其转移到一个替代细菌宿主。尽管E.大肠杆菌不能天然合成四氢嘧啶，但ECT启动子保留了其非常敏感的渗透控制，表明ECT转录的非线性调节是启动子及其侧翼序列的固有特征。这些序列被缩小到一个116 bp的DNA片段。Ectoines具有有趣的商业应用。基于ect启动子定点突变研究的数据，我们设计了一个合成细胞工厂，可以分泌四氢嘧啶、羟基四氢嘧啶或两种化合物的混合物到生长培养基中。
Ectoine and hydroxyectoine are widely synthesized by members of the Bacteria and a few members of the Archaea 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 Pseudomonas stutzeri by transferring it into Escherichia coli, an enterobacterium that does not produce ectoines naturally. Using ectlacZ reporter fusions, we found that the heterologous ect 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 ect promoter in setting promoter strength and response to osmotic stress was assessed through site-directed mutagenesis. Moderate changes in the ect promoter sequence that increase its resemblance to housekeeping sigma-70-type promoters of E. coli afforded substantially enhanced expression, both in the absence and in the presence of osmotic stress. Building on this set of ect promoter mutants, we engineered an E. coli 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.IMPORTANCE 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 ect promoter from an ectoine/hydroxyectoine-producing soil bacterium for such a study by transferring it into a surrogate bacterial host. Despite the fact that E. coli does not synthesize ectoines naturally, the ect promoter retained its exquisitely sensitive osmotic control, indicating that osmoregulation of ect 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 ect promoter, we designed a synthetic cell factory that secretes ectoine, hydroxyectoine, or a mixture of both compounds into the growth medium.