Although high hydrostatic pressure (HHP) is an interesting parameter to be applied in bioprocessing, its potential is currently limited by the lack of bacterial chassis capable of surviving and maintaining homeostasis under pressure. While several efforts have been made to genetically engineer microorganisms able to grow at sublethal pressures, there is little information for designing backgrounds that survive more extreme pressures. In this investigation, we analyzed the genome of an extreme HHP-resistant mutant of E. coli MG1655 (designated as DVL1), from which we identified four mutations (in the cra, cyaA, aceA and rpoD loci) causally linked to increased HHP resistance. Analysing the functional effect of these mutations we found that the coupled effect of downregulation of cAMP/CRP, Cra and the glyoxylate shunt activity, together with the upregulation of RpoH and RpoS activity, could mechanistically explain the increased HHP resistance of the mutant. Using combinations of three mutations, we could synthetically engineer E. coli strains able to comfortably survive pressures of 600–800 MPa, which could serve as genetic backgrounds for HHP-based biotechnological applications.

尽管高静水压 (HHP) 是一个有趣的生物加工参数，但其潜力目前受到缺乏能够在压力下生存和维持体内平衡的细菌底盘的限制。虽然已经做出了一些努力来对能够在亚致死压力下生长的微生物进行基因工程改造，但几乎没有信息可以设计在更极端压力下存活的背景。在这项研究中，我们分析了大肠杆菌MG1655的极端 HHP 抗性突变体（指定为 DVL1）的基因组，从中我们确定了四个突变（在cra、cyaA、aceA和rpoD位点）与 HHP 抗性增加有因果关系。分析这些突变的功能影响，我们发现 cAMP/CRP、Cra 和乙醛酸分流活性下调的耦合效应，以及 RpoH 和 RpoS 活性的上调，可以从机制上解释突变体的 HHP 抗性增加。使用三个突变的组合，我们可以合成工程大肠杆菌菌株能够在 600-800 MPa 的压力下舒适地存活，这可以作为基于 HHP 的生物技术应用的遗传背景。