Compatible solutes are key for the ability of halophilic bacteria to resist high osmotic stress. They have received wide attention from researchers for their excellent osmotic protection properties. Hydroxyectoine is a particularly important compatible solute, but its production by microbes faces several challenges, including low titer/yield, the presence of the byproduct ectoine, and the requirement of high salinity. Here, we aimed to metabolically engineer Escherichia coli to efficiently produce hydroxyectoine in the absence of osmotic stress without accumulating the byproduct ectoine. First, combinatorial optimization of the expression strength of key genes in the ectoine synthesis module and hydroxyectoine synthesis module was conducted. After optimization of the expression of these genes, 12.12 g/L hydroxyectoine and 0.24 g/L ectoine were obtained at 36 h in shake-flask fermentation with the addition of the co-substrate α-ketoglutarate. Further optimization of the addition of α-ketoglutarate achieved the sole production of hydroxyectoine (i.e., no ectoine accumulation), indicating that the supply of α-ketoglutarate is critically important for sole hydroxyectoine production. Finally, quorum sensing-based auto-regulation of intracellular α-ketoglutarate pool was implemented as an alternative to α-ketoglutarate addition by coupling the expression of sucA with the esaI/esaR circuit, which led to 14.93 g/L hydroxyectoine with a unit cell yield of 1.678 g/g and no ectoine accumulation in the absence of osmotic stress. This is the highest reported titer of sole hydroxyectoine production under salinity-free fermentation to date.

中文翻译：

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大肠杆菌的代谢工程用于高效无渗透压力生产相容的溶质羟基四氢嘧啶

相容的溶质是嗜盐细菌抵抗高渗透压力能力的关键。它们因其出色的渗透保护特性而受到研究人员的广泛关注。羟基四氢嘧啶是一种特别重要的相容性溶质，但其通过微生物生产面临几个挑战，包括低滴度/产量、副产物四氢嘧啶的存在以及对高盐度的要求。在这里，我们的目标是对大肠杆菌进行代谢工程改造在没有渗透压力的情况下有效地生产羟基四氢嘧啶，而不会积累副产物四氢嘧啶。首先，对四氢嘧啶合成模块和羟基四氢嘧啶合成模块中关键基因的表达强度进行组合优化。优化这些基因的表达后，加入共底物α-酮戊二酸，在摇瓶发酵36 h时分别获得12.12 g/L的羟基四氢嘧啶和0.24 g/L的四氢嘧啶。进一步优化α-酮戊二酸的添加实现了羟基四氢嘧啶的单独生产（即没有四氢嘧啶的积累），表明α-酮戊二酸的供应对于单独的羟基四氢嘧啶生产至关重要。最后，sucA与esaI / esaR电路，产生 14.93 g/L 羟基四氢嘧啶，单胞产量为 1.678 g/g，并且在没有渗透应力的情况下没有四氢嘧啶积累。这是迄今为止在无盐发酵下唯一报道的羟基四氢嘧啶生产的最高滴度。