Hobmeier Karina, Goëss Marie C, Sehr Christiana, Schwaminger Sebastian, Berensmeier Sonja, Kremling Andreas, Kunte Hans Jörg, Pflüger-Grau Katharina, Marin-Sanguino Alberto
Professorship for Systems Biotechnology, Technical University of Munich, Munich, Germany.
Bioseparation Engineering Group, Department of Mechanical Engineering, Technical University of Munich, Munich, Germany.
Front Microbiol. 2020 Sep 25;11:561800. doi: 10.3389/fmicb.2020.561800. eCollection 2020.
Salt tolerance in the γ-proteobacterium is linked to its ability to produce the compatible solute ectoine. The metabolism of ectoine production is of great interest since it can shed light on the biochemical basis of halotolerance as well as pave the way for the improvement of the biotechnological production of such compatible solute. Ectoine belongs to the biosynthetic family of aspartate-derived amino-acids. Aspartate is formed from oxaloacetate, thereby connecting ectoine production to the anaplerotic reactions that refill carbon into the tricarboxylic acid cycle (TCA cycle). This places a high demand on these reactions and creates the need to regulate them not only in response to growth but also in response to extracellular salt concentration. In this work, we combine modeling and experiments to analyze how these different needs shape the anaplerotic reactions in . First, the stoichiometric and thermodynamic factors that condition the flux distributions are analyzed, then the optimal patterns of operation for oxaloacetate production are calculated. Finally, the phenotype of two deletion mutants lacking potentially relevant anaplerotic enzymes: phosphoenolpyruvate carboxylase (Ppc) and oxaloacetate decarboxylase (Oad) are experimentally characterized. The results show that the anaplerotic reactions in are indeed subject to evolutionary pressures that differ from those faced by other gram-negative bacteria. Ectoine producing halophiles must meet a higher metabolic demand for oxaloacetate and the reliance of many marine bacteria on the Entner-Doudoroff pathway compromises the anaplerotic efficiency of Ppc, which is usually one of the main enzymes fulfilling this role. The anaplerotic flux in is contributed not only by Ppc but also by Oad, an enzyme that has not yet been shown to play this role . Ppc is necessary for to grow normally at low salt concentrations but it is not required to achieve near maximal growth rates as long as there is a steep sodium gradient. On the other hand, the lack of Oad presents serious difficulties to grow at high salt concentrations. This points to a shared role of these two enzymes in guaranteeing the supply of oxaloacetate for biosynthetic reactions.
γ-变形菌中的耐盐性与其产生相容性溶质四氢嘧啶的能力有关。四氢嘧啶的代谢备受关注，因为它能揭示耐盐性的生化基础，也为改善这种相容性溶质的生物技术生产铺平道路。四氢嘧啶属于天冬氨酸衍生氨基酸的生物合成家族。天冬氨酸由草酰乙酸形成，从而将四氢嘧啶的产生与回补反应联系起来，这些反应将碳重新补充到三羧酸循环（TCA循环）中。这对这些反应提出了很高的要求，不仅需要根据生长情况进行调节，还需要根据细胞外盐浓度进行调节。在这项工作中，我们结合建模和实验来分析这些不同需求如何塑造[细菌名称未给出]中的回补反应。首先，分析影响通量分布的化学计量和热力学因素，然后计算草酰乙酸产生的最佳操作模式。最后，通过实验表征了两个缺失可能相关的回补酶的缺失突变体的表型：磷酸烯醇丙酮酸羧化酶（Ppc）和草酰乙酸脱羧酶（Oad）。结果表明，[细菌名称未给出]中的回补反应确实受到与其他革兰氏阴性菌不同的进化压力。产生四氢嘧啶的嗜盐菌必须满足对草酰乙酸更高的代谢需求，并且许多海洋细菌对Entner-Doudoroff途径的依赖损害了Ppc的回补效率，而Ppc通常是履行这一作用的主要酶之一。[细菌名称未给出]中的回补通量不仅由Ppc贡献还由Oad贡献，Oad这种酶尚未被证明发挥这一作用。Ppc是[细菌名称未给出]在低盐浓度下正常生长所必需的，但只要存在陡峭的钠梯度，实现接近最大生长速率则不需要它。另一方面，缺乏Oad会给在高盐浓度下生长带来严重困难。这表明这两种酶在保证为生物合成反应提供草酰乙酸方面具有共同作用。
