BACKGROUND The obligate aerobe Azotobacter vinelandii is a model organism for the study of biological nitrogen fixation (BNF). This bacterium regulates the process of BNF through the two component NifL and NifA system, where NifA acts as an activator, while NifL acts as an anti-activator based on various metabolic signals within the cell. Disruption of the nifL component in the nifLA operon in a precise manner results in a deregulated phenotype that produces levels of ammonium that far surpass the requirements within the cell, and results in the release of up to 30 mM of ammonium into the growth medium. While many studies have probed the factors affecting growth of A. vinelandii, the features important to maximizing this high-ammonium-releasing phenotype have not been fully investigated. RESULTS In this work, we report the effect of temperature, medium composition, and oxygen requirements on sustaining and maximizing elevated levels of ammonium production from a nitrogenase deregulated strain. We further investigated several pathways, including ammonium uptake through the transporter AmtB, which could limit yields through energy loss or futile recycling steps. Following optimization, we compared sugar consumption and ammonium production, to attain correlations and energy requirements to drive this process in vivo. Ammonium yields indicate that between 5 and 8% of cellular protein is fully active nitrogenase MoFe protein (NifDK) under these conditions. CONCLUSIONS These findings provide important process optimization parameters, and illustrate that further improvements to this phenotype can be accomplished by eliminating futile cycles.

中文翻译：

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影响为生物固氮放松管制的葡萄固氮菌菌株生产铵的关键因素。

背景技术专性的厌氧性拟南芥（Azotobacter vinelandii）是用于研究生物固氮（BNF）的模型生物。该细菌通过NifL和NifA两部分系统调节BNF的过程，其中NifA充当激活剂，而NifL基于细胞内各种代谢信号充当抗激活剂。以精确的方式破坏nifLA操纵子中的nifL成分会导致表型失控，产生的铵水平远远超过细胞内的需求，并导致向生长培养基释放高达30 mM的铵。虽然许多研究已经探究了影响葡萄曲霉生长的因素，但尚未充分研究最大化这种高铵态释放表型的重要特征。结果在这项工作中，我们报告了温度的影响，中等的成分，以及维持和最大程度提高氮酶失活菌株产生的氨的水平所需的氧气。我们进一步研究了几种途径，包括通过转运蛋白AmtB吸收铵盐，这些途径可能通过能量损失或徒劳的回收步骤限制产量。经过优化后，我们比较了糖的消耗和铵的产生，以获得相关性和能量需求以在体内驱动该过程。铵的产量表明，在这些条件下，细胞蛋白中有5％至8％是完全活性的固氮酶MoFe蛋白（NifDK）。结论这些发现提供了重要的工艺优化参数，并说明通过消除无效周期可以进一步改善该表型。以及维持和最大程度提高氮酶失活菌株产生的氨的水平所需的氧气。我们进一步研究了几种途径，包括通过转运蛋白AmtB吸收铵盐，这些途径可能通过能量损失或徒劳的回收步骤限制产量。经过优化后，我们比较了糖的消耗和铵的产生，以获得相关性和能量需求以在体内驱动该过程。铵的产量表明，在这些条件下，细胞蛋白中有5％至8％是完全活性的固氮酶MoFe蛋白（NifDK）。结论这些发现提供了重要的工艺优化参数，并说明通过消除无效周期可以进一步改善该表型。以及维持和最大程度提高氮酶失活菌株产生的氨的水平所需的氧气。我们进一步研究了几种途径，包括通过转运蛋白AmtB吸收铵盐，这可能通过能量损失或徒劳的回收步骤限制产量。经过优化后，我们比较了糖的消耗和铵的产生，以获得相关性和能量需求以在体内驱动该过程。铵的产量表明，在这些条件下，细胞蛋白中有5％至8％是完全活性的固氮酶MoFe蛋白（NifDK）。结论这些发现提供了重要的工艺优化参数，并说明通过消除无效周期可以进一步改善该表型。