Methylotuvimicrobium buryatense 5GB1C, a fast-growing gammaproteobacterial methanotroph, is equipped with two glycolytic pathways, the Entner-Doudoroff (ED) pathway and the Embden-Meyerhof-Parnas (EMP) pathway. Metabolic flux analysis and ¹³C-labeling experiments have shown the EMP pathway is the principal glycolytic route in M. buryatense 5GB1C, while the ED pathway appears to be metabolically and energetically insignificant. However, it has not been possible to obtain a null mutant in the edd-eda genes encoding the two unique enzymatic reactions in the ED pathway, suggesting the ED pathway may be essential for M. buryatense 5GB1C growth. In this study, the inducible P_BAD promoter was used to manipulate gene expression of edd-eda, and in addition, the expression of these two genes was separated from that of a downstream gltA gene. The resulting strain shows arabinose-dependent growth that correlates with ED pathway activity, with normal growth achieved in the presence of ∼0.1 g/liter arabinose. Flux balance analysis shows that M. buryatense 5GB1C with a strong ED pathway has a reduced energy budget, thereby limiting the mutant growth at a high concentration of arabinose. Collectively, our study demonstrates that the ED pathway is essential for M. buryatense 5GB1C. However, no known mechanism can directly explain the essentiality of the ED pathway, and thus, it may have a yet unknown regulatory role required for sustaining a healthy and functional metabolism in this bacterium.

中文翻译：

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Entner-Doudoroff途径是甲基蛇毒微藻5GB1C的重要代谢途径。

快速增长的γ-蛋白细菌甲烷营养生物伯氏隐球菌5GB1C具有两个糖酵解途径，即Entner-Doudoroff（ED）途径和Embden-Meyerhof-Parnas（EMP）途径。代谢通量分析和¹³ C标记实验表明，EMP途径是buryatense 5GB1C中主要的糖酵解途径，而ED途径在代谢和能量上似乎无关紧要。但是，在ED途径中编码两个独特的酶促反应的edd - eda基因中不可能获得无效突变体，这表明ED途径可能对伯氏杆菌5GB1C的生长至关重要。在这项研究中，诱导型P _BAD启动子用于操纵edd-eda的基因表达，此外，这两个基因的表达与下游gltA基因的表达分开。所得菌株显示出与ED途径活性相关的阿拉伯糖依赖性生长，在约0.1 g / L阿拉伯糖存在下可实现正常生长。助焊剂平衡分析表明，具有较强ED途径的伯氏疟原虫5GB1C的能量收支减少，从而限制了高浓度阿拉伯糖条件下突变体的生长。总体而言，我们的研究表明ED途径对于buryatense支原体至关重要5GB1C。但是，没有已知的机制可以直接解释ED途径的必要性，因此，在该细菌中维持健康和功能性代谢所需的调节作用可能还未知。