Department of Chemical Engineering, University of Washington, Seattle, Washington, USA
Department of Chemical Engineering, University of Washington, Seattle, Washington, USA.
Appl Environ Microbiol. 2021 Jan 15;87(3). doi: 10.1128/AEM.02481-20.
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 C-labeling experiments have shown the EMP pathway is the principal glycolytic route in 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 - genes encoding the two unique enzymatic reactions in the ED pathway, suggesting the ED pathway may be essential for 5GB1C growth. In this study, the inducible P promoter was used to manipulate gene expression of , and in addition, the expression of these two genes was separated from that of a downstream 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 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 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. The gammaproteobacterial methanotrophs possess a unique central metabolic architecture where methane and other reduced C carbon sources are assimilated through the ribulose monophosphate cycle. Although efforts have been made to better understand methanotrophic metabolism in these bacteria via experimental and computational approaches, many questions remain unanswered. One of these is the essentiality of the ED pathway for 5GB1C, as current results appear contradictory. By creating a construct with and genes controlled by P and P , respectively, we demonstrated the essentiality of the ED pathway for this obligate methanotroph. It is also demonstrated that these genetic tools are applicable to 5GB1C and that expression of the target genes can be tightly controlled across an extensive range. Our study adds to the expanding knowledge of methanotrophic metabolism and practical approaches to genetic manipulation for obligate methanotrophs.
5GB1C 是一种快速生长的γ变形菌甲烷营养菌，配备有两条糖酵解途径，即 Entner-Doudoroff（ED）途径和 Embden-Meyerhof-Parnas（EMP）途径。代谢通量分析和 C 标记实验表明，EMP 途径是 5GB1C 的主要糖酵解途径，而 ED 途径在代谢和能量上似乎并不重要。然而，迄今为止，无法获得编码 ED 途径中两种独特酶促反应的基因的缺失突变体，这表明 ED 途径可能对 5GB1C 的生长是必不可少的。在这项研究中，诱导型 P r 启动子被用于操纵基因表达，此外，这两个基因的表达与下游基因分离。由此产生的菌株表现出依赖阿拉伯糖的生长，这与 ED 途径的活性相关联，在存在约 0.1 g/L 阿拉伯糖的情况下可实现正常生长。通量平衡分析表明，具有强 ED 途径的 5GB1C 具有降低的能量预算，从而限制了突变体在高浓度阿拉伯糖下的生长。总的来说，我们的研究表明 ED 途径对 5GB1C 是必不可少的。然而，没有已知的机制可以直接解释 ED 途径的必要性，因此，它可能具有未知的调节作用，对于维持这种细菌的健康和功能代谢是必需的。γ变形菌甲烷营养菌具有独特的中央代谢结构，其中甲烷和其他还原 C 碳源通过核酮糖单磷酸循环同化。尽管已经通过实验和计算方法努力更好地理解这些细菌中的甲烷营养代谢，但仍有许多问题尚未得到解答。其中之一是 ED 途径对 5GB1C 的必要性，因为目前的结果似乎相互矛盾。通过创建一个带有和基因的构建体，这些基因分别由 P r 和 P r 控制，我们证明了 ED 途径对这种严格的甲烷营养菌是必不可少的。还表明，这些遗传工具适用于 5GB1C，并且可以在广泛的范围内对靶基因的表达进行严格控制。我们的研究增加了对甲烷营养代谢的扩展知识和对严格的甲烷营养菌的遗传操作的实用方法。
