Department of Chemical and Biomolecular Engineering, Metabolic Engineering and Systems Biology Laboratory, University of Delaware, Newark, DE 19716, USA.
Department of Chemical and Biomolecular Engineering, Metabolic Engineering and Systems Biology Laboratory, University of Delaware, Newark, DE 19716, USA.
Metab Eng. 2017 Nov;44:182-190. doi: 10.1016/j.ymben.2017.10.007. Epub 2017 Oct 14.
Thermophilic organisms are being increasingly investigated and applied in metabolic engineering and biotechnology. The distinct metabolic and physiological characteristics of thermophiles, including broad substrate range and high uptake rates, coupled with recent advances in genetic tool development, present unique opportunities for strain engineering. However, poor understanding of the cellular physiology and metabolism of thermophiles has limited the application of systems biology and metabolic engineering tools to these organisms. To address this concern, we applied high resolution C metabolic flux analysis to quantify fluxes for three divergent extremely thermophilic bacteria from separate phyla: Geobacillus sp. LC300, Thermus thermophilus HB8, and Rhodothermus marinus DSM 4252. We performed 18 parallel labeling experiments, using all singly labeled glucose tracers for each strain, reconstructed and validated metabolic network models, measured biomass composition, and quantified precise metabolic fluxes for each organism. In the process, we resolved many uncertainties regarding gaps in pathway reconstructions and elucidated how these organisms maintain redox balance and generate energy. Overall, we found that the metabolisms of the three thermophiles were highly distinct, suggesting that adaptation to growth at high temperatures did not favor any particular set of metabolic pathways. All three strains relied heavily on glycolysis and TCA cycle to generate key cellular precursors and cofactors. None of the investigated organisms utilized the Entner-Doudoroff pathway and only one strain had an active oxidative pentose phosphate pathway. Taken together, the results from this study provide a solid foundation for future model building and engineering efforts with these and related thermophiles.
嗜热微生物在代谢工程和生物技术中得到了越来越多的研究和应用。嗜热微生物独特的代谢和生理特性，包括广泛的底物范围和高摄取率，加上遗传工具开发的最新进展，为菌株工程带来了独特的机会。然而，对嗜热微生物细胞生理学和代谢的理解不足，限制了系统生物学和代谢工程工具在这些生物体中的应用。为了解决这个问题，我们应用高分辨率 C 代谢通量分析来量化来自不同门的三个不同的极端嗜热细菌的通量：Geobacillus sp. LC300、Thermus thermophilus HB8 和 Rhodothermus marinus DSM 4252。我们进行了 18 个平行标记实验，使用每个菌株的所有单标记葡萄糖示踪剂，重建和验证代谢网络模型，测量生物质组成，并量化每个生物体的精确代谢通量。在此过程中，我们解决了许多关于途径重建中的空白的不确定性，并阐明了这些生物体如何维持氧化还原平衡和产生能量。总的来说，我们发现这三种嗜热微生物的代谢非常不同，这表明适应高温生长并没有有利于任何特定的代谢途径集。所有三种菌株都严重依赖糖酵解和三羧酸循环来产生关键的细胞前体和辅因子。没有一种被研究的生物利用 Entner-Doudoroff 途径，只有一种菌株具有活跃的氧化戊糖磷酸途径。总之，这项研究的结果为未来与这些和相关嗜热微生物的模型构建和工程努力提供了坚实的基础。
