Institute of Systems Biotechnology, Saarland University, Campus A 1.5, 66123 Saarbrücken, Germany.
Institute of Systems Biotechnology, Saarland University, Campus A 1.5, 66123 Saarbrücken, Germany.
Metab Eng. 2019 Jul;54:35-53. doi: 10.1016/j.ymben.2019.01.008. Epub 2019 Mar 1.
The genus Pseudomonas comprises approximately 200 species with numerous isolates that are common inhabitants of soil, water, and vegetation and has been of particular interest for more than one hundred years. Here, we present a novel approach for accurate, precise and convenient C metabolic flux analysis of these and other microbes possessing periplasmic glucose oxidation and a cyclic hexose metabolism, which forms the recently discovered EDEMP cycle. This complex cyclic architecture cannot be resolved by common metabolic flux workflows, which rely on GC-MS-based labelling analysis of proteinogenic amino acids. Computational analyses revealed that this limitation can be overcome by three parallel labelling experiments on specific tracers, i.e., [1-C], [6-C] and 50% [C] glucose, with additional consideration of labelling information from glucose and glucosamine. Glucose and glucosamine display building blocks from cellular glycogen, peptidoglycan and lipopolysaccharides, reflect the pools of glucose6-phosphate and fructose6-phosphate in the heart of the EDEMP cycle and as we show, can be precisely assessed in biomass hydrolysates by GC-MS. The developed setup created 534 mass isotopomers and enabled high-resolution flux analysis of the cell factory Pseudomonas putida KT2440 and the human pathogen P. aeruginosa PAO1. The latter strain oxidized approximately 90% of its glucose into gluconate via the periplasmic route, whereas only a small fraction of substrate was phosphorylated and consumed via the cytoplasmic route. The oxidative pentose phosphate pathway was completely inactive, indicating the essentiality of the Entner-Doudoroff pathway and recycling of triose units into anabolic precursors. In addition to pseudomonads, many microbes operate a cyclic hexose metabolism, which becomes more accessible to flux analysis with this approach. In this regard, the presented approach displays a valuable extension of the available set of flux methods for these types of bacteria.
假单胞菌属包含大约 200 个种，其中许多分离株是土壤、水和植被中的常见居民，并且已经引起了一百多年的关注。在这里，我们提出了一种新的方法，用于对这些和其他具有周质葡萄糖氧化和环状己糖代谢的微生物进行准确、精确和方便的 C 代谢通量分析，该方法形成了最近发现的 EDEMP 循环。这种复杂的循环结构无法通过依赖于基于 GC-MS 的蛋白氨基酸标记分析的常见代谢通量工作流程来解决。计算分析表明，通过对特定示踪剂（即 [1-C]、[6-C] 和 50%[C]葡萄糖）进行三个平行的标记实验，并额外考虑来自葡萄糖和葡糖胺的标记信息，可以克服这一限制。葡萄糖和葡糖胺显示来自细胞糖原、肽聚糖和脂多糖的构建块，反映了 EDEMP 循环核心中的葡萄糖 6-磷酸和果糖 6-磷酸池，并且正如我们所展示的，可以通过 GC-MS 精确评估生物质水解物中的这些物质。所开发的设置创建了 534 个质量同位素，并且能够对细胞工厂 Pseudomonas putida KT2440 和人类病原体 P. aeruginosa PAO1 进行高分辨率通量分析。后者菌株通过周质途径将大约 90%的葡萄糖氧化为葡萄糖酸，而只有一小部分底物通过细胞质途径被磷酸化和消耗。氧化戊糖磷酸途径完全不活跃，表明 Entner-Doudoroff 途径的必要性以及三碳单位循环为合成前体提供物质。除了假单胞菌之外，许多微生物还运行环状己糖代谢，而这种方法使通量分析更容易进行。在这方面，所提出的方法是对这些类型细菌的现有通量方法集的有价值扩展。
