GC-MS-based 13C metabolic flux analysis resolves the parallel and cyclic glucose metabolism of Pseudomonas putida KT2440 and Pseudomonas aeruginosa PAO1

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 13 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– 13 C], [6– 13 C] and 50% [ 13 C 6 ] 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.