Time-resolved, deuterium-based fluxomics uncovers the hierarchy and dynamics of sugar processing byPseudomonas putida

Summary Pseudomonas putida , a soil bacterium widely used for synthetic biology and metabolic engineering, processes glucose through convergent peripheral pathways that ultimately yield 6-phosphogluconate. Such a periplasmic gluconate shunt (PGS), composed by glucose and gluconate dehydrogenases, sequentially transforms glucose into gluconate and 2-ketogluconate. Although the secretion of these organic acids by P . putida has been extensively recognized, the mechanism and spatiotemporal regulation of the PGS remained elusive thus far. To address this challenge, we have developed a novel methodology for metabolic flux analysis, D-fluxomics , based on deuterated sugar substrates. D-Fluxomics demonstrated that the PGS underscores a highly dynamic metabolic architecture in glucose-dependent batch cultures of P . putida , characterized by hierarchical carbon uptake by the PGS throughout the cultivation. Additionally, we show that gluconate and 2-ketogluconate accumulation and consumption can be solely explained as a result of the interplay between growth rate-coupled and decoupled metabolic fluxes. As a consequence, the formation of these acids in the PGS is inversely correlated to the bacterial growth rate—unlike the widely studied overflow metabolism of Escherichia coli and yeast. Our findings, which underline survival strategies of soil bacteria thriving in their natural environments, open new avenues for engineering P . putida towards efficient, sugar-based bioprocesses.