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

Abstract

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 byP.putidahas 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 ofP.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 ofEscherichia coliand yeast. Our findings, which underline survival strategies of soil bacteria thriving in their natural environments, open new avenues for engineeringP.putidatowards efficient, sugar-based bioprocesses.