Abstract
SummaryPseudomonas 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.
Publisher
Cold Spring Harbor Laboratory