Engineering of Pseudomonas putida for accelerated co-utilization of glucose and cellobiose yields aerobic overproduction of pyruvate explained by an upgraded metabolic model
Abstract:Pseudomonas putida KT2440 is an attractive bacterial host for biotechnological production of valuable chemicals from renewable lignocellulosic feedstocks as it can valorize lignin-derived aromatics or cellulosic glucose. P. putida EM42, a genome-reduced variant of P. putida KT2440 endowed with advantageous physiological properties, was recently engineered for growth on cellobiose, a major cellooligosaccharide product of enzymatic cellulose hydrolysis. Co-utilization of cellobiose with glucose was achieved in a… Show more
“…Quantitative measurements of D-enrichment in downstream metabolites, e.g. intermediates from the tricarboxylic acid cycle and the PP pathway [91], can be also included to increase resolution and coverage. In this sense, adopting D-based isotopic tracers enables either elucidation of fluxes that could not be resolved with 13 C-substrates or it improves analytical accuracy.…”
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.
“…Quantitative measurements of D-enrichment in downstream metabolites, e.g. intermediates from the tricarboxylic acid cycle and the PP pathway [91], can be also included to increase resolution and coverage. In this sense, adopting D-based isotopic tracers enables either elucidation of fluxes that could not be resolved with 13 C-substrates or it improves analytical accuracy.…”
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.