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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 mutant lacking periplasmic glucose dehydrogenase Gcd (PP_1444). However, the cause of the observed co-utilization was not understood and the Δgcd strain suffered from a significant growth defect. In this study, we aimed to investigate the basis of the simultaneous uptake of the two sugars and accelerate the growth of P. putida EM42 Δgcd mutant for the bioproduction of valuable compounds from glucose and cellobiose. We show that the gcd deletion abolished the inhibition of the exogenous β-glucosidase BglC from Thermobifida fusca by the intermediates of the periplasmic glucose oxidation pathway. The additional deletion of the hexR gene, which encodes a repressor of the upper glycolysis genes, failed to restore the rapid growth on glucose. The reduced growth rate of the Δgcd mutant was partially compensated by the implantation of heterologous glucose (Glf from Zymomonas mobilis) and cellobiose (LacY from Escherichia coli) transporters. Remarkably, this intervention resulted in the accumulation of pyruvate in aerobic P. putida cultures. We demonstrated that the excess of this key metabolic intermediate can be redirected to the enhanced biosynthesis of ethanol and lactate. The overproduction of pyruvate was then unveiled by an upgraded genome-scale metabolic model constrained with proteomic and kinetic data. The model pointed to the saturation of glucose catabolism enzymes due to unregulated substrate uptake and it predicted improved bioproduction of pyruvate-derived chemicals by the engineered strain. This work sheds light on the co-metabolism of cellulosic sugars in an attractive biotechnological host and introduces a novel strategy for pyruvate overproduction in bacterial cultures under aerobic conditions.HighlightsCo-utilization of glucose and cellobiose achieved in P. putida EM42 Δgcd mutant.Growth defect of the mutant compensated by implanting exogenous sugar transporters.Enhanced influx of carbon caused aerobic overproduction of pyruvate and acetate.Carbon from excess pyruvate streamed into ethanol or L-lactate.Pyruvate overproduction unveiled by a mathematical model of P. putida metabolism. |
