Bujdos, D. | Search bioRxiv

bioRxiv2023

Genomic and metabolic plasticity drive alternative scenarios for adapting Pseudomonas putida to non-native substrate D-xylose.

D-Xylose, a major constituent of plant biomass and second most abundant sugar on Earth, holds a considerable potential as a substrate for sustainable bio-production. Pseudomonas putida KT2440 is an attractive bacterial host for valorizing biogenic feedstocks but lacks a xylose utilization pathway. While several attempts to engineer P. putida for growth on xylose have been reported, a comprehensive understanding of xylose metabolism in this bacterium is lacking, hindering its further improvement and rational tailoring for specific biotechnological purposes. In this study, we elucidated the xylose metabolism in the genome-reduced P. putida strain, EM42, endowed with xylose isomerase pathway (xylAB) and transporter (xylE) from Escherichia coli and used the obtained knowledge in combination with adaptive laboratory evolution to accelerate the bacteriums growth on the pentose sugar. Carbon flux analyses, targeted gene knock-outs, and in vitro enzyme assays portrayed xylose assimilation in P. putida and confirmed a partially cyclic upper xylose metabolism. Deletion of the local transcriptional regulator gene hexR de-repressed genes of several key catabolic enzymes and reduced the lag phase on xylose. Guided by metabolic modeling, we augmented P. putida with additional heterologous pentose phosphate pathway genes and subjected rationally prepared strains to adaptive laboratory evolution (ALE) on xylose. The descendants showed accelerated growth and reduced growth lag. Genomic and proteomic analysis of engineered and evolved mutants revealed the importance of a large genomic re-arrangement, transaldolase overexpression, and balancing gene expression in the synthetic xylABE operon. Importantly, omics analyses found that similar growth characteristics of two superior mutants were achieved through distinct evolutionary paths. This work provides a unique insight into how cell metabolism adjusts to a non-native substrate; it highlights the remarkable genomic and metabolic plasticity of P. putida and demonstrates the power of combining knowledge-driven engineering with ALE in generating desirable microbial phenotypes. HighlightsO_LIElucidated xylose catabolism via exogenous isomerase pathway in P. putida EM42. C_LIO_LIDeletion of transcriptional regulator HexR improved growth on xylose. C_LIO_LIKnowledge-guided interventions and adaptive evolution accelerated growth. C_LIO_LIOmics analyses of selected mutants highlighted the genomic and metabolic plasticity of P. putida. C_LIO_LITwo mutants with superior characteristics emerged from distinct evolutionary paths. C_LI

bioengineering↗

bioRxiv2022

Engineering of Pseudomonas putida for accelerated co-utilization of glucose and cellobiose yields aerobic overproduction of pyruvate explained by an upgraded metabolic model

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 {Delta}gcd 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 {Delta}gcd mutant for the bioproduction of valuable compounds from glucose and cellobiose. We show that the gcd deletion abolished the inhibition of the exogenous {beta}-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 {Delta}gcd 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. HighlightsO_LICo-utilization of glucose and cellobiose achieved in P. putida EM42 {Delta}gcd mutant. C_LIO_LIGrowth defect of the mutant compensated by implanting exogenous sugar transporters. C_LIO_LIEnhanced influx of carbon caused aerobic overproduction of pyruvate and acetate. C_LIO_LICarbon from excess pyruvate streamed into ethanol or L-lactate. C_LIO_LIPyruvate overproduction unveiled by a mathematical model of P. putida metabolism. C_LI

bioengineering↗