To broaden the substrate scope of microbial cell factories towards renewable substrates, rational genetic interventions are often combined with adaptive laboratory evolution (ALE). However, comprehensive studies enabling a holistic understanding of adaptation processes primed by rational metabolic engineering remain scarce. The industrial workhorse Pseudomonas putida was engineered to utilize the non-native sugar D-xylose, but its assimilation into the bacterial biochemical network via the exogenous xylose isomerase pathway remained unresolved. Here, we elucidate the xylose metabolism and establish a foundation for further engineering followed by ALE. First, native glycolysis is derepressed by deleting the local transcriptional regulator gene hexR. We then enhance the pentose phosphate pathway by implanting exogenous transketolase and transaldolase into two lag-shortened strains and allow ALE to finetune the rewired metabolism. Subsequent multilevel analysis and reverse engineering provide detailed insights into the parallel paths of bacterial adaptation to the non-native carbon source, highlighting the enhanced expression of transaldolase and xylose isomerase along with derepressed glycolysis as key events during the process.

APC Microbiome Ireland University College Cork College Rd Cork T12 YT20 Ireland

Australian Institute for Bioengineering and Nanotechnology The University of Queensland Cnr College Rd and Cooper Rd St Lucia QLD QLD 4072 Australia

Department of Biochemistry and Synthetic Metabolism Max Planck Institute for Terrestrial Microbiology Karl von Frisch Straße 10 35043 Marburg Germany

Department of Experimental Biology Faculty of Science Masaryk University Kamenice 753 5 62500 Brno Czech Republic

Institute of Applied Microbiology RWTH Aachen University Worringer Weg 1 52074 Aachen Germany

Institute of Inorganic and Analytical Chemistry University of Münster Corrensstraße 48 48149 Münster Germany

School of Microbiology University College Cork College Rd Cork T12 Y337 Ireland

Systems and Synthetic Biology Program Centro Nacional de Biotecnología CNB CSIC Cantoblanco Darwin 3 28049 Madrid Spain

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Narisetty, V. et al. Valorisation of xylose to renewable fuels and chemicals, an essential step in augmenting the commercial viability of lignocellulosic biorefineries. PubMed PMC

Baral, N. R. et al. Approaches for more efficient biological conversion of lignocellulosic feedstocks to biofuels and bioproducts.

Sun, L. et al. Complete and efficient conversion of plant cell wall hemicellulose into high-value bioproducts by engineered yeast. PubMed PMC

Ling, C. et al. Muconic acid production from glucose and xylose in PubMed PMC

Taha, M. et al. Commercial feasibility of lignocellulose biodegradation: possibilities and challenges. PubMed

Görke, B. & Stülke, J. Carbon catabolite repression in bacteria: many ways to make the most out of nutrients. PubMed

Volk, M. J. et al. Metabolic engineering: methodologies and applications. PubMed

Dragosits, M. & Mattanovich, D. Adaptive laboratory evolution – principles and applications for biotechnology. PubMed PMC

Sandberg, T. E., Salazar, M. J., Weng, L. L., Palsson, B. O. & Feist, A. M. The emergence of adaptive laboratory evolution as an efficient tool for biological discovery and industrial biotechnology. PubMed PMC

Porcar, M., Danchin, A. & de Lorenzo, V. Confidence, tolerance, and allowance in biological engineering: the nuts and bolts of living things. PubMed

Mavrommati, M., Daskalaki, A., Papanikolaou, S. & Aggelis, G. Adaptive laboratory evolution principles and applications in industrial biotechnology. PubMed

Nikel, P. I., Martínez-García, E. & de Lorenzo, V. Biotechnological domestication of pseudomonads using synthetic biology. PubMed

Sullivan, K. P. et al. Mixed plastics waste valorization through tandem chemical oxidation and biological funneling. PubMed

Dvořák, P., Nikel, P. I., Damborský, J. & de Lorenzo, V. Bioremediation 3.0: Engineering pollutant-removing bacteria in the times of systemic biology. PubMed

Salvachúa, D. et al. Outer membrane vesicles catabolize lignin-derived aromatic compounds in PubMed PMC

Liu, Z.-H. et al. Transforming biorefinery designs with ‘Plug-In Processes of Lignin’ to enable economic waste valorization. PubMed PMC

Kohlstedt, M. et al. Biobased PET from lignin using an engineered PubMed

Dvořák, P. & de Lorenzo, V. Refactoring the upper sugar metabolism of PubMed

Elmore, J. R. et al. Engineered PubMed

Meijnen, J.-P., de Winde, J. H. & Ruijssenaars, H. J. Engineering PubMed PMC

Lim, H. G. et al. Generation of

Bator, I., Wittgens, A., Rosenau, F., Tiso, T. & Blank, L. M. Comparison of three xylose pathways in PubMed PMC

Wang, Y. et al. Growth of engineered

Nikel, P. I., Chavarría, M., Fuhrer, T., Sauer, U. & de Lorenzo, V. PubMed PMC

Martínez-García, E., Nikel, P. I., Aparicio, T. & de Lorenzo, V. PubMed PMC

Meijnen, J.-P., Winde, J. Hde & Ruijssenaars, H. J. Metabolic and regulatory rearrangements underlying efficient d-xylose utilization in engineered PubMed PMC

Dvořák, P., Kováč, J. & de Lorenzo, V. Biotransformation of D-xylose to D-xylonate coupled to medium-chain-length polyhydroxyalkanoate production in cellobiose-grown PubMed PMC

Bujdoš, D. et al. Engineering of PubMed

Weimer, A., Kohlstedt, M., Volke, D. C., Nikel, P. I. & Wittmann, C. Industrial biotechnology of PubMed PMC

Salis, H. M., Mirsky, E. A. & Voigt, C. A. Automated design of synthetic ribosome binding sites to control protein expression. PubMed PMC

Ceroni, F., Algar, R., Stan, G.-B. & Ellis, T. Quantifying cellular capacity identifies gene expression designs with reduced burden. PubMed

Wiechert, W. PubMed

Kohlstedt, M. & Wittmann, C. GC-MS-based PubMed

Beckers, V., Poblete-Castro, I., Tomasch, J. & Wittmann, C. Integrated analysis of gene expression and metabolic fluxes in PHA-producing PubMed PMC

Kukurugya, M. A. et al. Multi-omics analysis unravels a segregated metabolic flux network that tunes co-utilization of sugar and aromatic carbons in PubMed PMC

Wilkes, R. A., Waldbauer, J. & Aristilde, L. Analogous Metabolic Decoupling in PubMed PMC

Sudarsan, S. et al. Dynamics of benzoate metabolism in PubMed PMC

La Rosa, R., Nogales, J. & Rojo, F. The Crc/CrcZ-CrcY global regulatory system helps the integration of gluconeogenic and glycolytic metabolism in PubMed

Sasnow, S. S., Wei, H. & Aristilde, L. Bypasses in intracellular glucose metabolism in iron-limited PubMed PMC

Nikel, P. I. et al. Reconfiguration of metabolic fluxes in PubMed PMC

Orth, J. D., Thiele, I. & Palsson, B. Ø. What is flux balance analysis? PubMed PMC

Cheng, K.-K. et al. Global metabolic network reorganization by adaptive mutations allows fast growth of PubMed

Breckell, G. L. & Silander, O. K. Growth condition-dependent differences in methylation imply transiently differentiated DNA methylation states in PubMed PMC

Udaondo, Z., Ramos, J., Segura, A., Krell, T. & Daddaoua, A. Regulation of carbohydrate degradation pathways in PubMed PMC

Rojo, F. Carbon catabolite repression in PubMed

del Castillo, T., Duque, E. & Ramos, J. L. A set of activators and repressors control peripheral glucose pathways in PubMed PMC

Bentley, G. J. et al. Engineering glucose metabolism for enhanced muconic acid production in PubMed

Volke, D. C., Olavarría, K. & Nikel, P. I. Cofactor specificity of Glucose-6-Phosphate dehydrogenase isozymes in PubMed PMC

Noor, E., Eden, E., Milo, R. & Alon, U. Central carbon metabolism as a minimal biochemical walk between precursors for biomass and energy. PubMed

Zhang, M., Eddy, C., Deanda, K., Finkelstein, M. & Picataggio, S. Metabolic engineering of a pentose metabolism pathway in ethanologenic PubMed

Martínez-García, E., Aparicio, T., de Lorenzo, V. & Nikel, P.I. New transposon tools tailored for metabolic engineering of gram-negative microbial cell factories. PubMed PMC

Espeso, D. R., Dvořák, P., Aparicio, T. & de Lorenzo, V. An automated DIY framework for experimental evolution of PubMed PMC

Silby, M. W., Winstanley, C., Godfrey, S. A. C., Levy, S. B. & Jackson, R. W. PubMed

El-Kirat-Chatel, S., Beaussart, A., Boyd, C. D., O’Toole, G. A. & Dufrêne, Y. F. Single-cell and single-molecule analysis deciphers the localization, adhesion, and mechanics of the biofilm adhesin LapA. PubMed PMC

Chu, E. K., Kilic, O., Cho, H., Groisman, A. & Levchenko, A. Self-induced mechanical stress can trigger biofilm formation in uropathogenic PubMed PMC

Mutalik, V. K. et al. Precise and reliable gene expression via standard transcription and translation initiation elements. PubMed

Paget, M. S. Bacterial sigma factors and anti-sigma factors: structure, function and distribution. PubMed PMC

Conrad, T. M. et al. RNA polymerase mutants found through adaptive evolution reprogram PubMed PMC

Tenaillon, O. et al. The molecular diversity of adaptive convergence. PubMed

Choe, D. et al. Adaptive laboratory evolution of a genome-reduced PubMed PMC

Tomko, T. A. & Dunlop, M. J. Expression of heterologous sigma factor expands the searchable space for biofuel tolerance mechanisms. PubMed

Tomatis, P. E., Schütz, M., Umudumov, E. & Plückthun, A. Mutations in sigma 70 transcription factor improves expression of functional eukaryotic membrane proteins in PubMed PMC

Pang, C. et al. Engineering sigma factors and chaperones for enhanced heterologous lipoxygenase production in PubMed PMC

Wagner, S., Bader, M. L., Drew, D. & de Gier, J.-W. Rationalizing membrane protein overexpression. PubMed

Schmitz, S., Nies, S., Wierckx, N., Blank, L. M. & Rosenbaum, M. A. Engineering mediator-based electroactivity in the obligate aerobic bacterium PubMed PMC

Hassan, H. M. & Fridovich, I. Mechanism of the antibiotic action pyocyanine. PubMed PMC

DeBritto, S. et al. Isolation and characterization of nutrient dependent pyocyanin from PubMed PMC

Tan, Z. L. et al. In vivo continuous evolution of metabolic pathways for chemical production. PubMed PMC

Wirth, N. T. et al. A synthetic C2 auxotroph of PubMed

Abril, M. A., Michan, C., Timmis, K. N. & Ramos, J. L. Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway. PubMed PMC

Martínez-García, E. & de Lorenzo, V. Transposon-based and plasmid-based genetic tools for editing genomes of gram-negative bacteria. PubMed

Horton, R. M., Cai, Z., Ho, S. N. & Pease, L. R. Gene splicing by overlap extension: Tailor-made genes using the polymerase chain reaction. PubMed

Bennette, N. B., Eng, J. F. & Dismukes, G. C. An LC–MS-Based chemical and analytical method for targeted metabolite quantification in the model cyanobacterium PubMed

Millard, P., Letisse, F., Sokol, S. & Portais, J.-C. IsoCor: correcting MS data in isotope labeling experiments. PubMed

Schmitz, A., Ebert, B. E. & Blank, L. M. GC-MS-Based Determination of Mass Isotopomer Distributions for

Poskar, C. H. et al. iMS2Flux– a high–throughput processing tool for stable isotope labeled mass spectrometric data used for metabolic flux analysis. PubMed PMC

Young, J. D. INCA: a computational platform for isotopically non-stationary metabolic flux analysis. PubMed PMC

Nogales, J. et al. High-quality genome-scale metabolic modelling of Pseudomonas putida highlights its broad metabolic capabilities. PubMed PMC

Heirendt, L. et al. Creation and analysis of biochemical constraint-based models using the COBRA Toolbox v.3.0. PubMed PMC

Swain, P. S. et al. Inferring time derivatives including cell growth rates using Gaussian processes. PubMed PMC

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