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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