This article describes the synthetic priming of *Pseudomonas putida* to utilize the non-native sugar D-xylose through a combination of rational genetic engineering and adaptive laboratory evolution (ALE). The study aimed to enhance the metabolism of *P. putida* to efficiently utilize D-xylose, a major component of lignocellulosic biomass, which is important for biotechnological applications. The researchers first derepressed native glycolysis by deleting the *hexR* gene, which regulates glucose metabolism. They then introduced exogenous transketolase and transaldolase from *E. coli* to enhance the pentose phosphate pathway and allowed ALE to further optimize the rewired metabolism. The study revealed that the adaptation of *P. putida* to D-xylose involved the enhanced expression of transaldolase and xylose isomerase, along with derepressed glycolysis. The researchers used a combination of carbon flux analysis, enzyme activity assays, and reverse engineering to uncover the metabolic pathways involved in D-xylose metabolism. They found that the metabolism of D-xylose in *P. putida* involved a partially cyclic upper xylose metabolism, with the majority of the carbon entering the pentose phosphate pathway and being converted into fructose 6-phosphate and further to 6-phosphogluconate. The study also demonstrated that the growth of *P. putida* on D-xylose was limited by the carbon cycling via the 6-phosphogluconate dehydrogenase (Gnd) reaction, which was previously suggested to be involved in the adaptation of engineered *P. putida* strains to D-xylose. The researchers then performed ALE on strains with or without the *gnd* gene and with implanted exogenous PPP genes, which doubled the growth rate and reduced the lag phase up to 5-fold. The results showed that the bacterium's genomic and metabolic plasticity enabled it to find alternative solutions that led to improved phenotypes of selected mutants. The study also identified a large genomic region that was amplified in the evolved strains, which included the *tal* gene encoding transaldolase. The duplication of this region upstream of the *xyA* gene in the plasmid increased the expression of the *xyA* gene, which is essential for the xylose isomerase pathway. The researchers also found that the mutation in the *rpoD* gene, which encodes the RNA polymerase sigma factor σ^70, was responsible for the increased expression of the synthetic xyABE operon, which is crucial for the xylose isomerase pathway. The study highlights the importance of combining rational genetic engineering with ALE to improve the utilization of non-native substrates in *P. putida*.This article describes the synthetic priming of *Pseudomonas putida* to utilize the non-native sugar D-xylose through a combination of rational genetic engineering and adaptive laboratory evolution (ALE). The study aimed to enhance the metabolism of *P. putida* to efficiently utilize D-xylose, a major component of lignocellulosic biomass, which is important for biotechnological applications. The researchers first derepressed native glycolysis by deleting the *hexR* gene, which regulates glucose metabolism. They then introduced exogenous transketolase and transaldolase from *E. coli* to enhance the pentose phosphate pathway and allowed ALE to further optimize the rewired metabolism. The study revealed that the adaptation of *P. putida* to D-xylose involved the enhanced expression of transaldolase and xylose isomerase, along with derepressed glycolysis. The researchers used a combination of carbon flux analysis, enzyme activity assays, and reverse engineering to uncover the metabolic pathways involved in D-xylose metabolism. They found that the metabolism of D-xylose in *P. putida* involved a partially cyclic upper xylose metabolism, with the majority of the carbon entering the pentose phosphate pathway and being converted into fructose 6-phosphate and further to 6-phosphogluconate. The study also demonstrated that the growth of *P. putida* on D-xylose was limited by the carbon cycling via the 6-phosphogluconate dehydrogenase (Gnd) reaction, which was previously suggested to be involved in the adaptation of engineered *P. putida* strains to D-xylose. The researchers then performed ALE on strains with or without the *gnd* gene and with implanted exogenous PPP genes, which doubled the growth rate and reduced the lag phase up to 5-fold. The results showed that the bacterium's genomic and metabolic plasticity enabled it to find alternative solutions that led to improved phenotypes of selected mutants. The study also identified a large genomic region that was amplified in the evolved strains, which included the *tal* gene encoding transaldolase. The duplication of this region upstream of the *xyA* gene in the plasmid increased the expression of the *xyA* gene, which is essential for the xylose isomerase pathway. The researchers also found that the mutation in the *rpoD* gene, which encodes the RNA polymerase sigma factor σ^70, was responsible for the increased expression of the synthetic xyABE operon, which is crucial for the xylose isomerase pathway. The study highlights the importance of combining rational genetic engineering with ALE to improve the utilization of non-native substrates in *P. putida*.