This study explores the synthetic priming of Pseudomonas putida to utilize the non-native sugar D-xylose. P. putida, a well-known industrial microorganism, was engineered to metabolize D-xylose using an exogenous xylose isomerase pathway. However, its ability to assimilate D-xylose into its metabolic network remained unresolved. The study combines rational genetic engineering with adaptive laboratory evolution (ALE) to enhance D-xylose utilization. Key findings include the derepression of native glycolysis by deleting the hexR gene, enhancement of the pentose phosphate pathway through the introduction of exogenous transketolase and transaldolase, and the identification of key metabolic events during adaptation, such as the enhanced expression of transaldolase and xylose isomerase. The study also reveals that the adaptation of P. putida to D-xylose involves complex metabolic reprogramming, with the EDEMP cycle playing a central role. The results demonstrate that the deletion of hexR and the introduction of synthetic genetic constructs significantly improve D-xylose utilization. Additionally, the study highlights the importance of the glyoxylate shunt and the role of the transaldolase enzyme in the pentose phosphate pathway. The findings contribute to a better understanding of bacterial adaptation to non-native substrates and provide insights into the metabolic engineering of P. putida for the efficient utilization of lignocellulosic sugars.This study explores the synthetic priming of Pseudomonas putida to utilize the non-native sugar D-xylose. P. putida, a well-known industrial microorganism, was engineered to metabolize D-xylose using an exogenous xylose isomerase pathway. However, its ability to assimilate D-xylose into its metabolic network remained unresolved. The study combines rational genetic engineering with adaptive laboratory evolution (ALE) to enhance D-xylose utilization. Key findings include the derepression of native glycolysis by deleting the hexR gene, enhancement of the pentose phosphate pathway through the introduction of exogenous transketolase and transaldolase, and the identification of key metabolic events during adaptation, such as the enhanced expression of transaldolase and xylose isomerase. The study also reveals that the adaptation of P. putida to D-xylose involves complex metabolic reprogramming, with the EDEMP cycle playing a central role. The results demonstrate that the deletion of hexR and the introduction of synthetic genetic constructs significantly improve D-xylose utilization. Additionally, the study highlights the importance of the glyoxylate shunt and the role of the transaldolase enzyme in the pentose phosphate pathway. The findings contribute to a better understanding of bacterial adaptation to non-native substrates and provide insights into the metabolic engineering of P. putida for the efficient utilization of lignocellulosic sugars.