ABSTRACTBiotransformation of plastics or their depolymerization monomers as raw materials would offer a better end-of-life solutions to the plastic waste dilemma. 1,4-butanediol (BDO) is one of the major depolymerization monomers of many plastics polymers. BDO valorization presents great significance for waste plastic up-recycling and fermenting feedstock exploitation. In the present study, atmospheric pressure room temperature plasma (ARTP)-induced mutation combined with adaptive laboratory evolution (ALE) was used to improve the BDO utilization capability ofPseudomonas putidaKT2440. The excellent mutantP. putidaNB10 was isolated and stored in the China Typical Culture Preservation Center (CCTCC) with the deposit number M 2021482. Whole-genome resequencing and transcriptome analysis revealed that the BDO degradation process consists of β-oxidation, glyoxylate carboligase (GCL) pathway, glyoxylate cycle and gluconeogenesis pathway. The imbalance between the two key intermediates (acetyl-CoA and glycolyl-CoA) and the accumulation of cytotoxic aldehydes resulted in the weak metabolism performance of KT2440 in the utilization of BDO. The balance of the carbon flux and enhanced tolerance to cytotoxic intermediates endow NB10 with great BDO degradation capability. This study deeply revealed the metabolic mechanism behind BDO degradation and provided an excellent chassis cell for BDO further up-cycling to high-value chemicals.IMPORTANCEPlastic waste represents not only a global pollution problem but also a carbon-rich, low-cost, globally renewable feedstock for industrial biotechnology. BDO is the basic material for polybutylene terephthalate (PBT), poly butylene adipate-co-terephthalate (PBAT), poly (butylene succinate) (PBS), etc. Herein, the construction of BDO valorization cell factory presents great significance for waste plastic up-recycling and novel fermentation feedstock exploitation. However, BDO is hard to be metabolized and its metabolic pathway is unclear. This study presents aP. putidamutant NB10, obtained through the integration of ARTP and ALE, displaying significant growth improvement with BDO as the sole carbon source. Further genome resequencing, transcriptome analysis and genetic engineering deeply revealed the metabolic mechanism behind BDO degradation inP. putida, this study offers an excellent microbial chassis and modification strategy for plastic waste up-cycling.