Degradation pathway of (R)-1,3-butanediol in Pseudomonas putida KT2440 and development of its biosensor.

UNLABELLED: 1,3-Butanediol (1,3-BDO) is widely used in consumer and industrial products; however, its microbial degradation remains poorly understood. Here, we dissect the catabolic and regulatory mechanisms of (R)-1,3-BDO utilization in Pseudomonas putida KT2440 and develop (R)-1,3-BDO-responsive transcriptional biosensors. Transcriptomics and qRT-PCR revealed strong induction of the ped gene cluster, which oxidizes (R)-1,3-BDO to (R)-3-hydroxybutyrate [(R)-3-HB], and of the LysR-regulated operon PP_2047-2051, which channels (R)-3-HB toward acetoacetate and acetyl-CoA. Gene deletion and enzyme assays identified pedE and PP_2049 as essential for (R)-1,3-BDO catabolism, with PP_2049 playing a more critical role than the canonical β-hydroxybutyrate dehydrogenase HbdH (PP_3073). Regulatory analysis showed that PedR1 directly activates catabolic genes independently of PedR2-challenging the widely accepted indirect-only model-while PedS1 proved dispensable, implying an alternative sensor kinase. Promoter-GFP fusions demonstrated that full activation of the pedE promoter requires both PedR1 and the PedS2-PedR2 two-component system, whereas the pedS2R2 promoter requires only PedR1 and functions in both native and heterologous hosts, including Escherichia coli. These results define the genetic and regulatory architecture of (R)-1,3-BDO degradation in P. putida and establish pedE- and pedS2R2-based promoters as (R)-1,3-BDO-responsive biosensors.

IMPORTANCE: 1,3-Butanediol is an increasingly prevalent industrial chemical whose environmental fate depends on microbial degradation. Understanding how microorganisms sense and metabolize this compound is therefore essential for both environmental microbiology and biotechnology. This study clarifies the genetic and regulatory basis of (R)-1,3-BDO catabolism in Pseudomonas putida, identifying key enzymes and transcriptional regulators that control its utilization. By coupling pathway elucidation with promoter characterization, this work enables the development of (R)-1,3-BDO-responsive biosensors that function in both native and heterologous hosts. These insights are important for understanding the environmental fate of 1,3-BDO and designing inducible regulatory systems for biotechnological applications.