Redefining HexR regulatory landscape in Pseudomonas putida KT2440 through integrative systems biology

Pseudomonas putida strains are prized biocatalysts, renowned for their versatility in degrading diverse chemicals, tolerating organic solvents, and withstanding environmental stressors. Central to their adaptive success is the precise regulation of primary carbon metabolism, with HexR emerging as a key regulator. While previous research has explored HexR binding through in vitro assays and comparative transcriptomics, the in vivo binding sites and genome-scale regulon remain uncharted. This study presents a comparative analysis of P. putida KT2440, comparing expression profiles of wild-type and hexR deletion mutant strains across distinct growth substrates: glucose (glycolytic), acetate, succinate (gluconeogenic), and glycerol (inducing both metabolic responses). Our findings revealed an extensive regulatory role of HexR in acetate metabolism, simultaneously suppressing the glycolytic pathway while enhancing pyruvate metabolism, glyoxylate shunt, and gluconeogenesis to support growth. Integration of ChIP-exo data identified 29 HexR binding locations in the KT2440 strain grown on acetate, directly regulating 75 genes. Complementing these findings, model-based in silico simulations provided contextual insight into metabolic flux states, deepening our understanding of carbon metabolism orchestrated by this transcription factor. This study thus offers a holistic view of the HexR regulatory landscape, highlighting its relevance in P. putida KT2440 metabolism and laying the groundwork for future metabolic engineering efforts in this versatile organism.