The carbon catabolite repression system - polyhydroxyalkanoate metabolism link in Pseudomonas putida KT2440

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polyesters with potential to replace petroleum-based plastics. However, their industrial production is constrained by high costs and the lack of precise control over polymer composition and molecular weight. Pseudomonas putida KT2440 is a model organism for medium-chain-length (mcl) PHA production, yet the regulatory networks shaping PHA metabolism remain incompletely understood. In particular, the influence of the carbon catabolite repression (CCR) system—including the proteins Hfq and Crc and the small RNAs CrcY and CrcZ—on PHA biosynthesis has not been systematically investigated. Here, we combined genetic, proteomic, and translational reporter approaches to elucidate the role of CCR elements in PHA metabolism. Deletion and overexpression mutants of hfq, crc, crcY, and crcZ were constructed and tested under different carbon and nitrogen conditions, and proteomic profiling was performed to capture global changes in protein abundance. Translational fusions of phaC1, phaF, and phaI with real-time monitoring revealed dynamic, condition-specific patterns of regulation. In addition, chromosomal integration strategies were explored to modulate CrcY and CrcZ expression. Our results demonstrate that overexpression of CrcY and CrcZ enhances mcl-PHA accumulation and reduces polymer molecular weight, providing a potential tool to tune material properties. More broadly, we show that CCR plays a complex role extending beyond simple repression. Nutrient context, particularly carbon identity and nitrogen availability, strongly influenced the CCR–PHA link. Among CCR components, Hfq emerged as the dominant regulator: its deletion abolished PHA synthesis on glucose and broadly altered protein expression, particularly in central carbon metabolism. These findings highlight the multifaceted impact of CCR on PHA metabolism. To refine current regulatory models, future studies employing comprehensive multi-omics approaches will be required. A deeper understanding of CCR regulation will ultimately enable rational chassis-strain engineering to improve PHA yield and tailor polymer properties for industrial applications.