Adaptive Laboratory Evolution Unlocks Membrane Permeability as a Key Limitation in Long‐Chain Alcohol Metabolism by Pseudomonas putida KT2440

ABSTRACT

Pseudomonas putida KT2440, renowned for its diverse metabolic capabilities, is a promising platform for downstream processing and revalorisation of recalcitrant molecules. In this study, we examined and optimised P. putida KT2440's ability to utilise products of the degradation of polyethylene (PE), the most used and disposed plastic. PE degradation creates over 200 molecules that vary in oxidation level and, thus, chemical properties. Among those, long‐chain alcohols represent one of the most challenging fractions to process due to their poor solubility. Using them as feedstock for microbial growth would close the plastic‐derived carbon cycle, reducing environmental impact. First, we discovered that P. putida KT2440 can use the long‐chain alcohols, 1‐hexadecanol and 1‐eicosanol, as the sole carbon and energy source. Using adaptive laboratory evolution (ALE), we generated variants with improved growth rates on such substrates. Mutations that became fixed during ALE provided insights into the mechanism, highlighting the importance of cell–substrate interaction. By heterologously expressing a hydrocarbon transporter‐encoding gene, we successfully reproduced the ALE‐derived phenotype, suggesting that the bottleneck in long‐chain alcohol utilisation lies in uptake rather than substrate transformation. These findings lay the groundwork for the potential application of P. putida KT2440 for the valorisation of PE degradation products.