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Adaptive laboratory evolution unlocks membrane permeability as a key limitation in long-chain alcohol metabolism by Pseudomonas putida KT2440
Summary
Adaptive laboratory evolution of Pseudomonas putida KT2440 revealed that the bottleneck in metabolizing long-chain alcohols—byproducts of polyethylene degradation—is membrane uptake rather than enzymatic transformation, a finding confirmed by heterologous expression of a hydrocarbon transporter gene that reproduced the evolved phenotype. This mechanistic insight advances the engineering of bacteria capable of closing the plastic-derived carbon cycle by consuming PE degradation products, a promising avenue for biological microplastic remediation.
Abstract Pseudomonas putida KT2440, renowned for its diverse metabolic capabilities, is a promising platform for downstream processing and revalorization of recalcitrant molecules. In this study, we examined and optimized P. putida KT2440’s ability to utilize long-chain alcohols. These molecules are byproducts of the degradation of polyethylene (PE), the most widely used plastic. Using them as feedstock for microbial growth would close the plastic-derived carbon cycle, reducing environmental pollution. First, we discovered that P. putida KT2440 can use long-chain alcohols as the sole carbon and energy source. Using adaptive laboratory evolution (ALE), we generated variants with improved growth rates on long-chain alcohols, specifically 1-hexadecanol and 1-eicosanol. 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, demonstrating that the bottleneck in long-chain alcohol utilization is not substrate transformation but uptake. These findings lay the groundwork for the potential application of P. putida KT2440 for the degradation of PE.