Systems Metabolic Engineering of Genome-Reduced <i>Pseudomonas putida</i> for Efficient Production of Polyhydroxyalkanoate from <i>p</i>-Coumaric Acid

<i>Pseudomonas putida</i> KT2440, which harbors native aromatic catabolic pathways, has emerged as a cell factory for funnelling lignin derivatives to medium-chain-length polyhydroxyalkanoates (mcl-PHA). To enhance this bioconversion, we engineered the genome-reduced strain <i>P. putida</i> KTU-U27 (with higher PHA productivity than its parental strain KT2440) to further enhance mcl-PHA synthesis from the lignin-derived aromatic compound <i>p</i>-coumaric acid (<i>p</i>-CA). Three targeted strategies were employed: (i) blocking PHA degradation via deletion of <i>phaZ</i>; (ii) suppressing β-oxidation by deleting <i>fadBA1</i> and <i>fadBA2</i>; and (iii) enhancing biosynthesis through overexpression of <i>phaC1</i> and <i>alkK</i>, resulting in the engineered strain KTU-U27Δ<i>Z2BA</i>-P46C1K. Subsequent optimization of the carbon-to-nitrogen (C/N) ratio and high-density fed-batch fermentation further improved PHA productivity. To adapt the substrate toxicity, strain tolerance toward <i>p</i>-CA was augmented by overexpressing the <i>ttg2</i>ABCDE operon and the <i>vacJ</i> gene. Under optimized fed-batch fermentation conditions (initial C/N ratio of 8:4), the final strain KTU-U27Δ<i>Z2BA</i>-P46C1K-P46TJ achieved a cell dry weight of 2050 mg/L with a PHA content of 82.19 wt %, corresponding to a PHA yield of 1685 mg/L, which is the highest reported to date using <i>p</i>-CA as the sole carbon source. This integrated approach of combining genome reduction, metabolic engineering, and bioprocess optimization, provides a scalable platform for mcl-PHA production from lignin-derived aromatics, highlighting the potential of KTU-U27-based chassis for cost-effective lignin valorization.