Multiomics Insights into Valorization of Vanillic Acid and Oil Palm Residues for Sustainable Polyhydroxyalkanoate Production by <i>Pseudomonas putida</i>

Polyhydroxyalkanoates (PHA) are promising biodegradable alternatives to petroleum-based plastics, but high production costs limit their deployment. In this study, we evaluated a cosubstrate strategy combining vanillic acid (VA), representative of lignin-derived aromatics, with palm oil (PO) recovered from oil palm fruit residues to enhance PHA synthesis in <i>Pseudomonas putida</i>. Cofeeding 4 g/L VA and 4 g/L PO increased PHA titer to 1.13 g/L, a 52.7% improvement over VA alone, and yielded a new monomer, 3-hydroxyhexanoate (3HHx). Integrated transcriptome and metabolomic analyses revealed that cosubstrate metabolism attenuated TCA flux, enhanced β-oxidation and hydroxyacyl precursor pools, and altered NAD(P)H turnover and membrane lipids. These shifts were correlated with expanded monomer diversity. This study demonstrates that cofeeding lignin-derived aromatics with agricultural oils provides a synergistic route to improve PHA yield and diversify monomer composition, linking biomass valorization with circular, low-carbon material production.