Unveiling plastic biodegradation pathways through ¹³C-DNA stable isotope probing and metagenomics

Polyhydroxyalkanoates (PHAs) are promising biobased and biodegradable alternatives to conventional plastics, yet their degradation mechanisms and the diversity of microorganisms involved remain poorly characterized in marine ecosystems. Here, we used ¹³C-labeled poly(3-hydroxybutyrate) (PHB) and combined DNA-stable isotope probing (DNA-SIP) with metagenomic to identify and functionally characterize active PHB-degrading bacteria in seawater. We identified three metagenome-assembled genomes (MAGs) affiliated with the genus Agarilytica that exhibited an exceptional expansion of preficted extracellular short-chain-length PHA depolymerase genes (ephaZ_scl) with up to 14 copies per genome, far exceeding the one-to-two copies typically reported. Comparative genomic and structural analyses revealed gene duplication and fusion events, given rise to tandem or chimeric depolymerases that may enhance catalytic diversity and substrate accessibility. Three-dimensional structural modeling confirmed that these fusion proteins retained functional catalytic domains with potential cooperative or independent activity. Such genomic redundancy and structural diversification likely confer an adaptive advantage for PHB biodegradation in marine environment. Collectively, our findings provide new insights into the ecological and evolutionary strategies of marine PHB degraders and highlight the power of DNA-SIP metagenomic for elucidating active plastic biodegradation pathways under natural conditions.