Yap, Z. L.; Lucas, G. G.; Safklou, M.; Darragh, R.; Hogan, A. M.; Motnenko, A.; Blunt, W.; Sparling, R.; Levin, D. B.; Fernandez Do Porto, D. A.; Cardona, S. T.

Bioplastics represent promising alternatives to petroleum-based plastics, yet their biodegradation remains insufficiently understood. Identifying bacteria capable of degrading bioplastics extracellularly could enhance end-of-life management practices. To investigate Burkholderia\'s capacities for the degradation of medium-chain-length polyhydroxyalkanoate (mcl-PHA), we screened a panel of Burkholderia strains and identified such capacity in some strains of B. gladioli, B. multivorans, and B. vietnamiensis. To elucidate the genetic basis of this activity, we performed transposon mutagenesis followed by activity-based screening and Tn-seq on B. vietnamiensis LMG 16232. Transposon insertions with negative phenotype were identified in genes encoding a triacylglycerol lipase, a lipase chaperone, type II secretion system components, HTH-type transcriptional activators, and a polyhydroxyalkanoate synthesis repressor. To validate the involvement of these genetic elements in mcl-PHA degradation, we generated insertional mutants using a CRISPR-associated transposase system (CAST). Complete loss or reduced extracellular mcl-PHA depolymerase activity was observed in the CAST mutants, validating their involvement in mcl-PHA degradation. In addition, the interruption of these genetic elements showed a loss of lipase activity, suggesting that the enzymes responsible for mcl-PHA depolymerase are lipases with substrate ambiguity; moreover, docking experiments supported these findings. Together, we identify B. vietnamiensis as a source of enzymes capable of degrading extracellular mcl-PHA and demonstrate the power of combining activity-based screening, Tn-seq, and CAST to rapidly establish gene-to-function links.