Burkholderia Genes Involved in Extracellular Bioplastic Degradation Revealed by Tn-seq and CRISPR-Cas

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 Burkholderias capacities for the degradation of medium-chain-length polyhydroxyalkanoate (mcl-PHA), we screened a panel of Burkholderia strains and identified such capacity in 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. Disrupted genetic elements in transposon mutants with negative phenotypes were further investigated using a CRISPR-associated transposase (CAST) system. These included a lipase production gene cluster, encoding two putative triacylglycerol lipases and a chaperone, and genes coding for a a A24 family peptidase, a TetR/AcrR family transcriptional regulator and a type II secretion system (T2SS) protein. Complete loss or reduced extracellular mcl-PHA depolymerase activity was observed in the CAST mutants, validating their involvement in mcl-PHA degradation. Notably, only one of the two lipases encoded in the lipase production gene cluster was responsible for mcl-PHA degradation, suggesting that while lipases may show substrate promiscuity, lipase functional annotation does not necessarily imply mcl-PHA depolymerization. Docking experiments using the amino acid sequences of the two lipases supported these findings. Together, we identify a gene coding for an active mcl-PHA depolymerase in B. vietnamiensis and demonstrate the power of combining activity-based screening, Tn-seq, and CAST to rapidly establish gene-to-function links. ImportanceDue to their versatile metabolism, Burkholderia strains play critical roles in degradation of multiple compounds in the environment. Here we show that several Burkholderia species can extracellularly degrade medium-chain-length polyhydroxyalkanoates (mcl-PHAs), a promising class of bioplastics. By integrating transposon mutagenesis, Tn-seq, and CRISPR-associated transposase (CAST) technologies, we identify and validate key genetic determinants involved in mcl-PHA degradation in B. vietnamiensis. These genes encode a lipase, a secretion system component, and regulatory factors, underscoring the complexity and specificity of microbial bioplastic degradation pathways. These findings not only advance our understanding of PHA biodegradation but also identifies B. vietnamiensis as as a source of enzymes capable of degrading extracellular mcl-PHA.