Engineering Paracoccus denitrificans PD1222 for Fusarium solani cutinase-mediated biodegradation of poly(butylene...

Poly(butylene adipate-co-terephthalate) (PBAT) is an aromatic-aliphatic copolyester widely used in packaging and consumer products. Its aromatic rings confer high resistance to hydrolysis, limiting biological degradation. To enhance PBAT biodegradation, we engineered Paracoccus denitrificans PD1222, a metabolically versatile and genetically tractable bacterium that can accumulate poly(3-hydroxybutyrate) (PHB). A novel plasmid (pV1) was constructed to express the broad-specificity cutinase FsCut under the constitutive Ptuf promoter and fused to the PorG signal peptide for extracellular secretion. Using an optimized transformation protocol, we stably transformed P. denitrificans PD1222 with pV1, enabling secretion of active FsCut and efficient PBAT hydrolysis. In degradation assays, the engineered strain exhibited significantly higher depolymerization rates than the strain carrying the pV0 vector, used as a control. Furthermore, the FsCut-expressing strain accumulated 1.16-fold more PHB than the pV0 strain and exhibited degradation rates of PBAT 2.27-fold higher in enriched medium and 1.9-fold higher in defined mineral medium. These findings demonstrate that targeted expression of a secreted cutinase substantially improves PBAT degradation by P. denitrificans, supporting its potential as a microbial platform for plastic bioremediation.IMPORTANCEThe widespread use of poly(butylene adipate-co-terephthalate) (PBAT) is limited by its low hydrolytic degradation rate, resulting from its aromatic structure, which makes it highly resistant to biological degradation. Developing strategies to accelerate PBAT depolymerization is critical for advancing sustainable plastic waste management. In this study, a novel plasmid, pV1, was successfully constructed to enable the heterologous expression and extracellular secretion of the broad-specificity cutinase FsCut in Paracoccus denitrificans PD1222. The engineered strain effectively hydrolyzes PBAT, demonstrating the plasmid's capability for correct synthesis and export of functional protein. Furthermore, the implementation of a newly developed bacterial transformation protocol in P. denitrificans represents a significant methodological advancement, reducing the time and complexity required compared with traditional conjugation approaches. Together, these findings highlight a dual achievement: improving the enzymatic degradation of a polymer with a low hydrolytic degradation rate and establishing an efficient genetic engineering strategy in a metabolically versatile bacterium, providing a promising platform for future bioremediation and biopolymer valorization studies.