Synthetic biology-guided modular engineering of the secreted cutinase FoCut5a with a carbohydrate-binding module improves poly(ester-urethane) depolymerization in Yarrowia lipolytica

Poly(ester-urethane) bioconversion requires efficient polymer-degrading enzymes and microbial hosts capable of processing the resulting monomers. However, the optimal modular design of secreted cutinases for depolymerizing insoluble polyester-polyurethanes remains largely unexplored. Here, we established a combinatorial synthetic biology strategy in Yarrowia lipolytica based on the fungal cutinase FoCut5a from Fusarium oxysporum. A library of 96 Golden Gate-assembled constructs, combining four secretion signals, four interdomain linkers, and six substrate-binding domains, was integrated site-specifically into the genome and screened for depolymerization of the polyester-polyurethane dispersion Impranil DLN-SD. The best-performing variant fused FoCut5a to the carbohydrate-binding module TrCBM1. LC-MS analysis demonstrated release of adipic acid, 1,6-hexanediol, and neopentyl glycol, confirming ester-bond hydrolysis within the polyester domains of the polymer and additionally detected 6-hydroxycaproate, indicating oxidation of polyurethane-derived 1,6-hexanediol by Y. lipolytica. Purified FoCut5a-TrCBM1 displayed unchanged activity on p-nitrophenyl butyrate but reduced Impranil turbidity 5.6-fold faster and increased release of 1,6-hexanediol and adipic acid by 76-fold and 220-fold, respectively, compared to the unfused enzyme. These results highlight modular enzyme engineering as an effective strategy to enhance polyester-polyurethane deconstruction and support Y. lipolytica as a platform for polymer bioconversion and upcycling.