Work

Synthetic biology-guided modular engineering of the secreted cutinase Fo Cut5a with a carbohydrate-binding module improves poly(ester-urethane) depolymerization in Yarrowia lipolytica

Year: 2026

Type: preprint

Abstract: Summary 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 Fo Cut5a to the carbohydrate-binding module Tr CBM1. 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 Fo Cut5a- Tr CBM1 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.

Source: bioRxiv (Cold Spring Harbor Laboratory)

DOI: 10.64898/2026.10.15.759203