Synthetic biology-guided modular engineering of the secreted cutinase FoCut5a with a carbohydrate-binding module improves poly(ester-urethane) depolymerization in Yarrowia lipolytica
- 1Department of Biosciences, Biotechnologies and Environment, University of Bari Aldo Moro, Via E. Orabona 4, 70125 Bari, Italy
Abstract
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.
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BibTeX
@misc{messina2026synthetic,
title = {{Synthetic biology-guided modular engineering of the secreted cutinase FoCut5a with a carbohydrate-binding module improves poly(ester-urethane) depolymerization in Yarrowia lipolytica}},
author = {E. Messina and M. Papi and S. Barile and R. De Rosa and P. Scarcia and A. Biundo and I. Pisano and E. Topakas and G. Agrimi},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.15.759203},
url = {https://www.biorxiv.org/content/10.64898/2026.10.15.759203v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Synthetic biology-guided modular engineering of the secreted cutinase FoCut5a with a carbohydrate-binding module improves poly(ester-urethane) depolymerization in Yarrowia lipolytica
AU - E. Messina
AU - M. Papi
AU - S. Barile
AU - R. De Rosa
AU - P. Scarcia
AU - A. Biundo
AU - I. Pisano
AU - E. Topakas
AU - G. Agrimi
PY - 2026
DA - 2026/10/15
PB - bioRxiv
DO - 10.64898/2026.10.15.759203
UR - https://www.biorxiv.org/content/10.64898/2026.10.15.759203v1
AB - 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.
ER -