Verschoor_2025_Microbiol.Spectr__e0217725

Cutinases are versatile enzymes with substrate promiscuity, making them promising candidates for the degradation of both natural and synthetic polyesters. While bacterial cutinases have been extensively studied, fungal cutinases remain underexplored, particularly in their enzymatic activity beyond their role in plant virulence. In this study, we investigated four cutinases from two Fusarium strains. Both strains displayed activity on Impranil-DLN, revealing their polyester-degrading potential. The strains were identified as Fusarium oxysporum (strain 38) and Fusarium redolens (strain 62). We characterized two cutinases per strain, tentatively called Cut1 and Cut5. Phylogenetic analysis revealed that both Cut1 clustered together in one branch where the Cut5 variants are closely related to the previously characterized bis(2-hydroxyethyl) terephthalate (BHET)-hydrolyzing enzyme FoCut5, providing structural insights and insights into their catalytic potential. We successfully expressed the Cut5 cutinases in Aspergillus niger using a CRISPR-Cas9-based multicopy integration system, resulting in enhanced degradation of Impranil-DLN and tributyrin. Using the same multicopy integration approach, transformants containing multicopy Cut1 variants were obtained but found to produce considerably lower amounts of Cut1, resulting in less activity and disabling further purification. The optimal substrate length, temperature, and pH for both Cut5 enzymes were determined. Additionally, we show activity of the purified Cut5 enzymes on synthetic substrates Impranil-DLN and BHET, suggesting that these fungal cutinases may be valuable for bioremediation. Accelerating the discovery of fungal cutinases and optimizing their expression systems holds promise for future strategies for polymer degradation to reduce agricultural and plastic waste. IMPORTANCE: Cutinases are promising enzymes for a spectrum of applications due to their substrate promiscuity toward both natural and synthetic polymers. This makes them strong candidates for the development of sustainable solutions to battle environmental pollution. Therefore, the successful production and characterization of novel cutinases is fundamental for understanding their mechanisms and potential applications. In this study, we have identified, produced, and characterized two cutinases from different Fusarium species using a multicopy integration system in Aspergillus niger. Structural characteristics and in vivo and in vitro enzyme activity provide a unique insight into the polyester-degrading activity of these enzymes and how they can contribute to more sustainable solutions to our current waste management and pollution challenges.