Structure-guided engineering of a fungal feruloyl esterase enhances both MHET conversion and lignocellulose breakdown

The persistent accumulation of plastic waste has intensified the demand for sustainable waste management strategies. Enzymatic degradation has emerged as a promising approach, especially for polymers with hydrolysable bonds like polyethylene terephthalate (PET). PET-degrading enzymes (PETases) catalyze the breakdown of PET into water-soluble intermediates, primarily mono(2-hydroxyethyl) terephthalate (MHET), while further hydrolysis of MHET to terephthalic acid (TPA) is essential for efficient degradation. Structural analysis of Is MHETase, the benchmark MHETase from Ideonella sakaiensis , reveals structural similarities to feruloyl esterases (FAEs) of the tannase family, involved in lignocellulose deconstruction. Building on the structural homology between Fo FaeC, a FAE from Fusarium oxysporum , and Is MHETase, specific single-point mutations were designed to enhance Fo FaeC's MHETase activity, mimicking Is MHETase active site architecture. Fo FaeC-G122S variant exhibited a 1.3- and 4.4-fold increase in specific activity (0.13 Units/mg enz ) and catalytic efficiency on MHET (26.9 mM −1 min −1 ), respectively, and 2.0-fold higher activity on PET trimer (110.3 μM prod /mg M(HET)3 ). Regarding typical FAE substrates, the variant displayed 2.0- and 1.2-fold higher catalytic efficiency on methyl p -coumarate (M p CA, 6703.1 mM −1 min −1 ) and methyl caffeate (MCA, 6917.8 mM −1 min −1 ), respectively. In PET degradation, supplementation of Is PETase with Fo FaeC-G122S led to a 17-fold increased MHET hydrolysis, achieving its near-complete conversion to TPA (31.4 μΜ TPA /mg PET ). Concerning lignocellulose breakdown, Fo FaeC-G122S exhibited a 2.0-fold higher ferulic acid release rate (26.5 mg FA mg enz −1 mg DSWB −1 h −1 ) from destarched wheat bran compared to the WT, when combined with a GH10 xylanase. • G122S mutation increases Fo FaeC catalytic efficiency on MHET by 4.4-fold. • G122S mutation changes the catalytic profile of Fo FaeC on methyl hydroxycinnamates. • Compared to WT, Fo FaeC-G122S degrades bulky PET model substrate more efficiently. • Fo FaeC-G122S completely degrades MHET released by Is PETase during PET breakdown. • Combined with xylanase, Fo FaeC-G122S releases FA from DSWB 2-fold faster than WT.