Structure-guided engineering of a fungal feruloyl esterase enhances both MHET conversion and lignocellulose breakdown
Konstantinos Makryniotis, Markella Papi, Efstratios Nikolaivits, Evangelos Topakas
Abstract
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.