An Engineered Laccase from Fomitiporia mediterranea Accelerates Lignocellulose Degradation

Abstract

Laccases from white-rot fungi catalyze lignin depolymerization, a critical first step to upgrading lignin to valuable biodiesel fuels and chemicals. In this study, a wildtype laccase from the basidiomycete Fomitiporia mediterranea (Fom_lac) and a variant engineered to have a carbohydrate-binding module (Fom_CBM) were studied for their ability to catalyze cleavage of β-O-4' ether and C-C bonds in phenolic and non-phenolic lignin dimers using a nanostructure-initiator mass spectrometry-based assay. Fom_lac and Fom_CBM catalyze β-O-4' ether and C-C bond breaking, with higher activity under acidic conditions (pH < 6). The potential of Fom_lac and Fom_CBM to enhance saccharification yields from untreated and ionic liquid pretreated pine was also investigated. Adding Fom_CBM to mixtures of cellulases and hemicellulases improved sugar yields by 140% on untreated pine and 32% on cholinium lysinate pretreated pine when compared to the inclusion of Fom_lac to the same mixtures. Adding either Fom_lac or Fom_CBM to mixtures of cellulases and hemicellulases effectively accelerates enzymatic hydrolysis, demonstrating its potential applications for lignocellulose valorization. We postulate that additional increases in sugar yields for the Fom_CBM enzyme mixtures were due to Fom_CBM being brought more proximal to lignin through binding to either cellulose or lignin itself.

Keywords: Fomitiporia mediterranea; Komagataella pastoris expression; laccase; lignin; nanostructure-initiator mass spectrometry (NIMS).

Figure 1

Product distribution from bond cleavage of fluorous-tagged GGE (A) and VGE (B) by wildtype Fom_lac. The reaction contained 1 mM of NIMS-tagged lignin dimer, 5 µM of Fom_lac enzyme, and 20 mM of 1-hydroxybenzotriole as mediator and was performed in sodium acetate buffer pH 2.0–10.0. Error bars are the standard deviation for three replicates.

Figure 2

The proposed scheme of Fom_lac laccase catalyzed depolymerization and ΔG. Relative Gibbs free energy of reaction (kcal/mol) was calculated for various bond types via cationic radical intermediate from phenolic lignin dimer (A) and from non-phenolic lignin dimer (B).

Figure 3

Bond-cleavage frequency computed by AIMD simulation of non-protonated and protonated OHs GGE (A) and VGE (B) cationic radicals for different linkages.

Figure 4

Three-dimensional visualization of Fom_CBM molecule in the solvent box for M.D. simulations (A) and per-residue root mean square fluctuation (RMSF) for different CBM domains when fused with Fom_lac through polykinker (B). CBM1—Endoglucanase I (gene egl1) from Trichoderma reesei; CBM2—Endoglucanase II (gene egl2) from Trichoderma reesei; CBM3—Endoglucanase V (gene egl5) from Trichoderma reesei; CBM4—Endoglucanase from Myceliophthora thermophila; CBM5—Exoglucanase I (gene CBHI) Trichoderma reesei; CBM6—Exoglucanase I (gene CBHI) from Phanerochaete chrysosporium; CBM7—Exoglucanase I (gene CBHI) Trichoderma viride; CMB8—Exoglucanase II (gene CBHII) from Trichoderma reesei; CBM9—Exoglucanase 3 (gene cel3) from Agaricus bisporus; CBM10—Exoglucanase 1 from Trichoderma reesei; CBM11—Exoglucanase 1 from Humicola grisea var. thermoidea.

Figure 5

Product distribution from bond cleavage of fluorous-tagged GGE (A) and VGE (B) by Fom_lac fused with CBM—Fom_CBM. The reaction contained 1 mM of NIMS-tagged lignin dimer, 5 µM of Fom_lac enzyme, and 20 mM of 1-hydrobenzotriole as mediator and was performed in sodium acetate buffer pH 2.0–10.0. Error bars are the standard deviation for three replicates.

Figure 6

Synergistic effect of laccase and cellulases/hemicellulases in saccharification of different pine biomasses. (A) Untreated pine biomass with particle size > 250 µm. (B) Untreated pine biomass with particle size < 250 µm. (C) [Ch][Lys]-treated pine. Saccharification conditions: loading 2.5 wt%, 10 mg enzyme (CTec3:HTec3, 9:1 v/v) per g pine, 5 uM For_lac or Fom_CBM, in 0.1 M sodium acetate at pH 5.0, 50 °C, 72 h. Mediator: HBT: 1-Hydroxybenzotriazole (5 mM). Pretreatment conditions: pine (20 wt%), IL (80 wt%), 140 °C, 3 h.

Figure 7

Proposed mechanism of lignin-catalyzed Fom_lac in the presence/absence of CBM. (A) Long-distance electron transfer through an aqueous solution is inefficient from lignin to mediator. (B) CBM brings Fom_lac in close contact with lignin and shortens the electron transfer pathway between mediator and lignin.