The chemical logic of enzymatic lignin degradation

Abstract

Lignin is an aromatic heteropolymer, found in plant cell walls as 20-30% of lignocellulose. It represents the most abundant source of renewable aromatic carbon in the biosphere, hence, if it could be depolymerised efficiently, then it would be a highly valuable source of renewable aromatic chemicals. However, lignin presents a number of difficulties for biocatalytic or chemocatalytic breakdown. Research over the last 10 years has led to the identification of new bacterial enzymes for lignin degradation, and the use of metabolic engineering to generate useful bioproducts from microbial lignin degradation. The aim of this article is to discuss the chemical mechanisms used by lignin-degrading enzymes and microbes to break down lignin, and to describe current methods for generating aromatic bioproducts from lignin using enzymes and engineered microbes.

Fig. 1. Schematic representation of lignocellulosic biorefinery.

Fig. 2. (A) Structures of lignin substructures, and their occurrence in softwood (SW) and hardwood (HW). (B) Structures of H, G, and S units found in polymeric lignin.

Fig. 3. Active sites of lignin-degrading enzymes, with their respective PDB codes. (A) Phanerochaete chrysosporium LiP (PDB 1LGA), showing heme cofactor (cyan), Fe centre (brown), catalytic residues His-47, Arg-43, and distal His-176 (magenta). (B) Phanerochaete chrysosporium MnP (PDB 1MNP), showing heme cofactor (cyan), Fe centre (brown), bound Mn²⁺ (purple), catalytic residues His-46, Arg-42, and distal His-173 (magenta). (C) Trametes versicolor laccase (PDB 1GYC), showing Cu_A and trinuclear Cu_B centres (cyan). (D) Rhodococcus jostii DypB (PDB 3QNR), showing heme cofactor (cyan), Fe centre (brown), catalytic residues His-153, Arg-244, and distal His-226 (magenta). (E) Streptomyces coelicolor laccase (PDB 7BDN), showing Cu_A and Cu_B centres (cyan). (F) Sphingobium sp. SYK-6 beta-etherase LigE (PDB 4YAN), showing reduced glutathione substrate (sticks) and non-polar active site residues (spacefill). Images prepared using Pymol software.

Fig. 4. Mechanism of Cα–Cβ bond cleavage via radical mechanism.

Fig. 5. Mechanism of LigFG β-aryl ether cleavage. GSH, reduced glutathione; GSSG, oxidised glutathione. β-Etherase LigE catalyses a similar reaction to LigF, on the R enantiomer.

Fig. 6. Other possible mechanisms for β-O-4 cleavage, via distal C–O cleavage (panel A), or radical elimination (panel B). Compounds 1 and 2 (cpd1, cpd2) are shown in Fig. 4. Electron acceptor for 2nd step would be compound 2.

Fig. 7. Mechanisms for aryl–Cα cleavage, by fungal LiP or bacterial DyP (panel A); or by Sphingobacterium sp. T2 SpMnSOD (panel B), also showing mechanism for demethylation. Compounds 1 and 2 (cpd1, cpd2) are illustrated in Fig. 4.

Fig. 8. Degradation of diarylpropane (β-1) unit and pinoresinol (β–β) unit via lignostilbene.

Fig. 9. Strategies to provide physical access for oxidants to lignin polymer. LiP, fungal lignin peroxidase; MnP, fungal manganese peroxidase; DyP, bacterial dye-decolorizing peroxidase; MCO, multi-copper oxidase; SpMnSOD, Sphingobacterium sp. T2 manganese superoxide dismutase; Med, mediator; Trp, surface tryptophan residue.

Fig. 10. Structures of condensed units found in Kraft lignin and lignosulfonate, formed from β-aryl ether units (X = O-aryl) and diarylpropane units (X = aryl).

Fig. 11. Mechanism of phenoxy radical trapping by FAD-dependent dihydrolipoamide dehydrogenase containing an active site disulfide. ArOH represents a second 1-electron reduction of a phenoxy radical to a phenol.

Fig. 12. Roles of accessory enzymes in lignin degradation.

Fig. 13. Production of bioproducts from lignin using engineered recombinant bacterial strains. Aromatic products shown in blue, non-aromatic products shown in brown.