Microbial degradation of lignin: how a bulky recalcitrant polymer is efficiently recycled in nature and how we can take advantage of this

Abstract

Lignin is the second most abundant constituent of the cell wall of vascular plants, where it protects cellulose towards hydrolytic attack by saprophytic and pathogenic microbes. Its removal represents a key step for carbon recycling in land ecosystems, as well as a central issue for industrial utilization of plant biomass. The lignin polymer is highly recalcitrant towards chemical and biological degradation due to its molecular architecture, where different non-phenolic phenylpropanoid units form a complex three-dimensional network linked by a variety of ether and carbon-carbon bonds. Ligninolytic microbes have developed a unique strategy to handle lignin degradation based on unspecific one-electron oxidation of the benzenic rings in the different lignin substructures by extracellular haemperoxidases acting synergistically with peroxide-generating oxidases. These peroxidases poses two outstanding characteristics: (i) they have unusually high redox potential due to haem pocket architecture that enables oxidation of non-phenolic aromatic rings, and (ii) they are able to generate a protein oxidizer by electron transfer to the haem cofactor forming a catalytic tryptophanyl-free radical at the protein surface, where it can interact with the bulky lignin polymer. The structure-function information currently available is being used to build tailor-made peroxidases and other oxidoreductases as industrial biocatalysts.

Figure 1

Three classical and two acylated lignin precursors or monolignols (top), and structural model for gymnosperm lignin (bottom). Gymnosperms produce the simplest lignin type formed only by guaiacyl units derived from coniferyl alcohol (2). In contrast, angiosperm lignin also include p‐hydroxyphenyl and sinapyl units derived from p‐coumaryl (1) and sinapyl (3) alcohols, as well as a variable amount of acylated lignin often derived from sinapyl alcohol γ‐esterified with acetic (4), p‐coumaric acid (5) or other organic acids (Ralph et al., 2004; Martínez et al., 2008). A variety of ether and carbon–carbon inter‐unit linkages are formed during monolignol radical polymerization resulting in β‐O‐4′ (A), phenylcoumaran (B), pinoresinol (C) and dibenzodioxocin (D) substructures, among others. Linkages to additional lignin chains are indicated (L‐containing circles). Other minor structures (in brackets) include vanillin, coniferyl alcohol and dimethylcyclohexadienone‐type units, the latter in new spirodienone substructures (Zhang et al., 2006) (courtesy of G. Gellerstedt).

Figure 2

Pictorial scheme of the enzymatic degradation of plant cell‐wall lignin (L‐containing circles represent the remaining lignin polymer) by Pleurotus VP, with contribution of extracellular flavooxidases (such as AAO) generating hydrogen peroxide during redox cycling of non‐phenolic aromatic aldehydes (such as the fungal metabolite p‐anisaldehyde) with participation of intracellular aryl‐aldehyde dehydrogenase. Peroxidase one‐electron oxidation of lignin units (the key step in the degradative process) results in an unstable cation radical that experiences different reactions including breakdown of C_α–C_β and C₄–ether linkages releasing the corresponding aromatic aldehydes (vanillin in the case of guaiacyl units) that can be intracellularly mineralized. In the case of P. chrysosporium LiP, lignin attack requires the presence of veratryl alcohol, probably as an enzyme‐bound mediator, and hydrogen peroxide is mainly generated by glyoxal oxidase.

Figure 3

General catalytic cycle of peroxidases (Dunford, 1999). The cycle includes two‐electron oxidation of the enzyme resting state (RS, containing Fe³⁺) by hydroperoxide to yield compound‐I (C‐I; containing Fe⁴⁺‐oxo and porphyrin cation radical), whose reduction in two one‐electron steps results in the intermediate compound‐II (C‐II; containing Fe⁴⁺=O after porphyrin reduction) and then the resting form of the enzyme, with concomitant oxidation of two substrate molecules (S; which could be low‐redox‐potential phenols and dyes, or Mn²⁺ in the cases of MnP and VP).

Figure 4

Two different views of the solvent access surface in a ligninolytic peroxidase (P. eryngii VP; PDB entry 2BOQ) revealing (left) the main haem access channel enabling hydrogen peroxide entrance for activation of the haem cofactor (in yellow) located in a central pocket (low‐redox‐potential phenols and dyes can also be oxidized at this channel albeit with low efficiency), and the Mn²⁺‐oxidation channel formed by three acidic residues (Glu‐36, Glu‐40 and Asp‐175); as well as an approximately 180° rotated view (right) of the same peroxidase showing the partially exposed side‐chain (yellow van der Waals spheres including hydrogen atoms) of the catalytic tryptophan (Trp‐164) involved in oxidation of high‐redox‐potential compounds, such as veratryl alcohol (VA) and lignin models, as well as in high‐efficiency oxidation of some phenols and dyes, by long‐range electron transfer (LRET) to the haem cofactor (surface colours correspond to electrostatic charge).

Figure 5

Details of haem environment and other structurally and catalytically relevant residues in P. eryngii VP. His‐169 (the fifth ligand of haem iron), Phe‐186 and Asp‐231 (corresponding to His‐176, Phe‐193 and Asp‐238 in LiP‐H8) are shown at the proximal side of the haem, while His‐47, Phe‐46, Arg‐43 and Asn‐78 (corresponding to His‐47, Phe‐46 and Arg‐43 in LiP‐H8) are shown at the distal side. Glu‐36, Glu‐40 and Asp‐175 (corresponding to Ala‐36, Glu‐40 and Asn‐182 in LiP‐H8) constitute the site of oxidation of Mn²⁺ (red van der Waals sphere) near the internal propionate of haem, while Trp‐164 (corresponding to Trp‐171 in LiP‐H8) is responsible for oxidation of lignin units and other aromatic compounds by LRET (red arrow) to the activated haem cofactor via Leu‐165 (corresponding to Leu‐172 in LiP‐H8). Finally, the ligands of the two structural Ca²⁺ ions (green spheres) are indicated at the proximal (Ser‐170, Asp‐187, Thr‐189, Val‐192 and Asp‐194) and distal (Asp‐48, Gly‐60, Asp‐62 and Ser‐64) sides (corresponding, respectively, to Ser‐177, Asp‐194, Thr‐196, Ile‐199 and Asp‐201; and Asp‐48, Gly‐66, Asp‐68 and Ser‐70 in LiP‐H8). Several water molecules are also shown including those completing Mn²⁺, Ca²⁺ and haem Fe³⁺ coordination.

Figure 6

Extended catalytic cycle proposed for ligninolytic peroxidases (LiP and VP). In addition to normal compound‐I and compound‐II of Fig. 3 (now C‐I_A and C‐II_A), C‐I_B (containing Fe⁴⁺=O and tryptophan radical) and C‐II_B (containing Fe³⁺ and tryptophan radical) are included, being involved in oxidation of high‐redox‐potential compounds such as veratryl alcohol (VA) and lignin units to their corresponding cation radicals. C‐I_B and C‐II_B are formed by LRET to the activated haem cofactor. Adapted from Pérez‐Boada and colleagues (2005).