Bacterial enzymes for lignin depolymerisation: new biocatalysts for generation of renewable chemicals from biomass
Graphical abstract
Introduction
Lignin is an aromatic heteropolymer comprising 15–30% of the lignocellulose cell wall of plant biomass, and it is the most abundant source of renewable aromatic carbon in the biosphere. Given the need to reduce greenhouse gas emissions in the 21st-century society, there is considerable academic and commercial interest in finding new sustainable biocatalytic routes to fuels and chemicals from renewable sources of carbon such as plant biomass [1]. For aromatic chemicals, lignin is an obvious starting point, but it is a very challenging polymer to deconstruct, owing to the presence of non-hydrolysable ether C–O and C–C bond linkages, poor solubility in aqueous solution, and other technical challenges [2].
The search for microbial enzymes to deconstruct lignin has until recently focussed on white-rot basidiomycete fungi, such as Phanerochaete chrysosporium, that produce extracellular lignin peroxidases, manganese peroxidases, and multi-copper laccases that can attack lignin (see Figure 1) [3]. However, these fungal enzymes are often challenging to express in high yield, and their fungal hosts are not readily amenable to genetic modification for metabolic engineering. Hence, since 2010, there has been a resurgence of interest in lignin-oxidising enzymes from soil bacteria. A number of soil bacteria have been identified that can depolymerise lignin, mainly in the actinobacteria and α- and γ-proteobacteria phyla, albeit less rapidly than the most active basidiomycete fungi [4,5]. This article will describe recent developments in the enzymology of bacterial lignin-degrading enzymes, our current understanding of how they attack lignin, and applications for biotransformation.
Section snippets
Identification and characterisation of bacterial enzymes for lignin depolymerisation
The first bacterial lignin-oxidising enzyme to be identified was peroxidase DypB from Rhodococcus jostii RHA1, a member of the dye-decolourising peroxidases, found in bacteria and fungi [6]. Dyp-type peroxidases have activity for dye decolourisation and also oxidation of a range of phenolic substrates [7]. There are four sub-classes, A–D, based on sequence alignment, of which classes A–C are found in bacteria, and class D is found in fungi [7]. Although many Dyp-type peroxidases have been
Biotransformation of lignin by lignin-depolymerising enzymes
Our understanding of exactly how lignin-degrading enzymes attack polymeric lignin is still very incomplete. As shown in Figure 3, for the β-aryl ether structure which is the major structural unit found in polymeric lignin, there are a number of possible sites for oxidation or oxidative cleavage. In the relatively few cases where the site of reaction has been studied, studies may be based on the use of lignin dimer model compounds, or the site of reaction implied by structure of low molecular
Lignin degradation accessory enzymes
One limitation of using recombinant lignin-oxidising enzymes for in vitro biotransformation of lignin substrates is that dimerization or repolymerisation is often observed, due to the formation of phenoxy radicals that spontaneously recombine. Therefore, it is likely that there are accessory enzymes in vivo that can trap phenoxy radicals via one-electron reduction. One candidate enzyme for this activity has been recently identified, a highly expressed extracellular dihydrolipoamide
Protein engineering studies
Two recent reports describe the application of directed evolution methods to bacterial Dyp-type peroxidase enzymes. Brissos et al. report the engineering of P. putida DyP using error-prone polymerase chain reaction, giving a mutant enzyme containing three mutations (E188K, A142V, H12V), each on the surface of the enzyme (see Figure 4), that enhance kcat/KM for 2,6-dimethoxyphenol by 100-fold, and shift the optimum pH to 8.5 [49]. Rahmanpour et al. report the use of focused libraries around the
Conflict of interest statement
Nothing declared.
Acknowledgements
Research in the author’s laboratory is supported by research grants from BBSRC (research grant BB/P01738X/1) and the European Union Horizon 2020 research and innovation programme (Bio-Based Industries Joint Undertaking, grant agreement 720303).
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