Contributed by Tim Bugg, Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.

 

Lignin, the aromatic polymer that binds together cellulose and hemicellulose in plant cell walls, is one of the big unsolved problems in the “biorefinery concept”, whereby plant biomass could be used to provide renewable fuels and chemicals for society and the chemicals industry. While great progress has been made in converting cellulose to cellulosic biofuels, and hemi-cellulose to useful fermentation products, lignin is still the problem child that no one quite knows what to do with. It is produced as a by-product of pulp/paper manufacture, and increasingly produced from cellulosic biofuel manufacture, but it is not susceptible to hydrolytic breakdown, and is a rather inert, insoluble material that is usually just burnt to provide heat. But its content of aromatic rings represents a potential source of renewable aromatic chemicals, so surely we could do something better with it than burning it?

 

Since the 1980’s scientists have studied micro-organisms that can break down lignin, the most active organism being white-rot fungus Phanerochaete chrysosporium, which produces an arsenal of extracellular lignin peroxidase and manganese peroxidase enzymes, with other fungi producing copper-dependent laccases. However, the discovery of these enzymes hasn’t yet translated into a commercial process for conversion of lignin into renewable chemicals, partly because fungal enzymes are hard to over-express in large quantities. Hence there has been renewed interest in soil bacteria that can attack lignin. For many years no bacterial lignin-oxidising enzyme had been identified, but in the last few years several bacterial enzymes have emerged. Members of the dye-decolorizing peroxidase (DyP) family of peroxidases have been identified that have activity for oxidation of lignin and lignin model compounds: DypB from Rhodococcus jostii RHA1 was identified by my research group in collaboration with Lindsay Eltis at UBC, and Michelle Chang’s group at UC Berkeley have identified a Dyp2 enzyme from Amycolatopsis sp. 75iv2. Bacterial laccases are also found, particularly in actinobacteria, and John Gerlt’s group at U Illinois have shown that laccase enzymes in Streptomyces A3(2) are involved in lignin breakdown.

 

Can we use these enzymes as biocatalysts to transform lignin into renewable chemicals? It seems so obvious, and yet, unfortunately, it’s not as simple as that, because the same enzymes that depolymerise lignin also polymerise the radical intermediates formed, generating higher molecular weight material. So you get competing depolymerisation and repolymerisation, in other words, one step forward and two steps back. Somehow Nature appears to solve this problem, we don’t know exactly how at present.

 

So could we use microbial fermentation to transform lignin into chemicals? This approach is starting to yield some interesting results. Although our knowledge of the metabolic pathways for lignin breakdown is very incomplete, there are indications that vanillic acid is one key intermediate in lignin breakdown. In Rhodococcus jostii RHA1, the group of Lindsay Eltis identified the genes responsible for oxidation of vanillin to vanillic acid, and then demethylation to protocatechuic acid, and generated gene deletion strains lacking these genes. When we tested the gene deletion strain for vanillin dehydrogenase, we found that when we grew this strain on minimal media containing chopped wheat straw, we could detect vanillin (for which there is a market in the food/flavour industry) as a metabolite. Under optimised conditions we obtained a yield of 96 mg/litre vanillin, not quite an industrially useful yield, but a big step forward, showing that synthetic biology could in principle be used to generate aromatic chemicals from lignin breakdown. We now hope to use this kind of approach to generate other aromatic compounds for industrial applications, and my group is collaborating with Biome Bioplastics to try to generate products from lignin that could be used to make new bio-based plastics to replace oil-based plastics, as described on:

 

 

There is still a long way to go: we still don’t fully understand the degradation pathways, we know little about how lignin degradation is regulated, and how lignin fragments are taken up into bacterial cells. But that also makes it an interesting area for study, and despite a strong dose of scepticism that I’ve encountered sometimes, I personally believe that it will be possible to convert lignin into renewable chemicals, maybe using biocatalysis, or chemocatalysis, or some combination of the two approaches. Not only do I think it’s possible, but I think we have to find a way to do this, to provide an alternative to petrochemicals for tomorrow’s Society.

 

References

 

Bugg TDH, Ahmad M, Hardiman EM, Singh R (2011). The emerging role for bacteria in lignin degradation and bio-product formation. Curr. Opin. Biotech., 22, 394-400.

 

Ahmad M, Roberts JN, Hardiman EM, Singh R, Eltis LD, Bugg TDH (2011). Identification of DypB from Rhodococcus jostii RHA1 as a lignin peroxidase. Biochemistry, 50, 5096-5107.

 

Brown ME, Barros T, Chang MCY (2012). Identification and characterization of a multifunctional dye peroxidase from a lignin-reactive bacterium. ACS. Chem. Biol., 7, 2074-2081.

 

Majumdar S, Lukk T, Solbiati JO, Bauer S, Nair SK, Cronan JE, Gerlt JA (2014). Roles of small laccases from Streptomyces in lignin degradation. Biochemistry, 53, 4047-4058.

 

Sainsbury PD, Hardiman EM, Ahmad M, Otani, H, Seghezzi N, Eltis LD, Bugg TDH (2013). Breaking down lignin to high-value chemicals: the conversion of lignocellulose to vanillin in a gene deletion mutant of Rhodococcus jostii RHA1. ACS Chem. Biol., 8, 2151-2156.

 

For podcast on lignin degradation see: http://www2.warwick.ac.uk/fac/sci/chemistry/research/bugg/bugggroup/research/lig nin/

 

 

 

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