How a bacteriological tour of northern British Columbia uncovered thermostable ligninases

Fungi decompose lignin. Everyone knows that—from introductory biology textbooks to The Magic School Bus cartoon that my kids watch after school. But have we overlooked the small but unique role of bacteria in lignin degradation?

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Lindsay Eltis, Bill Mohn and colleagues at the University of British Columbia have collected a growing body of evidence that bacteria contribute to lignin degradation [1]. This post details our genome-resolved investigation into thermostable ligninases in bacteria found in BC's remote thermal environments [2].

Sampling for thermal ligninases

Figure 1. Field sampling in northern British Columbia to identify lignin degrading bacteria using stable isotope probing.

As a postdoc in Bill’s lab, my goal was to uncover the role of bacteria in enzymatic deconstruction of lignin in thermal environments. A multi-year effort combining advanced multi-omics, microbiology, and biochemistry revealed potential thermostable bacterial biocatalysts, which we are characterizing to create the next generation of sustainable bio-products.

Why thermal environments? Bacterial delignification in geothermally warmed environments was unstudied, despite being a clear target for identification of thermostable enzymes. Heat-resistant biocatalysts are attractive for use in industrial processes to convert plant biomass to value added chemicals. Because plant biomass is 15-30% lignin, thermostable ligninases can aid in the valorization of plant-based waste streams [3]. Sustainable plant-based fuels and chemicals could unlock the economic potential of lignin, replacing petroleum feedstocks.

To collect the raw material for 13C-lignin incubations, I set out to sample several hot springs in northern British Columbia: including a thermal swamp complex, a 53 oC pool in an abandoned resort, and one we never ended up finding after a sketchy 2.4 km kayak across the Skeena River in a storm (Figure 1). At a paper mill in Crofton, BC, I sampled a giant, 62 oC pile of wood shavings and debris called “hog fuel," a waste product used in biomass-fired power plants. The thermal swamp samples were used to develop a framework for genome-guided metatranscriptomics, that we applied to identify bacterial pathways for catabolism of lignin-derived aromatic compounds [4]. The samples from other sites were used for stable isotope probing, to monitor the microbial assimilation of carbon from lignin.

The key to optimizing stable isotope probing was our in-house production of 13C-labelleled lignin dehydrogenation polymer (DHP) led by postdoc Morgan Fetherolf. We incubated the thermal samples with 13C-DHP at 53-62 oC (Figure 2). Recovery of 13C-enriched DNA using stable isotope probing facilitated genome-resolved metagenomics targeting the organisms that assimilated lignin.

Stable Isotope Probing

Figure 2. Stable Isotope Probing (SIP) using 13C-DHP lignin, demonstrating incorporation into DNA

I extracted, fractionated, and purified 13C-labeled DNA with Thomas Dalhuisen, an undergraduate volunteer in Bill’s lab. Shotgun sequencing of this precious DNA would allow us to reconstruct genomes and enzyme sequences that we hypothesized would belong to lignin degrading bacteria. I turned to Sunita Sinha at the University of British Columbia Sequencing and Bioinformatics Consortium to produce high quality sequences from the nanograms of 13C enriched DNA. A few weeks later, we had hundreds of GB of sequencing data on our lab server ready for assembly.

Recovery of 125 near-complete genomes was essential to identification and classification of novel ligninase candidates. I followed an ensemble approach for genome binning and refinement [5] that identified phylogenetic clusters of Actinomycetes, Firmicutes and Gammaproteobacteria. I was particularly interested in the Actinomycetes genomes, such as those from potentially novel species in family Thermoleophilaceae, which contained abundant laccase-like multicopper oxidases and previously characterized aromatic degradation pathways. We considered evidence for aromatic degradation essential for explaining incorporation of 13C from labelled lignin into a bacterium’s nucleic acids, so I developed a custom suite of protein models to aid in their identification.

Laccase discovery

Figure 3. Identification of potential laccases encoded by 13C-enriched bacterial genomes from thermophilic environments, and activity against lignin substrates.

Copper oxidases are appealing targets for thermostable ligninases because they are a relative simple enzymatic system, and several stable small laccases (SLAC) had previously been identified in mesophilic Streptomyces [6]. Postdoc Laura Navas expressed our target laccases using E. coli, and wrestled a full contingent of four copper atoms into them. Because of Laura’s enzymology expertise, our team was able to demonstrate the thermostability and lignin modification potential of a gammaproteobacterial 2-domain laccase (Figure 3).

There remains a mountain of research to fully harness bacterial ligninases for sustainable lignin valorization, and we are only scratching the surface of how bacteria interact with lignified substrates in the environment. Nevertheless, our multi-disciplinary team applied a well-developed metagenomics-based bioprospecting platform to uncover novel bacterial enzymes for a more sustainable world.

 

  1. Wilhelm RC, Singh R, Eltis LD, Mohn WW. Bacterial contributions to delignification and lignocellulose degradation in forest soils with metagenomic and quantitative stable isotope probing. ISME J. 2019;13: 413–429. doi:10.1038/s41396-018-0279-6
  2. Levy-Booth, D.J., Navas, L.E., Fetherolf, M.M. et al. Discovery of lignin-transforming bacteria and enzymes in thermophilic environments using stable isotope probing. ISME J 2022. https://doi.org/10.1038/s41396-022-01241-8
  3. Ragauskas AJ, Beckham GT, Biddy MJ, Chandra R, Chen F, Davis MF, et al. Lignin Valorization: Improving Lignin Processing in the Biorefinery. Science. 2014;344: 1246843. doi:10.1126/science.1246843
  4. Levy-Booth DJ, Hashimi A, Roccor R, Liu L-Y, Renneckar S, Eltis LD, et al. Genomics and metatranscriptomics of biogeochemical cycling and degradation of lignin-derived aromatic compounds in thermal swamp sediment. ISME J. 2021;15: 879–893. doi:10.1038/s41396-020-00820-x
  5. Sieber CMK, Probst AJ, Sharrar A, Thomas BC, Hess M, Tringe SG, et al. Recovery of genomes from metagenomes via a dereplication, aggregation and scoring strategy. Nat Microbiol. 2018;3: 836–843. doi:10.1038/s41564-018-0171-1
  6. Singh R, Hu J, Regner MR, Round JW, Ralph J, Saddler JN, et al. Enhanced delignification of steam-pretreated poplar by a bacterial laccase. Sci Rep. 2017;7: 42121. doi:10.1038/srep42121

David Levy-Booth

Bioinformatics Specialist, University of British Columbia