Learning from marine wood borers; enzymes and mechanisms of lignocellulose digestion

Lead Research Organisation: University of York
Department Name: Biology

Abstract

The development of sustainable biofuels based on woody (lignocellulosic) plant biomass is generally seen as an essential element in the move to a low carbon economy within the constraints of food security. The key to unlock this potential lies in developing technologies for the cost effective deconstruction of lignocellulose into sugars for fermentation. A number of animals have evolved to live on the large amounts of woody biomass that enter the marine environment from river estuaries. Such animals may provide useful understanding and enzymes for lignocellulose deconstruction, as they thrive on a diet of wood, and digest it under ambient temperatures and pressures.

The project builds on an existing programme funded as part of the BBSRC Sustainable Bioenergy Centre that has focused on Limnoria quadripunctata, a wood borer, which is unusual in having a digestive tract free of microbial life. Our studies indicate that this animal has a gut with an inert chitinous lining in which free radical chemistry is deployed as a pretreatment for enzymatic hydrolysis, and in which enzymes are produced in a separate organ and transposed to the gut, which functions as an enzyme reactor. In the programme we have established a wide range of specialist capabilities in the area of producing and characterising recombinant secreted Eukaryotic enzymes, as well as methods for studying digestive processes in vivo.

Building from this basis of knowledge, skills and resources, we wish to expand the work to take in a wider range of wood-eating marine invertebrates. Our initial understanding was that the microbe-free gut was peculiar to the Limnoriids, but recent studies at Portsmouth show a similar situation in a marine amphipod, Chelura telebrans. We have now obtained substantial EST databases from the digestive tracts of three arthropods, two Limnoriids (L. quadripunctata and L. lignorum), and C. telebrans. Initial scrutiny reveals some broad similarities in the suite of obvious digestive genes expressed in the three, but also considerable divergence in the range and relative expression levels of these genes. We will undertake detailed comparative gut and hepatopancreas transcriptomic and proteomic studies to identify the range of proteins involved in digestion and also study the biochemical processes in the gut. C. terebrans has the advantage of being considerably larger than the Limnoriid species, which will make these studies easier to undertake.

In addition to crustacean wood borers, we also propose to initiate studies in Lyrodus pedicellatus, a representative of the shipworms. Shipworms are large bivalve molluscs with little relationship to the arthropods. The bivalve shells of shipworms have evolved into a highly effective rasp at the anterior of the animal, with which they bore through wood, consuming particles as they go. These animals have a complex and poorly understood digestive that includes a large chamber where wood particles are stored and a region where wood particles are phagocytosed indicating intracellular digestion. In addition, shipworms harbour endo-symbiotic bacteria in their gills that have been suggested to aid in digestion, and have intestinal microflora that could also be involved. We will investigate genes and enzymes expressed, and biochemical processes in different regions of the shipworm digestive system in order to identify new enzymes for study. We will produce recombinant forms of hydrolytic and oxidative enzymes involved in wood digestion and characterise these in detail in terms of structure and activity. We will work closely with industry to examine the commercial potential of new enzymes and pretreatment approaches developed in the project.

Technical Summary

Our previous work has studied a marine wood boring crustacean (Limnoria quadripunctata) that has a digestive system that is free of microbial life This work has shown that L. quadripunctata has a two compartment digestive system, where enzymes for digestion are produced in the hepatopancreas, while wood digestion occurs in the hindgut, which is protected by the food mass being encased in a non-living lining. The hindgut environment has high levels of reactive oxygen species and is an oxygen sink, suggesting that oxidative chemistry is being used to attack the lignocellulose, and this may also be responsible for the lack of microbes. We have been characterising enzymes from the digestive system and studied a new animal GH7 cellulase in considerable detail. This enzyme shows remarkable robustness with higher activity in 3M NaCl than at lower concentrations. The enzyme has an unusually high density of acidic residues at its surface, and this feature is seen in other enzymes from this system and may be associated with robustness necessary in the inhospitable gut conditions.

We will build on our previous work and the resources established during it. We will extend our studies to two other crustacean wood borers, L. lignorum and Chelura terebrans, which we find to have microbe free digestive systems. We will also include a shipworm, a wood boring mollusc, that appears to use very different approach to wood digestion but has been little studied. We will use transcriptomic, proteomic, biochemical, microscopic and spectroscopic studies of the digestive systems in these organisms to provide understanding of the processes and identify interesting enzymes for deeper study. We will produce recombinant version of lignocellulose active enzymes that will be studied in structural and biochemical detail. We will work with our industrial partner to investigate the commercial potential of these enzymes, and test them at scale using pilot scale biorefinery facilities.

Planned Impact

This programme fits well with the BBSRCs strategic priorities in Industrial Biotechnology and Bioenergy. The provision of a source of sugars that can form the platform for fermentative production of biofuels and platform chemicals without having negative consequences for food security is a major challenge of our times. The work in this project is explicitly aimed at identifying new enzymes and processes for the saccharification of non-food lignocellulosic biomass.

The work described in this proposal will most immediately benefit the private commercial sector by identifying and developing new enzymes for use in industrial biotechnological applications. The primary beneficiaries will be those in the enzyme industry, however, if this work significantly improves lignocellulose saccharification, its impacts will trickle down to many areas of the developing knowledge-based bio-economy by providing cost-competitive lignocellulosic sugars for multiple applications. In addition, cellulases and related carbohydrate active enzymes are used very widely in other industrial sectors including, pulp and paper, textiles, laundry detergents and the food industry, and our work may have impacts in any or all of these.

Our work will benefit other researchers working in the areas of enzymes, polysaccharides, biofuels and industrial biotechnology and enzymes structure and function, by providing novel data on new examples of lignocellulose active enzymes from animals and particularly from the marine environment, a largely underexplored area.

Our work could have long-term benefits for the environment and society at large by helping to decrease our reliance on fossil resources and help provide lower carbon routes for the production of fuels and industrial chemical with minimal impacts on food security. This work therefore has potentially important impact from the political perspective, potentially helpingthe UK to reach its targets for reduced carbon emissions.
We have assembled an extremely strong group of scientists to maximise the chances of the full promise of the scientific research being realised. In addition, we are committed to seeing that the impacts of the work are maximised. This will in part be achieved by working closely with our industrial partner to ensure that enzymes have a route for rapid industrial uptake. In addition, the team has a good records in terms of public outreach and communication. For example SMM has appeared in two recent Research Council videos and is a member of the BBSRC Bioenergy Outreach Group. The research on marine wood borers provides an excellent means of catching public interest as witnessed by numerous popular press articles that have been published on the BSBEC Marine Wood Borer Programme.

The project management will use proven processes to protect IP and publish results in scientific journals and at conferences. We will also use existing UK networks (eg the Bioscience KTN, the NNFCC) to communicate progress through their events and web-based or printed media. When appropriate, discoveries will be disseminated by the University to the general media through press releases. To ensure professional management of intellectual property, CNAP operates regular IP reviews of all projects. CNAP has an outstanding track record in commercialisation of strategic research through on-going collaborations with companies throughout the biorenewable supply chain.
The programme will provide researchers and PhD students with wide-ranging skills relevant to the establishment of a vibrant industrial biotechnology and bioenergy research and innovation-led industrial sector in the UK. We will encourage the researchers and students to attend networking meetings organised by BSBEC and other relevant networks that may be established through the new BBSRC new initiatives in industrial biotechnology and bioenergy.

Publications

10 25 50
 
Description We have begun to uncover the genes encoding enzymes for wood digestion in the shipworm Lyrodus pedicellatus. Previous studies have described a major role for bacterial proteins in the digestive process of wood in these animals. We have used in-depth transcripromic and proteomic studies to reveal the proteins involved in digestion. The caecum is the major site of wood digestion in shipworms and in Lyrodus we found that more than 80% of the protein in this organ are produced by the animal itself and include a range of recognisable glycosyl hydrolases as well as a number of novel uncharacterised proteins likely to be involved in the digestive process. A notable number of bacterial proteins are present and these belong to a narrow group of glycosyl hydrolases. The animals digestive system also includes a number of other organs and the roles of these in wood digestion is beginning to emerge. We hope to submit two papers on this topic in the coming year. We have discovered a new class of lytic polysaccharide monooxygenases from Thermobia domestica and a paper has been published in Nature Communications. A first paper on the shipworm digestive system has been published in Biotechnology for Biofuels. A second paper on the shipworm digestive system is almost ready for submission. We are about to submit a manuscript revealing the ability of of hemocyanins from gribble to serve as phenoloxidases that pretreat wood and greatly enhance cellulase activity. This work has now been published in Nature Communications. A manuscript on the transmission of lignocellulose digestive enzymes from bacteruia in the shipworm gills is under review in PLOS Biology.
Exploitation Route We anticipate that some of the enzymes we are working on will eventually find applications in areas of industrial biotechnology, especially the production of fuels and chemicals from woody plant biomass.
Sectors Agriculture, Food and Drink,Energy,Manufacturing, including Industrial Biotechology

 
Description We work closely with Novozymes, a global enzyme company, who are actively expressing some of our target proteins and will be examining their commercial potential.Two notable new enzymes have been identified and characterised. The first is a new family of lytic polysaccharide monooxygenases (LPMOs), the first such enzymes described in animals. These were discovered in an ancient insect called Themobia domestica,a detritivore with a remarkable capability to live on crystalline cellulose. We found that almost 20% of its digestive proteome is made up of a family of novel LPMOs that are active on both cellulose and chitin and increase the digestion of these polymers by cellulases or chitinases by up to 100 fold. This recently led to a further discovery of another new LPMO family in pathogenic oomycetes, which are the first LPMOs described that are active on pectins. Our industry partner is investigating potential commercial applications of these enzymes. We also recently revealed that Limnoria tripunctata (gribble) use hemocyanins to disrupt lignin during wood digestion. Treatment of wood with hemocyanins greatly increses the digestibility of wood, providing a key step in wood digestion by this animal. We have published two papers in Nature Communications on this work so far, with other good papers in progress. Our work on shipworms has revealed for the first time, the means by which lignocellulose-active enzymes produced by bacteria in the animal's gills reach the site of wood digestion in the caecum. This involves sloughing of bacteriocytes from the animals gills, and transmission of the bacteriocytes and bacteria to the digestive system in a mucus stream along the food groove. A manuscript has been submitted on this topic.
First Year Of Impact 2018
Sector Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology
 
Description EN275 Working Group
Geographic Reach Europe 
Policy Influence Type Membership of a guideline committee
Impact Working group EN275 is undertaking the updating of the European Norm for testing wood products for use in the marine environment. The involvement of SC in the meetings of this working group have lead to plans to adopt a laboratory testing method founded on the insights into marine borer digestion developed through BBSRC-funded projects on lignocellulose degradation.
 
Description BBSRC GCRF IAA 'Impact Enhancement Award'
Amount £7,360 (GBP)
Funding ID BB/GCRF-IAA/23 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2017 
End 03/2017
 
Description Developing rice straw for animal feed
Amount £749,043 (GBP)
Funding ID BB/P022499/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2017 
End 04/2019
 
Description MaxBio - Maximizing Conversion Yields in Biorefining
Amount £443,648 (GBP)
Funding ID BB/N023269/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2016 
End 03/2020
 
Description Omnivore: a multi-feedstock biorefinery
Amount £225,085 (GBP)
Funding ID BB/P02372X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2017 
End 12/2018
 
Description Port of Rotterdam
Amount £16,000 (GBP)
Organisation Port of Rotterdam 
Sector Private
Country Netherlands
Start 11/2015 
 
Description Research Council of Norway
Amount £250,000 (GBP)
Organisation Research Council of Norway 
Sector Public
Country Norway
Start 07/2017 
End 07/2020
 
Description Sustainable production of compost, biofuels and chemicals from oil palm residues
Amount £119,256 (GBP)
Funding ID BB/P027717/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2017 
End 02/2019
 
Title GenBank Chelura GH7A 
Description Etxabe, A.G., Elias, L., Kern, M., Cragg, S.M. and McQueen-Mason, S.J. (2013) Chelura terebrans family 7 cellobiohydrolase (GH7A) mRNA, complete cds. GenBank: KC776193.1 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact none yet 
 
Title GenBank Chelura GH7D 
Description Etxabe, A., Elias, L., Kern, M., Cragg, S. and McQueen-Mason, S. (2013) Chelura terebrans family 7 cellobiohydrolase (GH7D) mRNA, complete cds. GenBank: KC776194.1 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact none yet 
 
Title Genome of Limnoria quadripunctata 
Description Partially assembled genome of Limnoria quadripunctata 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact Currently a work in progress but has already sparked significant interest from our industrial partner Novozymes 
 
Title PDB: Daphnia GH7 
Description Crystal Structure of a GH7 Family Cellobiohydrolase from Daphnia pulex 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact Manuscript in progress: Ebrahim, A., Hobdey, S.E., Podkaminer, K., Taylor II, L.E., Beckham, G.T., Decker, S.R., Himmel, M.E., Cragg, S.M., McGeehan, J.E. 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4XNN
 
Description Borer and fungal enzymes 
Organisation Virginia Tech
Country United States 
Sector Academic/University 
PI Contribution Generated experimental materials Provided X-ray diffractograms of materials Contribution to end of project workshop Synthesising workshop contributions for review manuscript Synthesized outline proposal for ffollow up project
Collaborator Contribution Developed a methodology for interpreting diffractograms of digested material Contribution to end of project workshop Plans and ideas for follow up project
Impact Multidisciplinary: mycology, zoology, structural biology, wood science Workshop at Linnean Society Outline project proposal to NSF/BIO BBSRC Pilot Conference presentation Manuscript published 2015
Start Year 2012
 
Description Comparative omics 
Organisation Linnaeus Centre for Marine Evolutionary Biology (CeMEB) Gothenburg
Country Sweden 
Sector Academic/University 
PI Contribution Invited presentation to annual review meeting of the Centre Contribution to discussions of future directions and evaluation of past activities. Specific discussions over collaboration regarding marine wood borer studies
Collaborator Contribution Support for travel, meals and accommodation for 3 members of research team for the CeMEB meeting.
Impact Contributed to application from CeMEB collaborators to the Swedish Research Council Formas Presented our proteomic and transcriptomic work to the CeMEB 14th Assembly in 2015
Start Year 2015
 
Description Enzyme ontogeny 
Organisation University of Vienna
Country Austria 
Sector Academic/University 
PI Contribution Preparation of larval and postlarval borers for investigation by confocal microscopy
Collaborator Contribution Multi-fluorophore staining of invertebrate larvae Anatomical interpretation
Impact One manuscript published, second manuscript in review
Start Year 2012
 
Description Hemicellulose degradation 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution Contribution to planning of shared programme.
Collaborator Contribution Contribution to planning of shared programme.
Impact Collaboration is at an early stage. Manuscript published in 2015
Start Year 2014
 
Description Kebony 
Organisation Kebony AS
Country Norway 
Sector Private 
PI Contribution Rapid evaluation by novel laboratory and field protocols of wood borer protection capacity of novel wood modification processes
Collaborator Contribution Development and assessment of of novel wood modification processes
Impact None yet
Start Year 2017
 
Description Kebony 
Organisation Norwegian University of Science and Technology (NTNU)
Department Department of Chemical Engineering
Country Norway 
Sector Academic/University 
PI Contribution Rapid evaluation by novel laboratory and field protocols of wood borer protection capacity of novel wood modification processes
Collaborator Contribution Development and assessment of of novel wood modification processes
Impact None yet
Start Year 2017
 
Description Kebony 
Organisation SP Technical Institute of Sweden
Country Sweden 
Sector Academic/University 
PI Contribution Rapid evaluation by novel laboratory and field protocols of wood borer protection capacity of novel wood modification processes
Collaborator Contribution Development and assessment of of novel wood modification processes
Impact None yet
Start Year 2017
 
Description Kebony 
Organisation Statistics Norway
Country Norway 
Sector Public 
PI Contribution Rapid evaluation by novel laboratory and field protocols of wood borer protection capacity of novel wood modification processes
Collaborator Contribution Development and assessment of of novel wood modification processes
Impact None yet
Start Year 2017
 
Description Kebony 
Organisation University of Copenhagen
Country Denmark 
Sector Academic/University 
PI Contribution Rapid evaluation by novel laboratory and field protocols of wood borer protection capacity of novel wood modification processes
Collaborator Contribution Development and assessment of of novel wood modification processes
Impact None yet
Start Year 2017
 
Description Mangrove wood borers 
Organisation Lee Kong Chian Natural History Museum
PI Contribution Marine wood borer taxonomy and ecology
Collaborator Contribution Expertise with field sites, geographical appreciation of ecosystems, transport and personpower.
Impact Multidisciplinary: zoology, conservation, geography, systematics. Outputs currently consist of collections, but work is ongoing on publication.
Start Year 2017
 
Description Mangrove wood borers 
Organisation National Parks Board
PI Contribution Marine wood borer taxonomy and ecology
Collaborator Contribution Expertise with field sites, geographical appreciation of ecosystems, transport and personpower.
Impact Multidisciplinary: zoology, conservation, geography, systematics. Outputs currently consist of collections, but work is ongoing on publication.
Start Year 2017
 
Description Mangrove wood borers 
Organisation National University of Singapore
Department Department of Geography
Country Singapore 
Sector Academic/University 
PI Contribution Marine wood borer taxonomy and ecology
Collaborator Contribution Expertise with field sites, geographical appreciation of ecosystems, transport and personpower.
Impact Multidisciplinary: zoology, conservation, geography, systematics. Outputs currently consist of collections, but work is ongoing on publication.
Start Year 2017
 
Description Serenissima 
Organisation National Research Council
Country Italy 
Sector Public 
PI Contribution Expertise on marine borers Sending students to assist with taxonomic work
Collaborator Contribution Supply of experimental materials Sharing of field data
Impact Erasmus link Plans for publication to stabilise the taxonomy of one of our experimental species
Start Year 2015
 
Description Shipworm enzymes 
Organisation Northeastern University - Boston
Country United States 
Sector Academic/University 
PI Contribution Identification of endogenous lignocellulose active enzymes in the shipworm Organisation of the workshop on lignocellulose degradation mechanisms at the Linnean Society. Contribution to a manuscript on shipworm taxonomy
Collaborator Contribution Characterisation of the proteome of the digestive tract of the shipworm Contribution to workshop at Linnean Society.
Impact Development of a joint proposal for funding under the NSF/BIO - BBSRC Pilot. Recruitment of a PhD student from Portsmouth to a postdoctoral position at Northeastern University. Manuscript submitted on shipworm taxonomy in 2016.
Start Year 2013
 
Description Entomology conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited presentation to show similarities and differences of crustacean wood digestion mechanisms compared with insect mechanisms
Year(s) Of Engagement Activity 2016
URL http://ice2016orlando.org/
 
Description European Conference on Tropical Ecology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A presentation on the role of marine borers and their wood digestion capabilities in the functioning of mangrove ecosystems.
Year(s) Of Engagement Activity 2018
URL https://www.iyor2018.org/event/2018-european-conference-of-tropical-ecology-challenges-in-tropical-e...
 
Description Janelia Conference: Crustacean Models in Cross-Disciplinary Biological Research 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A gathering of expertise from around the globe to debate under non-disclosure conditions research on genomics of crustaceans.
Year(s) Of Engagement Activity 2017
 
Description Marine Evolution 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Marine Evolution 2018 brought together speakers from across the globe to a meeting with the Linnaeus Centre for Marine Evolutionary Biology. SC presented work on the diversity of digestive enzymes in marine wood-boring crustaceans.
Year(s) Of Engagement Activity 2018
URL https://cemeb.science.gu.se/activities/marine-evolution-2018
 
Description NOC Association annual meeting 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation to 5th Annual Meeting of the National Oceanographic Centre Association entitled:
Marine wood borers in biodegradation, biotechnology and carbon fluxes
This is a forum for users and potential users of the NOC at Southampton where policy makers and funders are present.
Year(s) Of Engagement Activity 2015
URL http://noc.ac.uk/about-us/noc-association/5th-annual-meeting-noc-association-presentations-posters
 
Description Outreach at Botanic Gardens Festival of Plants 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Outreach at Botanic Gardens Festival of Plants - Paul Dupree
Year(s) Of Engagement Activity 2016
 
Description Public lecture (Tjarno Marine Station) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Presentation setting the research of a PhD candidate within the context of research on marine borers in a historical context
Year(s) Of Engagement Activity 2015
 
Description Wallenberg Wood Science Center Meeting, Sweden - June 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Presented a talk at Wallenberg Wood Science Center Meeting
Year(s) Of Engagement Activity 2017
 
Description Wood Borers Meeting - December 2016 (York) 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Wood Borers Meeting
Year(s) Of Engagement Activity 2016