New tools for the realization of cost-effective liquid biofuels from plant biomass (revised costs)

Lead Research Organisation: University of Portsmouth
Department Name: Sch of Biological Sciences

Abstract

The production of liquid biofuels from lignocellulosic biomass offers the potential to provide liquid transportation fuels in an environmentally benign manner without compromising food security. Lignocellulose is largely composed of polysaccharides that can be converted into simple sugars and used to produce alcohols such as ethanol and butanol by microbial fermentation. Production of such liquid biofuels from plant biomass is currently hampered by the cost of converting lignocellulose into fermentable sugars (saccharification). There is a clear need for new and better enzymes for lignocellulose saccharification. A number of animals such as termites can survive on a diet of lignocellulose, suggesting they have overcome the problem of obtaining sugars from this recalcitrant substrate. These organisms generally rely on a population of bacteria and protists in their digestive tract that help to digest the lignocellulose. An exception to this rule is found in the Limnoriidae (also known as gribble), small crustacean wood borers from the marine environment. These animals can survive on a diet of lignocellulose and are unusual in having an effectively sterile digestive tract. This suggests that not only can Limnoria digest lignocellulose with their own enzymes, but that conditions within the digestive tract, associated with lignocellulose digestion, prevent microbes from becoming established. The unusual nature of lignocellulose digestion in Limnoria indicates a great potential for uncovering new insights and approaches to saccharification and new enzymes and genes for industrial applications. By analogy, the termite digestive tract can be seen as a complex microbial reactor for lignocellulose digestion, whereas the Limnoria gut is an enzyme reactor, and thereby much closer in its nature to current industrial systems. We have used deep transcriptomic sequencing of the digestive tract of Limnoria in order to reveal the genes expressed during lignocellulose breakdown. We sequenced more than 280,000 cDNAs revealing a breathtaking insight into this process. The Limnoria gut transcriptome is dominated by genes encoding several major classes of protein. Genes encoding glycosyl hydrolases (enzymes that convert polysaccharides into sugars) account for almost 30% of cDNAs, and putative cellulases and cellobiohydrolases (including some never before seen before in animal genomes) account for almost 25% of the transcriptome. A number of other protein classes are represented at very high abundance suggesting they may be involved in the digestive process. The programme of work presented here aims to identify the key mechanisms and components of lignocellulose digestion in Limnoria, in order that we can apply principles and enzymes from this process in order to enhance industrial lignocellulose saccharification. To better determine the roles of particular proteins in the digestive process we will establish whether or not they are secreted into the gut lumen where digestion occurs. We will produce recombinant versions of the enzymes, characterise their enzymatic properties and determine their usefulness for lignocellulose saccharification both using individual enzymes as well as combinations. We will also establish whether expressing the genes encoding these enzymes can be used to improve the saccharification potential of lignocellulosic biomass in energy crops.

Technical Summary

The cost effective saccharification of lignocellulosic biomass is recognized as a pivotal technical hurdle that must be overcome before the production of liquid biofuels from lignocellulosic biomass becomes a commercial reality. The search for new enzymes to disrupt lignin and release sugars from lignocellulose is being pursued aggressively around the world. We have identified a unique opportunity for the UK to become a major contributor to this competitive field. Limnoria are crustacean wood borers that can digest lignocellulose, and are unusual in having an effectively sterile digestive tract. This indicates that they can digest lignocellulose without the aid of resident gut flora in contrast to animals such as termites, which depend on such microbes. We have established the potential for gene and enzyme discovery in this organism by the provision of a potentially comprehensive transcriptomic database comprising over 280,000 ESTs. Initial analysis of the data reveals a transcriptome dominated by abundant transcripts representing several broad classes of proteins. Glycosyl hydrolases from 12 different families account for more than 25% of the ESTs, with two classes of putative cellulases and cellobiohydrolases alone accounting for more than 20%. Sequences with high homology to hemocyanins and phenol oxidases account for a further 17% of the transcriptome, followed by sequences with homology to proteases, ferritin, oxygenases, and a group with low homology to fatty acid binding proteins. We will study the digestive process in Limnoria in detail using proteomic and metabolomic analyses, and will characterize the enzymatic activities of recombinant proteins representing many of the genes involved in the digestive process. These enzymes will be assessed for utility in industrial lignocellulose saccharification, and we will investigate the use of these genes expressed in transgenic plants to improve the saccharification potential of lignocellulosic feedstocks.

Publications

10 25 50
 
Description A complex suite of digestive enzymes and proteins of unknown function have been identified from the digestive gland of a crustacean that can break down wood without assistance from microorganisms. The structure of a key enzyme has been described and its activity has been modelled at the atomic level. An oxygen carrying protein that normally functions in blood was found to be active within the contents of the gut. The function of novel oxidative chemistry has been revealed to take place within the gut.
Exploitation Route The enzymes have already attracted attention from a major industrial supplier of enzymes and the National REnewable Energy Lab in the USA. Comparisons with existing commercial enzymes are being undertaken. Tests with model feedstocks for generation of biofuel have been initiated.
Sectors Agriculture, Food and Drink,Chemicals,Education,Energy,Transport

URL http://www.bbsrc.ac.uk/organisation/institutes/bsbec/marine-wood-borer-enzyme-discovery.aspx
 
Description The first enzyme that we fully characterised has now been incorporated by our collaborators at NREL into a chimeric protein with a fungal cellulose binding domain. The efficacy of the chimera is now being assessed in a pilot plant. Our ability to crystallize challenging proteins has lead NREL to send their proteins to us to crystallize and resolve the structure.
First Year Of Impact 2014
Sector Energy
 
Description Diamond Light Source Matched Funding
Amount £50 (GBP)
Organisation Diamond Light Source 
Sector Academic/University
Country United Kingdom
Start 10/2014 
End 09/2018
 
Description KBBE Strategic Theme
Amount £95,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2018
 
Description Norwegian Research Council
Amount £77,000 (GBP)
Organisation Research Council of Norway 
Sector Public
Country Norway
Start 10/2013 
End 09/2015
 
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 Subcontract between the University of Portsmouth and the US National Renewable Energy Laboratory
Amount $80,000 (USD)
Funding ID REV-5-52031 
Organisation U.S. Department of Energy 
Sector Public
Country United States
Start 10/2015 
End 09/2019
 
Description Template H (McGeehan)
Amount £43,121 (GBP)
Organisation University of Portsmouth 
Sector Academic/University
Country United Kingdom
Start 06/2014 
End 07/2016
 
Description USA Partnering
Amount £33,305 (GBP)
Funding ID BB/H531543/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2010 
End 10/2013
 
Description University of Portsmouth Template H (Cragg)
Amount £43,000 (GBP)
Organisation University of Portsmouth 
Sector Academic/University
Country United Kingdom
Start 10/2014 
End 12/2015
 
Description strategic LoLa
Amount £568,462 (GBP)
Funding ID BB/L001926/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2014 
End 03/2019
 
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 PDB: Limnoria GH7 
Description McGeehan, J.E., Martin, R.N.A., Streeter, S.D., Cragg, S.M., Guille, M.J., Schnorr, K.M., Kern, M., Bruce, N.C. & McQueen-Mason, S.J. (2012) Crystal Structure of a GH7 Family Cellobiohydrolase from Limnoria quadripunctata. Protein Database Submission PDB ID: 4GWA, RCSB ID : RCSB074712. 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact supported 2013 pNAS paper on GH7 structure and function 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4GWA
 
Title PDB: Limnoria GH7 with cellobiose 
Description Martin, R.N.A., McGeehan, J.E., Streeter, S.D., Cragg, S.M., Guille, M.J., Schnorr, K.M., Kern, M., Bruce, N.C., McQueen-Mason S.J. (2012) Crystal Structure of a GH7 family cellobiohydrolase from Limnoria quadripunctata in complex with cellobiose. Protein Database Submission PDB ID : 4HAP, RCSB ID : RCSB075231. 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact Supports 2013 PNAS paper on GH7 structure 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4HAP
 
Title PDB: Limnoria GH7 with cellobiose and triose 
Description Martin, R.N.A., McGeehan, J.E., Streeter, S.D., Cragg, S.M., Guille, M.J., Schnorr, K.M., Kern, M., Bruce, N.C., McQueen-Mason S.J. (2012) Crystal Structure of a GH7 family cellobiohydrolase from Limnoria quadripunctata in complex with cellobiose and cellotriose. Protein Database Submission PDB ID: 4HAQ, RCSB ID: RCSB075232 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact supported 2013 paper in PNAS on GH7 structure and function 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4HAQ
 
Title PDB: Limnoria GH7 with thiocellobiose 
Description Martin, R.N.A., McGeehan, J.E., Streeter, S.D., Cragg, S.M., Guille, M.J., Schnorr, K.M., Kern, M., Bruce, N.C., McQueen-Mason S.J. (2012) Crystal structure of a GH7 family cellobiohydrolase from Limnoria quadripunctata in complex with thiocellobiose. Protein Database Submission PDB ID: 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact supported 2013 PNAS paper on GH7 structure and function 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4IPM
 
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 Enzyme structures 
Organisation Novozymes
Country Denmark 
Sector Public 
PI Contribution Crystallized proteins generated by Novozymes and solved structures.
Collaborator Contribution Supplied large quantities of proteins of interest, including ones falling outside the BBSRC project range, though relevant to the project objectives.
Impact Protein structures have been generated which help us with interpretation of the function of similar proteins generated by our experimental organisms.
Start Year 2013
 
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 NREL Biofuels 
Organisation U.S. Department of Energy
Department National Renewable Energy Laboratory (NREL)
Country United States 
Sector Public 
PI Contribution Provision of high quality DNA samples for genomic analysis Prediction of enzyme structures Solving of enzyme structures Crystallizing of proteins
Collaborator Contribution Liaison with U Colorado over genome sequencing Substantial intellectual input into publication Technical assistance regarding protein expression
Impact Multidisciplinary: digestion physiology, gene expression, protein synthesis, structural biology
Start Year 2011
 
Title Patent application no. 1305487.9 
Description A method for the modification of plant biomass comprising the incubation of one or more Limnoriidae isolated glycosyl hydrolase polypeptides in a reaction mixture comprising plant biomass and a high ionic strength buffer. 
IP Reference GB1305487.9 
Protection Patent application published
Year Protection Granted 2013
Licensed No
Impact None to date. Commercial decision was taken not to proceed with the patent.
 
Description Cafe Scientifique 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Lively q & a about potential impact of the research and about the underlying science

I developed a better appreciation of how to put our message across and how to stimulate thinking in people without subject-specific expertise
Year(s) Of Engagement Activity 2011,2012
URL http://www.cafescientifique.org/
 
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 Images for textbooks and newspaper articles 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Images disseminated through web sites prompted requests for images for use in textbooks and other publications.

The subject of our work appeared in an A-level question. Images were requested for the renovation at Seattle seafront.
Year(s) Of Engagement Activity 2011,2012,2013,2014
 
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 Outgoing school visits 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Increased enthusiasm for science as a career evidenced in reports from teachers.

Raised awareness of careers in science
Year(s) Of Engagement Activity 2012,2014
 
Description Radio reports 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Interest spread from general to specialist media.

Some BSc applicants mentioned hearing about the research.
Year(s) Of Engagement Activity 2010,2011
 
Description Royal Society MP Pairing Scheme 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Engagement with local MPs on the impact of our science and the relationship to science policy.
Year(s) Of Engagement Activity 2015
URL http://www.port.ac.uk/uopnews/2015/11/25/research-scientist-heads-to-westminster/
 
Description TV interviews 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Radio stations made contact with press office. Contacts made by special interest groups.

Further requests for interviews.
Year(s) Of Engagement Activity 2010,2011
 
Description Web presence 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Wide variety of types of web site got the story of the research to a diverse audience. The rationale for second generation biofuels was explained.

Requests for images for publication in particular for educational texts keep coming in.
Year(s) Of Engagement Activity 2011,2012,2013,2014
URL http://www.bbsrc.ac.uk/news/industrial-biotechnology/2013/130603-pr-enzyme-could-turn-waste-into-bio...