Cell wall lignin programme: Manipulating lignin to improve biofuel conversion of plant biomass

Lead Research Organisation: University of Dundee
Department Name: College of Life Sciences

Abstract

An obvious way of improving plant materials for biofuel production would be to manipulate the structure of plant cell walls, since it is resistant polymers in these walls that prevent microbial enzymes from degrading the plant material into simple sugars and ethanol. One of the most important cell wall polymers in this respect is lignin. We already know quite a lot about how lignin is made and how it can be manipulated in woody plants. However much of the plant biomass that could be used to produce energy comes from grasses, and more research is therefore needed to enable us to manipulate lignin in these types of plant. Barley is a good model for the grasses that might be grown for energy applications (e.g. Miscanthus), and there are more research tools available for barley than for most other grasses. Barley and wheat straw waste could also be useful resources for biofuel production, and genetic discoveries in barley are usually transferable to wheat. This project aims to determine how lignin content and structure influence (1) the amount of sugars that can be released from barley straw; (2) how efficiently these sugars can be converted into biofuels; and (3) the amount of energy that can be released from barley straw by burning. This will indicate how the polymer can best be manipulated to make it easier to produce biofuels from plant biomass. We also aim to determine whether any lignin biosynthesis genes are important for barley disease resistance or stem strength, so that we can determine how to manipulate lignin while keeping plants healthy. The genes and genetic markers that we isolate can be used directly in energy crop improvement breeding programmes. Because we will be looking at a lot of different barley varieties, we will also be able to identify which current varieties are best for biofuel production and for burning for heat and energy.

Technical Summary

Plant biomass is made of cell walls of cellulose, hemicellulose, and lignin (lignocellulose). It is difficult release the lignocellulosic sugars for biofuel production because lignin is extremely resistant to degradation. We have already proven that manipulating lignin can make cellulose more accessible for papermaking and forage digestibility. It could just as easily be manipulated to improve saccharification of plant biomass, making biofuels more feasible and competitive. We will study the relationship between lignin content/composition and (1) saccharification/fermentation of straw; (2) combustion of different straws. We will work in barley, a good research model for biomass grasses. We will isolate barley genes, alleles, and genetic markers that associate with high saccharification. These can subsequently be used in MAS of improved energy crops. We will also investigate whether any lignin genes are associated with disease resistance or stem strength so that we know how to manipulate lignin while keeping plants healthy. We will achieve this by performing QTL mapping and novel association genetics using both a 'candidate gene' (lignin genes) and a 'hypothesis-free' genome-wide approach. This will tell us which lignin genes most influence saccharification and whether we can manipulate them without affecting disease resistance and stem strength. It will also point out other major loci affecting saccharification, as will eQTL analysis. If possible, we will identify and clone these genes which could be novel candidates for manipulating lignin to optimize biofuel production. Among the genotypes we investigate, there will be TILLING mutants and transgenics suppressed in lignin gene expression, enabling us to determine the effects of more extreme lignin gene alleles or manipulations. We will also characterize the natural diversity that exists in barley landraces for these genes. Useful mutant and landrace alleles can be directly incorporated into breeding programmes.

Publications

10 25 50
 
Title MOESM2 of BaRTv1.0: an improved barley reference transcript dataset to determine accurate changes in the barley transcriptome using RNA-seq 
Description Additional file 2: Figure S1. 3 Pass Star alignment details. Figure S2. GMAP parameter options to align the 22,651 Haruna Nijo full-length cDNAs to the barley genome. Figure S3. Optimal StringTie assembly. Figure S4. Transdecoder annotation and filtering short transcripts. Figure S5. Examples of significant alternative transcript switches between developing inflorescence (INF1) and leaf (LEA) samples. Figure S6. Pipeline describing the algorithm to compare HR-RT-PCR and RNA-seq alternatively spliced transcript proportions and correlations. 
Type Of Art Film/Video/Animation 
Year Produced 2019 
URL https://springernature.figshare.com/articles/MOESM2_of_BaRTv1_0_an_improved_barley_reference_transcr...
 
Title MOESM2 of BaRTv1.0: an improved barley reference transcript dataset to determine accurate changes in the barley transcriptome using RNA-seq 
Description Additional file 2: Figure S1. 3 Pass Star alignment details. Figure S2. GMAP parameter options to align the 22,651 Haruna Nijo full-length cDNAs to the barley genome. Figure S3. Optimal StringTie assembly. Figure S4. Transdecoder annotation and filtering short transcripts. Figure S5. Examples of significant alternative transcript switches between developing inflorescence (INF1) and leaf (LEA) samples. Figure S6. Pipeline describing the algorithm to compare HR-RT-PCR and RNA-seq alternatively spliced transcript proportions and correlations. 
Type Of Art Film/Video/Animation 
Year Produced 2019 
URL https://springernature.figshare.com/articles/MOESM2_of_BaRTv1_0_an_improved_barley_reference_transcr...
 
Description DISCOVERY OF GENES DETERMINING SUGAR YIELDS FROM LIGNOCELLULOSIC BIOMASS: The BSBEC Cell Wall Lignin Programme (UoD/UoY) evaluated the amount of sugar released from straw of 860 elite spring barley cultivars by alkali pretreatment and commercial enzymes. Even in this un-optimized processing, some cultivars consistently yielded 30% more sugars than others (Oakey et al 2013 Biotechnol Biofuel 6:185) with no impact on grain properties. A Genome-Wide Association Scan identified several genomic loci that influence sugar release and candidate genes were identified for two of them. Several of these genes were validated as influencing saccharification by transgenic manipulation including CRISPR-Cas9 knock-outs in barley. These discoveries enable advanced breeding techniques (marker assisted and genomic selection) to be used in MaxBio to bring beneficial QTL together in new barley lines, while recent availability of new TILLING mutant populations also allows translation into wheat, introducing useful straw traits to the UK's two most important cereals.
Exploitation Route Our discoveries are enabling advanced breeding techniques (marker assisted and genomic selection) to be used in the MaxBio to bring beneficial QTL together in new barley lines.
Sectors Agriculture

Food and Drink

Chemicals

Energy

Environment

Transport

 
Description We are currently using our findings to breed new and improved barley cultivars (with straw more suited to industrial biotechnology applications) using a genomic selection approach developed during the BSBEC project. We have trainined visiting researchers in our approach, most notably as part of a Partnership award with Vietnam. Several PhD students have received training through the project. Many public engagement activities were run during the project and well-received.
First Year Of Impact 2010
Sector Agriculture, Food and Drink,Education,Leisure Activities, including Sports, Recreation and Tourism
Impact Types Cultural

Economic

 
Description House of Lords Science and Technology Committee inquiry into waste and the bioeconomy
Geographic Reach National 
Policy Influence Type Contribution to a national consultation/review
 
Description Nuffield Council of Bioethics consultation and report on Biofuels: Ethical Issues
Geographic Reach Multiple continents/international 
Policy Influence Type Contribution to a national consultation/review
 
Description Developing Rice Straw for Animal Feed
Amount £705,293 (GBP)
Funding ID BB/P022499/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2017 
End 04/2019
 
Description Developing rice resources for resilience to climate change and mitigation of carbon emissions
Amount £96,958,042 (GBP)
Funding ID BB/N013689/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2016 
End 05/2019
 
Description IB Catalyst
Amount £2,253,398 (GBP)
Funding ID BB/N023455/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2016 
End 08/2021
 
Description Industrial Biotechnology Catalyst
Amount £2,715,668 (GBP)
Funding ID BB/N023455/1, BB/N022718/1 and BB/N023269/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2016 
End 05/2021
 
Description International Partnering Vietnam
Amount £50,000 (GBP)
Funding ID BB/J013838/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2012 
End 07/2013
 
Description International Partnership
Amount £4,200 (GBP)
Funding ID BB/I023305/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2013 
End 01/2015
 
Title Abbey Meiosis CRISPR 
Description Successful use of CRISPR-Cas9 in transgenic barley 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact Invitations to speak at scientific meetings e.g. forthcoming SEB Plant Satellite Meeting on New Breeding Technologies, Gothenberg, July 2017 
 
Title Transgenic barley with manipulated lignin 
Description Barley Transgenics: COMT RNAi lines; CCR RNAi lines; PAL RNAi lines; CAD RNAi lines; 4CL RNAi lines, C4H RNAi lines, C3H RNAi lines, HCT RNAi lines RNAi constructs were developed in pBRACT207 vectors and barley cv Golden Promise was transformed using Agrobacterium tumefaciens to produce at least 30 independent primary transformants per gene along with 15 pBRACT207 empty vector control lines. Plants were initially screened by enzyme assay or Western blot to identify those with reduced target enzyme activity/protein. Plants with reduced target enzyme were subjected to Southern blotting to identify lines with a single transgene insertion. Homozygous lines were produced from these plants for further study and full lignin analysis. 
Type Of Material Biological samples 
Provided To Others? No  
Impact Validation of the role of specific genes in lignin biosynthesis and of candidate genes underlying GWAS peaks 
 
Title FTIR Predictive model for lignin content and composition 
Description A model was used to correlate FT-IR spectral data with Klason lignin content data for barley straw. This allowed FT-IR to be used as a high-throughput method for predicting lignin content in barley straw. Similarly, thioacidolysis data was used to train models for predicting lignin composition (H, G and S monomer content) from FT-IR spectra. 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact Allowed thousands of samples to be analysed so that lignin content and composition could be used as phenotypes for GWAS to identify underlying genes. 
 
Title Genomic Selection model 
Description A model was generated for performing genomic selection from multi-trial data of barley 
Type Of Material Data analysis technique 
Year Produced 2016 
Provided To Others? Yes  
Impact Generation of the model was used to support a successful application for an industrial CASE studentship and led to interaction with industrial partner James Hutton Limited for use in barley breeding. 
 
Title MOESM1 of BaRTv1.0: an improved barley reference transcript dataset to determine accurate changes in the barley transcriptome using RNA-seq 
Description Additional file 1: Table S1. RNA-seq datasets from both public databases and in house unpublished data. Table S2. StringTie Parameters used in the different assemblies (sorted by transcript number). Table S3. Mean proportions of alternatively spliced products by HR-RT-PCR analyisis. Table S4. Correlation of HR RT-PCR data with BaRTv1.0, BaRTv1.0- QUASI and HORVU transcripts. Table S5. Splice Junctions and intron lengths. Table S6. Differentially expressed gene clusters and differential transcript usage gene clusters. Table S7. Pairwise significant changes in alternatively spliced transcripts detected by HR RT-PCR between different organs. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
URL https://springernature.figshare.com/articles/MOESM1_of_BaRTv1_0_an_improved_barley_reference_transcr...
 
Title MOESM1 of BaRTv1.0: an improved barley reference transcript dataset to determine accurate changes in the barley transcriptome using RNA-seq 
Description Additional file 1: Table S1. RNA-seq datasets from both public databases and in house unpublished data. Table S2. StringTie Parameters used in the different assemblies (sorted by transcript number). Table S3. Mean proportions of alternatively spliced products by HR-RT-PCR analyisis. Table S4. Correlation of HR RT-PCR data with BaRTv1.0, BaRTv1.0- QUASI and HORVU transcripts. Table S5. Splice Junctions and intron lengths. Table S6. Differentially expressed gene clusters and differential transcript usage gene clusters. Table S7. Pairwise significant changes in alternatively spliced transcripts detected by HR RT-PCR between different organs. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
URL https://springernature.figshare.com/articles/MOESM1_of_BaRTv1_0_an_improved_barley_reference_transcr...
 
Description ARC 
Organisation Australian Research Council
Country Australia 
Sector Public 
PI Contribution Partner in Australian Research Council Centre of Excellence in Plant Cell Wall Biology with specific collaboration on beta-glucan biosynthesis with Profs. Geoff Fincher and Rachel Burton, University of Adelaide. Presented lecture at Annual Meeting; Provided written feedback on project; secured funds for PhD student and collaborative research project at University of Dundee; forged links with BBSRC Sustainable Bioenergy Centre; sharing of data and resources and common publications.
Collaborator Contribution Matched funding for collaborative work in Adelaide; Seminars presented at University of Dundee by Prof Geoff Fincher (University of Adelaide); sharing of data and common papers.
Impact Research papers published; PhD students trained
Start Year 2011
 
Description International Scientific Interchange Scheme with Vietnam 'Developing a cereal straw biorefinery using rice as a model and a feedstock 
Organisation University of York
Country United Kingdom 
Sector Academic/University 
PI Contribution Training to visiting researcher who brought rice seeds, grew them in Dundee, received help and training in genome-by-sequencing, collected straw samples for phenotyping and performed GWAS This collaboration was later continued via different funding sources including GCRF funding from Dundee University.
Collaborator Contribution In York, straw samples were phenotypes for saccharification sugar yield. In Vietnam, seeds were assembled for a rice GWAS population.
Impact Further collaborative funding through the Newton Fund
Start Year 2012
 
Description International Scientific Interchange Scheme with Vietnam 'Developing a cereal straw biorefinery using rice as a model and a feedstock 
Organisation Vietnam Academy of Agricultural Science
Department Field Crops Research Institute
Country Viet Nam 
Sector Public 
PI Contribution Training to visiting researcher who brought rice seeds, grew them in Dundee, received help and training in genome-by-sequencing, collected straw samples for phenotyping and performed GWAS This collaboration was later continued via different funding sources including GCRF funding from Dundee University.
Collaborator Contribution In York, straw samples were phenotypes for saccharification sugar yield. In Vietnam, seeds were assembled for a rice GWAS population.
Impact Further collaborative funding through the Newton Fund
Start Year 2012
 
Description Cafe Science on 2nd Generation Biofuels 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Talk sparked extensive discussion afterwards

Talk sparked extensive discussion and more invitations to speak
Year(s) Of Engagement Activity 2013,2014
 
Description College of Life Sciences Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact School of Life Sciences Open Day to bring the public in to learn about our research. Very interactive event with many questions from the public and comments about how it has changed their attitudes to key topics.
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014,2015
 
Description Dundee Botanic Gardens Family Fun Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Dundee Botanic Garden Family Fun Day (usually April/May): This is an annual family activity day at the gardens which has now developed into Plant Power Day and Fascination of Plants Day. We provide interactive activities related to plant science such as DNA preparation from raspberries along with displays on barley cultivation and uses, and biofuels from plants.
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014,2015,2016
 
Description GWAS workshop and a field trip in Vietnam from 8th to12th May 2017. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Rice resilience meeting, gwas workshop and a field trip in Vietnam from 8th to 12th May 2017.
Year(s) Of Engagement Activity 2017
 
Description Newton Workshop Singapore 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Workshop on 'Biorefining of oil palm residues' at the University of Technology Malaysia 27th-30th August organised by colleagues at York University.
Year(s) Of Engagement Activity 2017
 
Description Society of Biology Annual Teachers Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Every year the Society holds a conference with teachers and researchers to share news, network, and inform on topical issues. Over 100 Biology teachers attend from all over Scotland. I gave a talk to the 2014 meeting covering the controversial "Food versus Fuel" debate, and spoke about using alternative crops, and the balance between the application of modern genetics and genetic manipulation of crops.
Year(s) Of Engagement Activity 2014
 
Description Society of Biology Scottish Branch Annual Symposium and AGM 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Prior to its AGM the Society holds some public talks and a debate on topical issues. In 2013 the topic was Renewable Energy and I spoke and subsequently debated on the development of second generation biofuels.
Year(s) Of Engagement Activity 2013
 
Description Vietnam Workshop: GCRF Hubs - Can Tho, 9-10th October 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact The objective of this workshop is to develop a research proposal for a funding competition in the UK entitled "GCRF Interdisciplinary Research Hubs to Address Intractable Challenges Faced by Developing Countries".
Year(s) Of Engagement Activity 2017