Bilateral BBSRC-FAPESP / Targeted analysis of microbial lignocellulolytic secretomes - a new approach to enzyme discovery.

Lead Research Organisation: University of York
Department Name: Biology


As fossil fuel supplies dwindle and concerns increase about the environmental impact of chemical waste streams, industrial biotechnologists are exploring ways to use plant based feedstocks in 'biorefineries' to generate biofuels and manufacture polymers, pharmaceuticals and commodity chemicals. The long-term success of biorefining is dependent on the development of economical methods for processing plant biomass to exploit the energy rich polysaccharides in cellulose for fermentation. The complex phenolic polymers present in lignin create a major bottleneck in the deconstruction of plant cell walls, as they are recalcitrant to degradation. Currently biorefineries require the use of acid and steam explosion to treat lignocellulose, which is inefficient and energy dependent, the released cellulose is then digested with a cocktail of cellulases. The costs involved in converting biomass into fermentable sugars currently make cellulosic fermentation too expensive. While the saccharification of lignocellulose remains a problem for industry, it is carried out effectively in the natural environment by microbial communities. Such communities are found in composting systems and soils. The major challenge in identifying the range of enzymes and other proteins used by communities of microorganisms during lignocellulose degradation lies in the complexity of the process itself. At present the vast majority of microbial biodiversity remains uncharacterised, because less than 1% of microorganisms in most environments are amenable to axenic cultivation, therefore, to date lignocellulose degradation has largely been studied in a few well characterised and culturable microorganisms. The research proposed here is concerned with discovering new enzymes and associated proteins for lignocellulose digestion from rotting cereal straw and sugar cane bagasse, and takes an integrated proteomics and metatranscriptomic approach for their identification. The innovative aspect to our proteomic approach arises from the fact that microbial cells cannot ingest pieces of undigested lignocellulose, but must first convert this material to simple sugars that can then be imported in to the cell, and this requires that they secrete the appropriate digestive enzymes. Thus, the majority of enzymes and accessory proteins involved in lignocellulose mobilisation are distinguished from those involved in housekeeping activities by the fact that they are secreted. The major challenge is that lignocellulose active proteins bind tightly to lignocellulose and require stringent extraction conditions to release them. Similarly, many of the microbes involved in lignocellulose digestion also bind to the substrate and cannot be washed out. The result of this is that if sufficiently stringent extraction is used to get the proteins off the substrate this inevitably leads to cell lysis and contamination of the extract with cellular enzymes; whilst milder extractions only release a small proportion of the target proteins. To overcome this problem we will use non-invasive extracellular protein tagging to identify secreted enzymes produced by microbial communities. The key to this is the use of a protein affinity tag that cannot cross biological membranes and can therefore only access and tag extracellular proteins. Once the tagging reaction has been quenched, the lignocellulose and microbial culture can be extracted under stringent conditions, with the tagged extracellular proteins simply separated by affinity purification prior to proteomic analysis. Combining the power of extracellular proteomics and metatranscriptomics will allow us to focus in on the proteins critical for lignocellulose deconstruction from microbial communities. This will greatly enhance our ability to identify completely new types of lignocellulose active proteins, both broadening our fundamental understanding of this process, as well as providing novel activities for research and industrial applications.

Technical Summary

From both a fundamental and industrial biotech viewpoint understanding the deconstruction of lignocellulose in soil and compost is of central importance. In the natural environments microbial communities can efficiently degrade or modify lignin to enable the effective enzymatic hydrolysis of the polysaccharides present in plant cell walls. The aim of this proposal is to use metatranscriptomics and proteomics to determine gene- and protein-centred details to determine new mechanisms and improved methods of lignocellulose deconstruction in mixed microbial communities from composting wheat straw and sugar cane bagasse. The novelty of our proteomic approach lies in the use of a biotin affinity tag to distinguish secreted proteins from intracelluar proteins that are released from lysed cells during the extraction process necessary to release the proteins that bind tightly to the decaying plant biomass. The secreted proteins will be tagged, affinity purified, digested with trypsin and the resulting peptide mixtures analyzed by LC-ESI-MS. In order to have a picture of the overall community dynamics in terms of species composition at the different stages in the composting process DNA will be extracted for SSU rRNA profiling. Saccharification of the lignocellulose will be monitored and the lignin content of the straw or bagasse analysed using FTIR spectroscopy and solid state NMR. Metatranscriptome analysis will be performed by preparing cDNA from samples taken at various time points from the lignocellulose enriched cultures, the cDNA will be bar coded pooled and sequenced using the Roche 454 GS FLX Titanium platform. The peptide sequences from the proteomics analysis will allow the identification of full and partial coding sequences in the library. These coding sequences will be cloned and expressed in established recombinant expression systems and the recombinant proteins screened for activity.

Planned Impact

The strategic relevance of this programme of work is high and falls within the BBSRC strategic priority in Bioenergy and Industrial Biotechnology described in the strategic plan 2010-2015. Exploring the digestion of lignocellulose by microbial communities using an integrated proteomics and metatranscriptomics has the potential to identify new approaches to lignocellulose utilisation along with new enzymes and intellectual property of benefit to the biofuel and biorefinery industries. Combining this potential with the power of high-throughput protein expression and top class analytical facilities in York and Sao Paulo will place the UK and Brazil in a strong position in this internationally competitive area. Establishing an efficient platform for sugar production from biomass feedstocks will underpin the establishment of competitive and sustainable biorefinery industries particularly in the area of transportation fuels. Other beneficiaries will include the farmers and breeders of energy crops. Waste management is a significant problem in the UK and our work is also aimed at providing underpinning tools for the conversion of waste biomass materials into sugars and biofuels. 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, the Biorenewables Network) to communicate progress through their events and web-based or printed media. A workshop will be held towards the end of year 2 to disseminate information to relevant industrial stakeholders and academic beneficiaries. The applicants have strong links with the University of York's Enterprise and Innovation office who will also ensure that any commercial opportunities are realised. The research will also be undertaken in the context of the Centre for Low Carbon Futures (CLCF), a translational research centre established by the four major Universities in Yorkshire and Humberside, supported by an investment of £5.7m by the Regional Development Agency. This centre has contractual responsibility to engage end-users with research conducted in the regional HEIs. 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. This involves the PIs, the University business development officer and a local patent company with expertise in biotechnology. CNAP has an outstanding track record in commercialisation of strategic research. This includes ongoing collaborations with companies throughout the biorenewable supply chain from major international seed companies, through processors to end users such as global pharmaceutical, personal care and feed companies as well as SMEs within the UK and EU.


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Description The exoproteome of microbial communities consists of secreted proteins involved in nutrient acquisition and interorganismal interactions, along with surface exposed proteins involved in recognition, adhesion, infection, transport and communication. The exoproteome, therefore, acts as a powerful signature of the processes peculiar to any particular microbial community. The challenge is how to identify these proteins of interest amongst a background pool of proteins that arises from cell lysis which occurs when protein are extracted from the microbial cultures, particularly when these extracellular proteins and microorganisms are both bound to solid surfaces.

We have addressed this challenge by developing a powerful new methodology that enables the identification of unique proteins in metaexoproteomes of complex microbial communities that would otherwise be uncharacterised. We demonstrated how this new approach can be targeted to: (1) identify and tease out proteins of interest from a large background pool; (2) identify proteins that are involved in insoluble substrate interactions; and (3) identify novel proteins that have potential commercial value for biomass processing. Specifically, we used sulfo-NHS-SS-biotin to label extracellular proteins under mild conditions so that, after stringent elution, they can be affinity enriched and separated from unlabelled intracellular proteins that arise from cell lysis during the elution process. MS/MS data analysis software requires a comprehensive sequence database from which to identify peptide matches and thus identify proteins present in the sample. This is straightforward for model organisms where the genome has been sequenced but not for complex microbial communities where the majority of the strains are unculturable and uncharacterised. To overcome this problem we carry out metatranscriptomic sequencing in parallel to proteomic analysis in order to provide a nucleotide sequence database suitable for MS-based peptide identification.

To enhance the practical relevance, we validated the methodology by analysing the exoproteome of lignocellulose degrading compost communties. In such communities, functional diversity is driven by multiple environmental factors including source of plant material, soil residues, water and oxygen content and seasonal temperature. The community is dependent on the presence of exoproteins involved in plant biomass degradation and transport proteins for nutrient uptake. Many of those exoproteins remain tightly bound to the substrate by specialised carbohydrate-binding domains. Hence we hypothesized that our combined targeted proteomics and metatranscriptomics approach would provide a detailed picture of unique and separate metaexoproteome pools and the dynamics of microbial communities acting on those insoluble substrates. Our strategy made it possible to identify many proteins involved in lignocellulose degradation and nutrient transport. The identification of large numbers of uncharacterized proteins offers an invaluable opportunity to expand our knowledge of lignocellulose degradation, with the potential to mine for new commercially valuable biomass processing enzymes.
Exploitation Route We believe that this work will be of wide interest due to both its novelty and topicality. The cell surface and extracellular space is the site where organisms first encounter and interact with one another, and we believe our proteomics method will find applications in many areas including environmental, medical and nutritional research. Our libraries of lignocellulose degrading activities offers the opportunity to develop new and improved enzyme cocktails for biomass processing. Our findings have contributed to a GCRF RCUK Hubs bid entitled "GCRF Insect biotechnology for sustainable food hub". Our aim is to help move crop residues from being a source of problems, to being a sustainable source of new economic activity for the production of insects as feed for animals, thus ensuring that there are tangible benefits for stakeholder farmers but also for businesses of all sizes.

Since the completion of this award we have characterised a novel ligninase from one of the fungal isolates identified in this study. This enzyme cleaves the structural ether linkages in lignin releasing the valuable flavonoid tricin from monocot lignin.
Sectors Chemicals,Energy,Environment

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 Biorefining oil palm waste residues
Amount £29,500 (GBP)
Funding ID BB/P025501/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2017 
End 03/2020
Description ISIS
Amount £10,000 (GBP)
Funding ID BB/M028445/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2015 
End 04/2015
Description LBNet Proof of Concept
Amount £19,781 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 02/2018
Description LBNet proof of concept project
Amount £40,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2015 
End 10/2016
Description Rice straw biorefining
Amount £99,000 (GBP)
Funding ID NF170883 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2018 
End 02/2020
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 Protein tagging 
Description Metagenomics and metatranscriptomics have significantly advanced our understanding of complex microbial communities containing numerous unculturable species; however, identifying functional extracellular proteins tightly bound to matrices, insoluble substrates and cell surfaces in these microbiomes remains a major challenge. The York group have developed a technique for targeting and enriching the extracellular proteome by tagging prior to stringent total extraction. The methodology combined with mass spectrometry-based proteomics and metatranscriptomics has enabled the identification of unique metaexoproteome pools from complex lignocellulose degrading communities. 
Type Of Material Biological samples 
Year Produced 2015 
Provided To Others? Yes  
Impact A wide variety of carbohydrate active enzymes bound to lignocellulosic biomass have been extracted and identified. Strikingly, amongst those proteins there was a high abundance of TonB-dependent transporters and periplasmic ligand-binding components of ATPase Binding Cassette transport systems. The study has also revealed a large number of uncharacterized and unknown proteins, suggesting that there are large gaps in our understanding of how microorganisms degrade lignocellulose. A selection of the carbohydrate active enzymes and proteins of unknown function are currently being cloned and expressed for further characterisation. The identification of large numbers of uncharacterized proteins offers an invaluable opportunity to expand our knowledge of lignocellulose degradation, with the potential to mine for new commercially valuable biomass processing enzymes. In addition, this protein-labelling approach could be applied to a variety of complex microbial ecosystems to provide details on major metabolic players and the function and contribution of the exoproteome in those communities. 
Title Wheat straw community Sequencing data 
Description All sequencing data are available from the European Nucleotide Archive database. Accession numbers for metatranscriptomics dataset and amplicon sequencing are PRJEB12382 and PRJEB21053, respectively. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact The results were published in 2018, no impact has yet arisen from the data. 
Description Oil Palm 
Organisation University of Technology, Malaysia
Country Malaysia 
Sector Academic/University 
PI Contribution Provided intellectual input, materials, hosted visits, training and held joint workshops.
Collaborator Contribution Provided intellectual input, materials, hosted visits and held joint workshops.
Impact Newton SEA award with Dr Chun Shiong Chong. Multidisciplinary involving biological science and social science.
Start Year 2016
Description Workshop at the Linnean Society 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact To assist the prospecting of biodiversity for lignocellulolytic mechanisms with potential for biotechnology applications, a discussion meeting was held in September 2013 at the Linnean Society in London, which reviewed the vast array of mechanisms across the Tree of Life. An article was published in Opinion in Chemical Biology that captures and updates the diverse chemical and organismal perspectives brought to the subject by the participants in the meeting.
Year(s) Of Engagement Activity 2013
Description YorNight European Researchers' Night 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact This activity was a great way to talk about our research in a simple and accessible way. Many participants questioned our methods which led to useful discussion afterwards. It also improved the researchers communication and presentation skills.

Subsequent requests for further information on the work.
Year(s) Of Engagement Activity 2014