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

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.

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.

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.