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Improving Plant Cell Walls for Use as a Renewable Industrial Feedstock

Periodic Report Summary - RENEWALL (Improving plant cell walls for use as a renewable industrial feedstock)

Project Context and objectives:

The need for renewable energy and raw materials for industry and society has become a pressing concern. The dependence of a burgeoning and rapidly industrialising world population on fossil fuels is clearly unsustainable due to dwindling reserves and the impact of Greenhouse gas (GHG) emissions on global climate. Industrial economies are also increasingly concerned about stability and security of fuel supply. Transportation fuels derived from petroleum account for more than 30 % of European energy consumption. Liquid biofuels such as bioethanol (produced by fermentation of plant-derived sugars) and biodiesel (produced from plant or animal oils) offer a renewable alternative for liquid transportation fuels and have the potential to lessen the dependence of the European Union states on oil imports. Bioethanol production is increasing worldwide, but expansion of the current production from sugar cane, sugar beet or cereal grain can add strain on world food supplies and prices. Development of 'second generation' biofuels that could be made from low-input non-food plant biomass can alleviate our dependence on oil in a sustainable carbon-neutral way without placing further stress on food supply.

Plant biomass (or 'lignocellulose') is one of the greatest untapped reserves on the planet and is mostly composed of cell walls. Energy-rich polysaccharide polymers make up about 75 % of plant cell walls and these can be broken down to produce sugars that can be fermented to produce bioethanol and other products. Even greater value can be added by using integrated processing systems that allow multiple products to be produced from the same biomass - the biorefinery concept. However, the complex structure of cell walls, consisting of a network of cellulose microfibrils and matrix polysaccharides (hemicellulose; pectin) encrusted by the phenolic polymer, lignin, makes them very resistant to degradation. Improving the ease and yield of cell wall saccharification (conversion to sugar) represents the major technological hurdle to overcome before the full vision of the plant-fuelled biorefinery can be realised.

The central aim of RENEWALL is to make game-changing breakthroughs in our understanding of plant cell walls and to use this knowledge to develop new strategies for breeding added-value plants with modified wall properties that are better suited for biorefining.

We will do this by identifying the molecular barriers to saccharification, and the genes that can be manipulated to lower these barriers. These may be plant genes involved in cell wall biosynthesis or other (often microbial) genes that can modify wall properties or degrade wall polymers when expressed in plants. These genes can then be directly used in Genetically modified (GM) approaches to breed improved plant feedstock for biorefining, or used as marker to improve crops by conventional breeding approaches.

The programme is divided into seven Work packages (WPs):

- WP1 delves into the biochemistry of saccharification to understand the processes underlying the saccharification bottleneck, and the influence of specific cell wall features on it. WP1 perform high-throughput screening of existing germplasm and new materials emerging from the other WPs.
- WPs2-4 generate fundamental knowledge into the genes, biochemistry and cell biology involved in assembling cell walls, focusing specifically on lignin, matrix polysaccharides and cellulose. State-of-the-art genomic, transcriptomic, proteomic and metabolomic approaches will be used. These WPs will also identify or generate new transgenics and mutants with modified cell walls for analysis by WP1.
- WP5 tests the value to subsequent saccharification of manipulating cell walls by expressing wall modifying or degrading enzymes in plants.
- WP6 develops rational engineering approaches to improve saccharification by integrating knowledge gained in the other WPs to devise efficient strategies for optimising biomass raw material quality. All participants will contribute intellectually to WP6.
- WP7 provides an efficient management structure for the project, ensuring smooth integration of the research effort, efficient reporting and dissemination of the results.

Project Results:

The first year of the programme was in part focussed on establishing the analytical tools and resources necessary for the successful operation of the overall project. This included the refocusing of cutting edge molecular methods in proteomics, transcriptomics and metabolomics onto the problem of wall digestibility. It was also critical to assemble the necessary populations of plant materials for the project. This included the generation of novel mutant populations of Brachypodium to be screened for digestibility mutants, as well producing tissues from previously characterised mutant and transgenic plants which had altered cell walls and had been produced by the project partners in other research activities, and now could be assessed for digestibility. Key to these investigations were the development of two major analytical platforms; one for high-throughput digestibility studies, and the other for more detailed analysis of the saccharification process.

During year 2 we managed to successfully complete the screening of existing Arabidopsis and poplar lines for digestibility and screen a population of Brachypodium mutants for digestibility. We also identified novel genes involved in lignin and secondary cell wall biosynthesis and generate new knowledge of lignin biosynthesis in grass species, such as Brachypodium. In addition we identified new gene targets for matrix polysaccharide biosynthesis in a range of plants and began making targeted modifications by genetic transformation in Brachypodium and other plants with new genetic constructs for modifying cell wall structure, cellulose biosynthesis, hemicelluloses and lignification.

During year 3 much of the initial work to identify genes that improve saccharification has come to maturity, such that we now have a large number of candidate genes identified and the impacts of knocking them out or overexpressing them has been determined. This work is now setting the stage for the final year of the project in which we will investigate the impact of stacking a number of these key genes in plants in order to obtain a step-change in digestibility. This work also has provided the platform for us to start to synthesise the knowledge generated from the research and reach are ultimate goal of providing a detailed understanding of what controls biomass digestibility in model plants and to define strategies by which improved saccharification can be introduced to biomass crops.

Potential impact:

The final outcomes that we anticipate from the work are to provide an in-depth understanding of what determines biomass digestibility at the genetic, molecular and biochemical levels. This will be achieved through combinations of forward and reverse genetic studies that will reveal the range of genetic modifications that can be made to improve saccharification. By studying the structure and composition of the biomass of these different plant materials we shall develop a much clearer understanding of the roles of different biomass components in determining digestibility. From this in-depth understanding we will be able to develop well-informed strategies that can be employed by crop breeders in order to produce biomass crops improved for biofuel and biorefinery applications.