The use of genome mining and protein engineering to generate novel xylan and cellulose degrading enzymes

The use of fossil fuels in the energy and chemical industries is no longer tenable; they represent a finite resource and their use results in carbon dioxide emissions, which is a major cause of global warming. There is, therefore, an urgent need to find alternative sources of liquid fuels that are renewable and do not have an adverse effect on the environment. Lignocellulosic biomass is a promising substrate for biofuel production as it is not a food source, is more abundant than starch, and its use is carbon dioxide neutral. A significant limitation in the use of lignocellulosic biomass in the biofuel industry is its recalcitrance to enzyme attack. Thus, cellulose, the major polysaccharide in lignocellulosic biomass, is chemically simple but its highly crystalline structure makes it inaccessible to enzymes that act as hydrolases. Recent studies, however, have identified novel enzymes that could improve the efficiency of plant cell wall deconstruction. Thus, several reports have shown that oxidases cleave bonds in crystalline regions of cellulose, leading to increased access to hydrolase attack. Significant advances have also been made in the degradation of xylan, the major matrix polysaccharide in lignocellulosic biomass. It was widely believed that degradation of the main chain of xylan required the removal of side chains prior to attack by xylanases. It is now apparent that a cohort of xylanases not only accommodate side chains, but actually display an absolute requirement for these decorations. We have also shown that it is possible to introduce novel functionalities into the active site of biotechnologically significant arabinofuranosidases that assist in removing the side chains from xylan. The generation of such multifunctional enzymes has the potential to simplify the biocatalysts required to deconstruct plant cell walls, and thus increase the economic potential of lignocellulosic biomass as a substrate for the biofuel industry. In this project we will explore the mechanism by which cellulose oxidases, arabinoxylanases and multifunctional arabinofuranosidase/xylanases recognize their target substrates. The data will provide a blueprint for further enhancing the efficiency of the plant cell wall degrading catalytic toolbox.

Exploiting lignocellulosic biomass in the biofuel industry is limited by its recalcitrance to enzyme attack. Xylan degradation requires an extensive repertoire of enzymes, while the hydrolysis of crystalline cellulose is limited by enzyme access. We have provided a proof of principle for introducing additional catalytic functions into biotechnologically significant arabinofuranosidase, and provide evidence that endo-acting xylanases can specifically target arabinoxylan. These enzymes, however, are relatively poor catalysts and thus have limited industrial utility. There is an urgent need, therefore, to understand the mechanism by which these enzymes display their respective specificities. Such information will provide a platform for generating highly active enzymes that can be used to improve the biocatalysts used to deconstruct plant biomass. The identification of cellulose oxidases also has the potential to improve the degradation of crystalline cellulose. It is evident, however, that there is a lack of understanding of how bacterial cellulose oxidases target crystalline cellulose. Such information is crucial if the full potential of these enzymes in biomass utilization is to be fully realized. Providing a mechanistic understanding of how natural and engineered hydrolases and oxidases attack decorated xylans and cellulose, respectively, has the potential to significantly increase the economic potential of lignocellulosic-based biofuel production. This project is designed to dissect and extend the specificity of CtXyl5A (arabinoxylanase), AXHd3-Xyl (arabinofuranosidase-xylanase) and cellulose oxidases to supply this much needed mechanistic understanding.

The proposed research programme has the potential to inform novel enzymatic strategies that improve the conversion of plant biomass into its constituent sugars. The sugars generated can then be deployed in industrial fermentations that yield liquid biofuels, such as ethanol and butanol, and substrates for the chemical industry. Currently the major economic limitation to the use of lignocellulosic biomass in biofuel production is the cost of the pre-treatments, which involved both the use of chemical and physical methods to open up the plant cell wall, and the subsequent enzymatic treatments used to generate the monosaccharides. By generating novel glycoside hydrolases and oxidases with improved activities against cell walls may reduce both the chemical and enzyme imputs into the process, and thus increase its economic viability. As such this research programme will be of interest to the bioenergy industry. Specifically this project is important to companies that are using plant biomass for industrial fermentations, such as bioethanol production, and companies (e.g. Novozymes, Genecore-Danisco-DuPont) that are developing enzymes for the bioenergy industry. The importance of this research programme is illustrated by the fact that this project is a BBSRC-IPA application in which the industrial partner, TMO renewables, is a leading player in the U.K. bioenergy industry. If successful we anticipate that that withn the 3 -year programme the enzyme developed will be protected by Newcastle University and commercialized, likely through licences with leading enzyme companies and through the development of Consolidated Bioprocessing Systems with TMO Renewables.

Increased employment: The research has the potential to deliver green jobs in the UK and further afield: The development of enzyme systems that contribute to the efficient deconstruction of lignocellulosic biomass will increase the take up of the technology, promoting growth within the clean technology sector.

Benefit to the environment: A primary driver for the move from fossil fuels to fuels and chemicals from waste or renewable sources of lignocellulose, is the production of greenhouse gas (GHG) emissions. An efficiently operated biorefinery using lignocellulose should be able to deliver an 80 % reduction in GHG emissions compared to its fossil fuel equivalent (based on ethanol production). This project will assist in reaching national and international targets for use of renewables and mitigation of climate change.