Towards Novel Glycoside Hydrolases

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One of the main challenges of industrial (white) biotechnology today is the production of fuel from biomass at a cost that ultimately must be competitive with fossil fuels - but potentially more sustainable in the long term as a renewable, carbon-neutral energy source providing energy security. "Green" (i.e. environmentally-friendly) industrial production lines - characterised by reduced energy consumption, waste and CO2-emissions - are clearly attractive, but are crucially reliant on the discovery, improvement and adaptation of robust and efficient biocatalysts. This means that methods and strategies have to be developed that allow identification of suitable catalysts. While the utility of enzymes for biocatalysis is clear, it is still not trivial to find or make such efficient, useful catalysts for a wide range of purposes by enzyme engineering (now arguably based more often on Darwinian cylces of 'directed evolution, rather than design - although the two approaches are not exclusive). We tackle in this proposal the challenge that each step in the 'bioenergy pipeline' (from growing biomass to fermentation for biofuels) can potentially become rate- or cost-limiting and that enzymes for those purposes are clearly needed. The natural resistance of plant cell walls to microbial and enzymatic deconstruction is largely responsible for the high cost of lignocellulosic biomass conversion. To date, only a small proportion (approximately 40%) of the energy content available from lignocellulose feedstocks (unusable portions of plant materials in the form of agricultural, industrial, domestic, and forest residues) is convertible to ethanol. We address this problem at a number of fronts: using a ultra-high throughput screening system we identify new protein catalysts (from metagenomic libraries) and we improve these and already characterised catalysts by multiple rounds of directed evolution. Our directed evolution is propelled by access to a new type of libraries (mimicking natural mechanisms - involving insertion and deletions) and by the availability of ultrahigh-throughput screens of very large libraries (>10e7 members). We hope that the analysis of the selected novel enzymes will unravel evolutionary relationships between cellulases/hemicellulases and the structural basis for substrate recognition as a basis to improve the engineering of new useful biocatalysts enabling efficient plant cell wall hydrolysis. Our final goal is to to make robust and multispecific biocatalysts that should be useful for the hydrolysis of recalcitrant lignocellulosic components available as well as general rules and experimental approaches to make this process more controllable and enable it to be widely used.