Unlocking the metabolic potential of the exceptional lignocellulose degrading fungus Parascedosporium putredinis N01

Lignocellulose provides structure and support to growing plants by enabling strong and durable cell walls. It is one of the most abundant forms of fixed carbon in the biosphere and its breakdown is a critical component of the global carbon cycle. Lignocellulose also represents a vast global resource of sugars and aromatics which can be liberated for the production of biofuels and chemicals. Biorefineries require the use of high yielding, low input biomass crops and/or waste biomass as feedstock; however, the complex phenolic polymers present in lignin make lignocellulose very resistant to enzymatic attack. Current industrial approaches for the breakdown of lignocellulose employ multi-component enzymatic mixtures. While this approach is effective in hydrolysing polysaccharides, it is inefficient and expensive due to the requirement of chemically-intensive pretreatments to disrupt lignin and reduce the crystallinity of cellulose. It is generally recognised that the breakdown of lignin forms the highest barrier to the development of a cost-competitive lignocellulose processing industry, and this barrier will be overcome in part by the discovery of new enzymes for more effective digestion of the feedstock. It is also generally recognised that valorisation of lignin will be a crucial component of integrated biorefineries if they are to be profitable, therefore, it is essential that new strategies for obtaining high value chemicals from lignin are developed.

The basis of this project is to gain a detailed understanding of lignocellulose degradation by the fungus Parascedosporium putredinis NO1 and begin to design more efficient industrial approaches to lignin degradation and valorisation. We will carry out a full characterisation of an exciting new ligninase that we have recently discovered that cleaves the major beta-ether structural units in lignin without a requirement of a cofactor. This enzyme not only boosts the breakdown of biomass by commercial cellulase cocktails, it also releases the pharmaceutically important flavonoid tricin from the lignin macromolecule, offering the possibility of producing a valuable product from lignin while reducing the processing costs. We will use 2-dimensional NMR to identify major changes in lignocellulose composition during digestion. We will use comparative proteomics to compare the secretome of P. putredinis NO1 growing on lignin to identify new lignin active enzymes for further study with a particular focus on possible biorefining applications. We will investigate the genes and enzymes expressed, and biochemical processes, in order to identify new enzymes for study. We will produce recombinant forms of these enzymes for characterisation studies. We will use high throughput assay systems to examine the commercial potential of new enzymes for biomass processing. These studies can help us to understand important environmental components of the global carbon cycle, as well as providing us with new understanding and tools to improve the industrial exploitation of lignocellulosic biomass.

We have isolated the soft rot Ascomycete Parascedosporium putredinis NO1 from a wheat straw composting community where it dominated the latter stages of growth after the readily accessible polysaccharides had been depleted and the lignin had become enriched. P. putredinis NO1 is adept at breaking down lignin and, surprisingly, is able to grow on kraft lignin as a sole carbon source. We have identified two exciting new activities in the secretome of P. putredinis NO1 when it grows on wheat straw, a previously unreported polyphenol oxidase (beta-etherase) which is able to cleave the major structural beta-O-4 linkage in lignin and a 'dioxygenase-like' enzyme that boosts saccharification of lignocellulose by commercial cellulase cocktails. Importantly, both enzymes do not require a cofactor for activity making them ideal for biorefining applications. We have also developed a powerful proteomics technique that has facilitated the identification a large number of unknown proteins that are specifically secreted when P. putredinis grows on lignocellulose feedstocks as the carbon source for growth. Our goal is to fully characterise these two new ligninases and determine the role the unknown proteins in the P. putredinis NO1 secretome play in lignin deconstruction. We will use our high-throughput assays systems to examine the synergies between enzymes and evaluate their use in lignocellulolytic cocktails.