Innovative enzyme technology for biomass processing

The goal of this project was to study a new type of enzymes that were discovered by NMBU in 2010 (see http://www.umb.no/forsiden/artikkel/nytt-enzym-gir-berekraftig-biodrivstoff). These enzymes are today called "LPMO" for "Lytic Polysaccharide Monooxygenase"; they make enzymatic decomposition of biomass, such as cellulose, much more efficient. There are many such enzymes in nature, but only few have been characterized in detail. The discovery of LPMOs has generated completely novel biochemical insights and has revolutionized the field of enzymatic biomass processing. Today, these enzymes are part of all major commercial cellulase preparations for biorefining. The overall goal of this project was to characterize a number of such LPMOs to identify which activities they have and to study the basic mechanisms behind these activities. Possible applications in biorefining of different types of biomasses were also be tested. An important question was whether some LPMOs may act on other biomass types ("substrates") than chitin and cellulose, which were the only two known substrates at the project start. We have produced and characterized several new LPMOs and this has led to new insights into how LPMOs work and, importantly, to the discovery of LPMOs acting on hemicellulose (xyloglucan, glucomannan). This last, very important discovery was published in the prestigious journal PNAS, in 2014. We are using a variety of advanced techniques, such as X-ray crystallography to study enzyme structures, Electron Para Magnetic Resonance (EPR) spectroscopy to understand the enzyme's catalytic mechanism, microscopy to understand how enzymes break up the substrate, and site-directed mutagenesis to map the structural determinants of activity, stability and substrate specificity. The results of such "in-depth characterization" of the hemicellulose-active enzyme were published in a massive and groundbreaking 15-page article in 2015. LPMOs use electrons, and from our applied projects project, we learned in 2014-2015 that access to electrons is an important success criterium in industrial biorefining processes. Therefore, in the project's final phase, and in cooperation with groups in Copenhagen and Vienna, we have looked at how enzymes obtain these electrons while they work in their natural surroundings, e.g. when a fungus breaks down wood. This has led to important new insights, also on the applied side (meaning: how can these enzymes best be utilized in a biorefinery). This work has contributed to two publications in top journals, namely Science and PNAS, both in spring 2016. The LPMOs that we have studied in this project are being tested in more applied projects that target effective enzymatic processing of biomass. Accumulating results suggest that the efficiency of LPMOs varies widely between substrates. Thus, certain LPMO seem best suited for certain substrates. This insight has important consequences for use of these enzymes in industry. One of the latest results from this project is that it may seem as if some LPMOs are optimized to work on "composites", i.e. mixtures of different polysaccharides, such as one finds them in plant cell walls. This feature is being studied further, in new projects because this has both great scientific and large industrial interest.

For many years, the enzymatic depolymerization of polysaccharides in plant biomass was thought to be accomplished by the complementary action of endo- and exo-acting hydrolases. Recently, however, it has been shown that there are additional enzymes, class ified as "CBM33" and "GH61", that aid in this conversion process using a hitherto unknown mechanism involving hydrolysis and oxidation. Genes encoding GH61 proteins are abundant in the genomes of biomass-degrading fungi and these enzymes are likely to act on a variety of biomass types. These recently discovered GH61 enzymes deserve massive attention since there are indications that they may be exploited to dramatically increase the efficiency of enzymatic biomass conversion. However, this enzyme class has hardly been explored and many crucial questions remain unanswered. In this project we will address such crucial questions by carrying out the following studies: (1) Cloning and expression of all or as many as possible GH61 enzymes from two biomass degrad ing fungi, Phanerochaete chrysosporium (17 genes) and Heterobasidion irregulare (10 genes). (2) Characterization of enzymatic activities, substrate-specificities and synergies with other enzymes (such as cellulases or other GH61s). (3) In-depth analysis o f the catalytic mechanism using atomic force microscopy to study enzyme-substrate interactions and using a variety of biochemical and biophysical techniques to unravel atomic details of the catalytic cycle; (4) Deepen understanding of GH61 functionality b y classical structure-function work (i.e determining crystal structures and testing effects of site-directed mutations); (5) Testing the applicability of GH61 for biomass conversion and designing optimal enzyme cocktails. The project will be based on the applicants' current leading position in research on these novel enzymes, including advanced analytical competence that is both unique and essential. There will be collaborations with groups in Sweden and Japan.