Research Area: Environment

Oxidative conversion of polysaccharides - enzyme discovery, functional characterization and novel applications

Project Number: 6510

Project Duration: 1. July 2014 - 30. June 2016

Project director: Vincent Eijsink, Norwegian University of Life Sciences (NMBU)

Division Head: Christian Collin-Hansen

Technical contact person, Statoil: Christian Collin-Hansen, e-mail chrc@statoil.com

This project belongs to VISTA's previous research area Biotechnology

Objective

The development of successful biorefineries relies in part on the ability to enzymatically depolymerize polysaccharides in biomass to sugars that subsequently may be converted to useful products such as biofuels or biomaterials. Although progress is being made, the efficiency of the current enzyme preparations used by industry hamper the process of making this a cost-effective operation. The recent discovery of lytic polysaccharide monooxygenases, first described in a 2010 paper in Science by the host research group, has led to considerable improvement of enzyme efficiency and has boosted the development of second generation (lignocellulosic) biofuels and biorefining in general. Importantly, in addition to providing new tools for biomass conversion and the production of biofuels, these enzymes display a completely novel type of oxidative surface chemistry with potential implications for other biochemical processes and industrial applications. The wide abundance of these LPMOs (up to > 30 genes per fungal genome and up to about 10 genes in bacterial genomes) suggests multiple functions, including possible actions on mucus, human epithelium and bacterial exopolysaccharide structures. Indeed, transcriptome and proteome studies indicate that LPMOs may be involved in processes such as virulence and biofilm formation. Notably, LPMOs need a copper ion, an external electron donor and molecular oxygen for activity, which makes their biochemical analysis quite challenging.

Building on VISTA project 6505, and other LPMO work done in recent years at NMBU, the present project has the following key objectives:

1. Functional analysis and improvement of LPMO activity in biomass processing.

2. Discovery of LPMO-type catalytic activities in anaerobic ecosystems.

3. Assessment of LPMO applications beyond biomass saccharification.

Key activities include:

- Extensive site-directed mutagenesis studies of LPMOs, combined with various functional and biophysical analyses.

- Expressing novel LPMOs and enzymes with potential LPMO-like activities, found in various ecosystems.

- Optimization of biomass conversion processes with optimized LPMOs and LPMO combinations, using natural and engineered variants.

- Analyze the potential of LPMO-based fiber modification.

- Look for novel LPMO-catalyzed reactions, e.g. related to biofilm formation and oil degradation.

Publications:

Courtade, G., Balzer, S., Forsberg, Z., Vaaje-Kolstad, G., Eijsink, V.G.H., Aachmann, F.L. (2015)¹H, ¹³C, ¹⁵N resonance assignment of the chitin-active lytic polysaccharide monooxygenase BlLPMO10A from Bacillus licheniformis; Biomol NMR Assign. 9 (2015) 207-210 (Nb. Also reported for 2014)

Aachmann, F. L., Vaaje-Kolstad, G., Forsberg, Z., Røhr, Å., Eijsink, V. G. H., and Sørlie, M. (2015) Lytic Polysaccharide Monooxygenase. in Encyclopedia of Inorganic and Bioinorganic Chemistry, John Wiley & Sons, Ltd. pp1–13.

Selig, M. J., Vuong, T. V., Gudmundsson, M., Forsberg, Z., Westereng, B., Felby, C., and Master, E. R. (2015) Modified cellobiohydrolase-cellulose interactions following treatment with lytic polysaccharide monooxygenase CelS2 (ScLPMO10C) observed by QCM-D. Cellulose 22, 2263-2270

Bacik, J. P., Mekasha, S., Forsberg, Z., Kovalevsky, A., Nix, J. C., Cuneo, M. J., Coates, L., Vaaje-Kolstad, G., Chen, J. C. H., Eijsink, V. G. H., and Unkefer, C. J. (2015) Neutron and high-resolution room-temperature X-ray data collection from crystallized lytic polysaccharide monooxygenase. Acta Crystallogr F 71, 1448-1452

Mekasha, S., Forsberg, Z., Dalhus, B., Bacik, J. P., Choudhary, S., Schmidt-Dannert, C., Vaaje-Kolstad, G., and Eijsink, V. G. H. (2016) Structural and functional characterization of a small chitin-active lytic polysaccharide monooxygenase domain of a multi-modular chitinase from Jonesia denitrificans. FEBS Letters, accepted for publication.

Forsberg, Z., Nelson, C.E., Dalhus, B., Mekasha, S., Loose J.S.M, Crouch, L.I., Røhr, Å.K., Eijsink, V.G.H., Gardner, J.G., Vaaje-Kolstad, G. (2016) Structural and functional analysis of a lytic polysaccharide monooxygenase important for efficient utilization of chitin in Cellvibrio japonicus; J Biol Chem, submitted for publication, November 2015.