Optimized oxidative enzyme systems for efficient conversion of lignocellulose to valuable products.

Highlights 2020

The project's contribution to the Centre for Digital Life Norway annual report 2020.

OXYMOD aims to discover, understand and apply redox enzyme systems for the conversion of biomass, such as lignocellulose, into valuable products. OXYMOD combines life sciences (enzyme technology, microbial biotechnology, high throughput screening, advanced analytics), bioinformatics (big data, enzyme systems modelling, process modelling) and engineering (enzyme evolution, synthetic biology) for developing novel biocatalytic systems.

In 2020 we have focused on characterisation of novel enzymes, in particular a multicopper oxidase and peroxidases, acting on lignin, and lytic polysaccharide monooxygenases (LPMOs), acting on cellulose. In addition, we have carried out an innovative functional screening of our unique collection of actinobacteria to select promising strains for enzyme discovery. Connections between lignin- and cellulose-degrading redox enzyme are being explored, including the roles of redox mediators and various oxidants. Individual enzymes are characterised in-depth, both experimentally and computationally. Kinetic modelling of complete enzyme systems will be done in 2021.

Highlights of 2020 include prestigious papers on LPMO catalysis and papers on enzyme engineering and on abiotic redox processes that affect LPMO activity. Much progress has been made in developing novel mass-spectrometry based screening methods for lignin-active enzymes. Another highlight was the identification of novel bacterial strains that can potentially break down lignin, providing a unique source for further enzyme discovery.

Being part of a transdisciplinary centre allows us to follow the interdisciplinary approach that we need to reach our goals. By combining techniques from different knowledge domains such as big data mining, atom-scale modelling, molecular biology, biochemical protein characterisation, and robotic variant screening, we have a unique possibility to understand and develop enzyme systems for degradation of lignocellulosic biomass and create bioeconomic value in Norway. The project has multiple direct and indirect (via adjacent projects) links to industries. In fact, technologies developed by OxyMod partners are already used in industrial settings.

Scientific publications 2020: 4

Short legend: NMR-based studies of the structure and dynamics of an LPMO, highlighting the catalytic copper binding site. Copper appears as an orange sphere.

Picture taken from “Mechanistic basis of substrate-O2 coupling within a chitin-active lytic polysaccharide monooxygenase: an integrated NMR/EPR study”, by G Courtade, L Ciano, A Paradisi, P Lindley, Z Forsberg, M Sørlie, R Wimmer, GJ Davies, VGH Eijsink, PH Walton, and FL Aachmann; Proc Natl Acad Sci U S A, 2020 Aug 11;117(32):19178-19189. doi: 10.1073/pnas.2004277117.

Blog about the publication: Biomass processing: Mechanistic aspects of lytic polysaccharide monooxygenases

Full figure legend from the journal

Structures of apo- and Cu(I)-BlLPMO10A. (A) Ensemble of the 10 lowest-energy conformers of apo-BlLPMO10A (PDB ID code 5LW4) in stereo representation. Helices are coloured red, loops are coloured green, and strands are coloured yellow; the lowest CYANA target energy conformer is coloured blue. The overall backbone rmsd of the ensemble is 2.41 Å, while the rmsd of the regions containing α-helices (residues 41–47, 57–61, 82–84, 89–94, and 159–162) and β-sheets (residues 33–36, 37–40, 103–107, 110–117, 125–132, 150–154, 164–168, 175–184, 185–190, and 191–201) is 1.48 Å. (B) Overlay of apo-BlLPMO10A (green) and Cu(I)-BlLPMO10A (PDB ID code 6TWE; blue). The copper atom is shown as an orange sphere, and the side chains of His32 and His121 are shown as sticks. The backbone (Cα, N, C′) rmsd between the apo ensemble and the Cu(I) ensemble is 0.9 Å. (C) Zoomed-in view of the overlay in B showing details of the copper site. (D) Ensemble of five lowest-energy conformers of Cu(I)-BlLPMO10A, showing the copper site. Average distances from each N atom to the Cu atom are indicated. (E) PRE effects upon adding Cu(II) to apo-BlLPMO10A. The black line shows the normalised HN, N signal intensity upon addition of Cu(II) to 13C- and 15N-labeled apo-BlLPMO10A in a 1:2 ratio, relative to the intensity for the apo-enzyme, with errors shown in gray. The red line shows PREs calculated using the Cu(I)-BlLPMO10A ensemble. Gaps in the data represent missing assignments for amino acid residues (e.g., Pro).

Project overview

Project lead: Vincent Eijsink
Institution: NMBU
Partners: SINTEF, NTNU, New University of Lisbon, VECTRON BIOSOLUTIONS AS



Announcement of four recent publiucations in OxyMod (2020)

Biomass processing: Mechanistic aspects of lytic polysaccharide monooxygenases (2020)

Searching for a redox partner for bacterial lytic polysaccharide monooxygenases (2020)
Camilla Demmene

Engineering lytic polysaccharide monooxygenases (LPMOs) (2020)
Zarah Forsberg, Anton Stepnov, Guro Kruge Nærdal, Geir Klinkenberg, Vincent Eijsink

Molecular mechanism of the chitinolytic peroxygenase reaction (2020)
Bastien Bissaro, Bennett Streit, Ingvild Isaksen, Vincent Eijsink, Gregg T. Beckham, Jennifer DuBois, Åsmund Røhr Kjendseth

Mechanistic basis of substrate–O2 coupling within a chitin-active lytic polysaccharide monooxygenase: An integrated NMR/EPR study (2020)
Gaston Courtade, Luisa Ciano, Alessandro Paradisi, Peter J. Lindley, Zarah Forsberg, Morten Sørlie, Reinhard Wimmer, Gideon J. Davies, Vincent Eijsink, Paul H. Walton, Finn Lillelund Aachmann

Discovery and characterization of novel redox enzymes from Trondheim fjord's actinobacteria strain collection for lignocellulosic biomass degradation (2019)
Giang-Son Nguyen, Malene Jønsson, Priscilla C. Neeraas, Vincent Eijsink, Alexander Wentzel

Oxidative monocopper enzymes that accelerate glycoside hydrolase conversion of lignocellulosic biomass (2019)
Vincent Eijsink

Lytic polysaccharide monooxygenases (LPMOs) - unique mono-copper oxidases for biomass processing and more (2019)
Vincent Eijsink

Mechanism of the chitinolytic peroxygenase reaction (2019)
Åsmund Røhr Kjendseth

All results in the CRIStin-database

Research group

The OXYMOD project will through a transdisciplinary approach define, develop and demonstrate applicability of new enzyme systems for the efficient biocatalytic conversion of lignocellulose from abundant Norwegian biomass into valuable products like sugars and aromatic building blocks. OXYMOD will focus on the still largely underexplored group of redox enzymes and their potential in the depolymerisation of cellulose, hemicellulose and lignin, including aspects such as redox enzyme interplay, co-factors and reaction partners, as well as their interplay with hydrolytic enzymes. OXYMOD will address these enzymes and enzyme systems as they occur and function in, among others, a unique in-house collection of approx. 1000 marine Actinobacteria isolates with genomes recently sequenced.

Redox enzymes require co-factors and redox partners, and there is a considerable degree of cooperativity between different enzyme classes. Enzyme systems-scale understanding and eventually engineering the efficient degradation of lignocellulose by these enzyme systems, requires an integrated transdisciplinary approach far beyond 'simple' enzyme discovery.

OXYMOD combines life sciences (enzyme biochemistry, enzyme production technology, microbial biotechnology, high throughput screening, advanced analytics), ICT (bioinformatics, big data handling), mathematical sciences (enzyme systems modelling, process modelling) and engineering (enzyme evolution, synthetic biology) for producing new and optimized biocatalytic systems for industrial application, primarily within the agricultural and forest sectors.

Besides the enzymes and enzyme systems themselves, additional innovations from OXYMOD concern the generation of well-defined products streams, primarily sugars from (hemi-)cellulose and aromatic building blocks from lignin for a variety of downstream applications (e.g. biofuels & bioplastics).