Background and activities

I am a PostDoc financed by a personal grant from the Novo Nordisk Foundation. The project consists of studying multi-domain lytic polysaccharide monooxgyenases with NMR spectroscopy, simulations, and other biophysical (e.g. SAXS) and biochemical techniques to understand the interplay between the folded domains and the role of unstructured linker regions.

I am a part of the Biopolymer NMR research group at the Norwegian Biopolymer Laboratory (NOBIPOL).

My main research interest is the use NMR spectroscopy, MD simulations and biochemistry techniques to study the structures and functions of enzymes. Currently, I am involved in the following activities.

1. Felxible protein linkers
Project: LinkD - Novo Nordisk Foundation

Linkers are intinsically disordered regions of proteins. Linkers of varying flexibility keep multiple domains in proteins joined together. Depending on their sequence and length, linkers may increase or decrease the number of contacts between the thethered domains [1]. Therefore, linkers have important roles in regulating the function of the enzyme domains they are attached to [2].

In collaboration with Prof. Kresten Lindorff-Larsen at the University of Copenhagen, we are using an integrative modelling approach [3] to study the function of linker in LPMOs. We perform MD simulations to generate conformational ensembles that are then validated with small-angle X-ray scattering (SAXS) and NMR spectroscopy (secondary chemical shift, diffusion) data to produce models of multi-modular enzymes.

2. Efficient enzyme production systems
Project: OXYMOD - Digital Life Norway/Research Council of Norway

Producing sufficient amounts (>1 g/L of culture) of pure, correctly folded enzymes is a pre-requirement for all biochemical investigations. Heterologous expression in E. coli is the most straight-forward way to produce enzymes. However, protein expression when using glucose as the sole carbon source, for example for 13C enrichment, often leads to low yields as a result of catabolite repression of promoters based on the carbon metabolism of E. coli, such as Plac. 

We have circumvented this problem by constructing an expression system for LPMOs based on the Pm/XylS promotor/activator system [4], in collaboration with Prof. Trygve Brautaset. In cooperation with SINTEF Industry, we were able to demonstrate the system’s usability in high-cell density cultivations, leading to very high protein yields in bioreactors.

3. LPMOs (lytic polysaccharide monooxygeneases) and CBMs (carbohydrate-binding modules)
Project: OXYMOD - Digital Life Norway/Research Council of Norway

LPMOs are copper-dependant enzymes that degrade carbohydrates through an oxidative mechanism [5–7]. The LPMO reaction involves the reduction of Cu(II) to Cu(I) in the active site by an electron donor that can either be another redox protein or small-molecule reductants [8]. After this initial reduction, the copper site activates either O2 [9] or H2O2 [10]. LPMOs use this activated oxygen species to oxidize carbohydrates, making tightly-bound polysaccharides like cellulose and chitin available to other enzymes [11, 12] like glycoside hydrolases, which convert polysaccharides to sugar. These products can then be fermented into bioethanol or converetd to high value-added products [13]. In addition to these polysaccharides, LPMOs have been shown to be active on a wide variety of carbohydrates such as amylose and hemicellulose. Moreover, CBMs [14] tethered to LPMOs through peptide linkers confer different substrate binding possibilities.

Overall, LPMOs are important factors in enabling the green shift from an oil-based to a sustainable society. To achieve this, a better understanding of their mode of action is needed. I started working on LPMOs in 2012 during my Master’s, and continued working with them as the main focus of my PhD (2014-2018). Through various collaborations, we have been able to gain valuable insights into LPMO functionality, such as:

  • Insights into the three-dimensional structure of LPMOs, including the effect of copper on the overall structure and active site conformation.
  • Structure-function relationships between LPMO structure and binding specificity to different substrates.
  • Interactions with redox partner enzymes.
  • Combining experiments and simulations to study substrate binding of CBM domains attached to LPMOs.

References

1. X. Chen, J. L. Zaro, W. C. Shen, Advanced Drug Delivery Reviews 65, 1357–1369 (2013). https://doi.org/10.1016/j.addr.2012.09.039

2. B. Ma, C. J. Tsai, T. Haliloglu, R. Nussinov, Structure 19, 907–917 (2011). https://doi.org/10.1016/j.str.2011.06.002

3. S. Bottaro, K. Lindorff-larsen, Science 361, 355–360 (2018). https://doi.org/10.1126/science.aat4010

4. Courtade G., Balzer S. L., Sætrom G. I., Brautaset T., Aachmann F. L.. A novel expression system, Carbohydrate Research 448, 212–219 (2017) https://doi.org/10.1016/j.carres.2017.02.003

5. G. Vaaje-Kolstad, S. J. Horn, D. M. F. van Aalten, B. Synstad, V. G. H. Eijsink, JBC 280, 28492–28497 (2005). https://doi.org/10.1074/jbc.M504468200

6. G. Vaaje-Kolstad et al., Science 330, 219–222 (2010). https://doi.org/10.1126/science.1192231

7. R. J. Quinlan et al., PNAS 108, 15079–15084 (2011). https://doi.org/10.1073/pnas.1105776108

8. D. Kracher et al., Science 352, 1098–1101 (2016). https://doi.org/10.1126/science.aaf3165

9. P. H. Walton, G. J. Davies, Current Opinion in Chemical Biology 31, 195–207 (2016). https://doi.org/10.1016/j.cbpa.2016.04.001

10. B. Bissaro et al., Nature Chemical Biology 13, 1123–1128 (2017). https://doi.org/10.1038/nchembio.2470

11. S. J. Horn, G. Vaaje-Kolstad, B. Westereng, V. G. H. Eijsink, Biotechnology for Biofuels 5, 45 (2012). https://doi.org/10.1186/1754-6834-5-45

12. Z. Forsberg et al., Protein Science 20, 1479–1483 (2011). https://doi.org/10.1002/pro.689

13. K. S. Johansen, Biochemical Society Transactions 44, 143–149 (2016). https://doi.org/10.1042/bst20150204

14. A. B. Boraston, D. N. Bolam, H. J. Gilbert, G. J. Davies, Biochem. J 382, 769–781 (2004). http://doi.org/10.1042/bj20040892

Scientific, academic and artistic work

Displaying a selection of activities. See all publications in the database

Journal publications

Part of book/report

  • Courtade, Gaston; Aachmann, Finn Lillelund. (2019) Chitin-Active Lytic Polysaccharide Monooxygenases. Targeting Chitin-containing Organisms.

Report/dissertation

  • Courtade, Gaston; Brautaset, Trygve; Aachmann, Finn Lillelund. (2018) An NMR investigation of lytic polysaccharide monooxygenases. 2018. ISBN 978-82-326-3462-0. Doktoravhandlinger ved NTNU (337).
  • Courtade, Gaston; Skjåk-Bræk, Gudmund; Aachmann, Finn Lillelund. (2014) Structural Investigation of a Lytic Polysaccharide Monooxygenase by NMR Spectroscopy. 2014.