AgreenSkills session, year: 1st session, 2017
Receiving laboratory: BBF (Biodiversity and Biotechnology of Fungi - UMR 1163), INRA PACA, Aix-Marseille University
Country of origin : Denmark
Specificity of key enzymes in plant biomass conversion
This project aims to dissect the sequence-structure-specificity relationships of Lytic Polysaccharide MonoOxygenases (LPMOs). LPMOs, which are key enzymes in plant biomass conversion, are classified in families AA9-AA11 and AA13 in the CAZy database. Genes encoding AA9 LPMOs are often highly abundant in wood-degrading fungal species (>30), and this multiplicity suggests distinct functions (e.g. substrate specificity), likely to match the diversity and complexity of plant biomass. However, determinants of LPMO specificity remain unclear. During my PhD (carried out in collaboration with several international partners) I studied an AA9 LPMO from the filamentous fungi Lentinus similis (LsAA9A). Here the first LPMO-carbohydrate complex structures were presented providing insights into LPMO interactions with cellulose fragments, and revealed structural features accounting for the specificity. Other AA9 LPMOs appear to have similar functionality (inferred from structure and sequence), thus the project aims to establish sequence-structure-specificity relationships for AA9 LPMOs. Bioinformatic analysis of AA9s based on fungal proteomic data are carried out at the Architecture et Fonction des Macromolécules Biologiques (AFMB) and Biodiversity and Biotechnology of Fungi (BBF) laboratory using the tools of the CAZy database. At the BBF the selected AA9 targets are expressed in Pichia pastoris, purified using chromatographic methods and biochemical characterized and assayed on various carbohydrate substrates. Substrate specificities and product formation after enzymatic degradation are assessed and analysed with fluorometric methods, ionic chromatography (Dionex HPEAC-PAD) and mass spectrometry. Lastly, some of these AA9 members will be subjected to protein crystallization trials to further investigate structure-activity relationships.
I obtained my MSc degree in July 2012 and my PhD degree in December 2016 both at the Department of Chemistry, University of Copenhagen in the research section of Biological Chemistry (supervision by section leader Leila Lo Leggio) using synchrotron radiation based X-ray crystallography as the primary method. During my MSc I studied the C transcription factor of the lactococcal temperate bacteriophage TP901-1 involved in a genetic switch related to human pathogens (e.g. MRSA) which resulted in a first author on a publication in Biochemistry (8). My PhD project focused on structural characterization of copper-containing metalloenzymes, called Lytic Polysaccharide MonoOxygenases (LPMOs), important in biomass conversion. The project was conducted as part of an international research consortium called CESBIC (http://www.era-ib.net/cesbic), which included the company Novozymes A/S. The outcomes of the project were the first LPMO-carbohydrate complex structures, resulting in a first author publication in Nature Chemical Biology (5), as well as two papers in Nature Communications (one shared first author (1) and one coauthor (7)) which were presented during the 11th Carbohydrate Bioengineering Meeting (CBM11) in Helsinki 10-13 May 2015 (wining a poster prize) and during an oral presentation at the 19th European Carbohydrate Symposium (EUROCARB 2017) 2-6 July 2017 in Barcelona. After my PhD I have been awarded Internationalisation Postdoc Fellowship grants by the Carlsberg Foundation in order carry out biochemical characterization of LPMOs at INRA, Aix-Marseille Université in the BBF lab under Senior Scientist Jean-Guy Berrin from May 2017 to May 2019.
1) Simmons, T. J., Frandsen, K. E. H., Ciano, L., Tryfona, T., Lenfant, N., Poulsen, J. C., Wilson, L. F. L., Tandrup, T., Tovborg, M., Schnorr, K., Johansen, K. S., Henrissat, B., Walton, P. H., Lo Leggio, L., and Dupree, P. (2017) Structural and electronic determinants of lytic polysaccharide monooxygenase reactivity on polysaccharide substrates. Nat. Commun. 8:1064
• 2) Frandsen, K.E., Poulsen, J.N., Tandrup, T., Lo Leggio, L. (2017) Unliganded and substrate bound structures of the cellooligosaccharide active lytic polysaccharide monooxygenase LsAA9A at low pH. Carbohydr Res. 448 187-190.
• doi: 10.1016/j.carres.2017.03.010.
• *3) Frandsen, K.E.H., Poulsen, J.C., Tovborg, M., Johansen, K.S. & Lo Leggio, L. (2017). Learning from oligosaccharide soaks of crystals of an AA13 lytic polysaccharide monooxygenase: crystal packing, ligand binding and active-site disorder. Acta crystallographica. Section D, Structural biology 73, 64-76.
• *4) Frandsen K.E., Lo Leggio, L. (2016) Lytic polysaccharide monooxygenases – a crystallographer’s view on a new class of biomass degrading enzymes. IUCrJ. 3, 448-467.
• *5) Frandsen, K.E.H., Simmons, T.J., Dupree, P., Poulsen, JC., Hemsworth, G.R., Ciano, L., Johnston, E.M., Tovborg, M., Johansen K.S., von Freiesleben, P., Marmuse, L., Fort, S., Cottaz, S., Driguez, H., Henrissat, B., Lenfant, N., Tuna, F., Baldansuren, A., Davies, G.J., Lo Leggio, L., Walton, P.H. (2016) The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases. Nat Chem Biol 12, 298-303.
• 6) Rasmussen, K.K., Frandsen, K.E.H., Erba, E.B., Pedersen, M., Varming, A.K., Hammer, K., Kilstrup, M., Thulstrup, P.W., Blackledge, M., Jensen, M.R., Lo Leggio, L. (2016) Structural and dynamics studies of a truncated variant of CI repressor from bacteriophage TP901-1. Scientific reports 6; 29574
• *7) Lo Leggio, L., Simmons, T.J., Poulsen, JC., Frandsen, K.E., Hemsworth, G.R., Stringer, M.A., von Freiesleben, P., Tovborg, M., Johansen, K.S., De Maria, L., Harris, P.V., Soong, C.L., Dupree, P., Tryfona, T., Lenfant, N., Henrissat, B., Davies, G.J., Walton, P.H. (2015) Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase. Nat Commun 6; 5961.
• 8) Frandsen, K.H., Rasmussen, K.K., Jensen, M.R., Hammer, K., Pedersen, M., Poulsen, JC., Arleth, L., Lo Leggio, L. (2013) Binding of the N-Terminal Domain of the Lactococcal Bacteriophage TP901-1 CI Repressor to Its Target DNA: A Crystallography, Small Angle Scattering, and Nuclear Magnetic Resonance Study. Biochemistry 52, 6892-6904.
• 9) Lo Leggio, L., Frandsen, K.E.H., Poulsen, J.-C.N., Tovborg, M., De Maria, L., & Johansen, K. S. (2015). Structural diversity of lytic polysaccharide monooxygenases. Proceedings of “Mie Bioforum 2014 – Lignocellulose Degradation and Biorefinery”, 148–156
• 10) MAX IV Laboratory synchrotron radiation activity report 2015
Frandsen K.E.H., Poulsen J-C. N., Tovborg M., Johansen K.S., von Freiesleben P., Lo Leggio L. Crystal structures of lytic polysaccharide monooxygenases (LPMOs) from families AA9 and AA13. (https://www.maxlab.lu.se/node/2032#MX_)
• Carlsberg Foundation Internationalisation Fellowship (Postdoctoral grant for outstanding PhDs for a 12-month stay at a leading international research institution). Grant CF17-0533 of DKK 425.000 (equivalent of € 57.000)
• Carlsberg Foundation Internationalisation Fellowship (Postdoctoral grant for outstanding PhDs for a 12-month stay at a leading international research institution). Grant CF16-0673 of DKK 350.000 (equivalent of € 47.000)
• Top 3 candidate for the MAX IV Best PhD Thesis Award 2017
• 2nd Prize of Poster Session I at 11th Carbohydrate Bioengineering Meeting (CBM11) | http://www.cbm11.org
Website(s): https://www6.paca.inra.fr/umrbcf | http://orcid.org/0000-0002-7136-9820 | https://www.researchgate.net/profile/Kristian_Frandsen | https://www.growkudos.com/profiles/146972 | https://www.scopus.com/authid/detail.uri?authorId=57073675100 | https://loop.frontiersin.org/people/309457/overview | https://scholar.google.com/citations?user=wPHpnfIAAAAJ&hl=en | https://www.linkedin.com/in/kristian-e-h-frandsen-68303124