Research on fungal pathogen – host plant relationship was historical research of the laboratory at the Institute of Biochemistry of the Faculty of Medicine, University of Ljubljana, preceding the core MCMB research unit. It was based on investigation of steroid biotransforming micoorganisms and their enzymes in order to construct relevant microbial strain(s) for biotechnological production of steroid drugs. The CYP-enzyme-harbouring filamentous fungi in the context of fungal pathogen – host plant relationship were studied first at the Faculty of Medicine, but in 2002 major experimentation was transfered from MCMB to the National Institute of Chemistry (partner of the interinstitutional programme Functional Genomics and Biotechnology for Health, P1-0104), where furthermore benzoate 4-monooxygenase (BPH) was cloned from the experimental fungus and research was then oriented to considering this cytochrome P450 as potential new target for antifungal treatment and therapy. With mathematical model and bioinformatic tools we made a selection of possible inhibitors, performed experiments “in vitro” and “in vivo”, and suggested leading compounds for the development of new antifungals.
Our strategy of targeting benzoate 4-monooxygenase (BPH) from the filamentous fungus Cochliobolus lunatus, a plant and facultative human pathogen, with a low-dose combinantion of natural plant defence compounds and selected BPH inhibitor (which was highlighted as an promising approach in 2008 by SciBX, a joint edition of Nature and BioCentury journals), was in the last years revived at MCMB in the context of plant defence against fungal pests. Namely, with the recent climate changes we are confronted with the large-scale destruction of spruce forests due to bark beetle and associated fungus Grosmannia clavigera invasion. In this context we have done, in collaboration with the University of British Columbia in Vancouver, some research on associated fungal P450 systems that may represent promising targets for our antifungal strategy.
Research projects:
- Interinstitutional national research program P1-0104 Functional Genomics and Biotechnology for Health (R. Komel, MF/NIC); 2004-2008 and 2009-2014; WP5 – Cytochromes P450 and design of new antifungal compounds.
- J4-2212 Fungal cytochromes P450 involved in detoxification of plant defence compounds, as targets of new antifungals; national basic research project (N. Kraševec, NIC & R. Komel NIC/MF), 1.5.2009 – 30.4.2012.
- L4-4353 Steroid 11beta hydroxylase from the filamentous fungus Cocliobolus lunatus; applied research project (R. Komel, NIC), 1.7.2002 – 30.6.2005.
Industrial partner:
- LEK Pharmaceutical Company d.d., Slovenia.
Collaborating research/faculty institutes:
- National Institute of Chemistry (NIC), L-11: Laboratory for Biosynthesis and Biotransformation (Head: Prof. Radovan Komel, PhD), Ljubljana.
- Institute of Biochemistry, Faculty of Medicine UL, Ljubljana (Prof. Jure Stojan, M.D.).
- Faculty of Pharmacy UL, Chair of Pharmaceutical Chemistry (Prof. Stanislav Gobec, PhD).
- Faculty of Agriculture and Life Sciences, University of Maribor, Slovenia (Dr. Andreja Urbanek Krajnc).
International collaboration:
- Michael Smith Laboratories, University of British Columbia, Vancouver, Canada (Prof. Joerg Bohlmann, PhD).
- Department of Wood Science, Faculty of Forestry, University of British Columbia, Vancouver, Canada (Prof. Joerg Bohlmann, PhD).
- Formerly also: Krebs Institute for Biomolecular Research, Sheffield University, U.K. (Dr. Steven L. Kelly).
Researchers involved:
| Researcher | Position |
| Radovan Komel | Program/project leader |
| Sabina Berne | PostDoc |
| Marko Vitas | PhD student |
| At NIC: | |
| Ljerka Lah | PhD student |
| Metka Novak | PhD student |
| Barbara Podobnik | PhD student from LEK |
| Erika Šuligoj | Postgraduate trainee |
| Nada Kraševec | Senior researcher |
| Branka Korošec | PostDoc |
Education:
| Student | Thesis | Title | Year | Supervisors |
| Metka Novak | PhD | Cytochromes P450 from blue-stain fungi involved in monoterpene biotransformation. | 2014 |
Radovan Komel Joerg Bohlmann |
| Erika Šuligoj | Dipl | Testing of putative benzoate 4-monooxygenase inhibitors as new antifungal compounds. | 2012 |
Radovan Komel Igor križaj |
| Barbara Podobnik | PhD | Functional analysis of cytochrome P450, para-benzoate hydroxylase from the filamentoud fungus Cochliobolus lunatus. | 2010 | Radovan Komel |
| Judita Merzdovnik | Dipl | Preparation of recombinant benzoate para-hydroxylase for crystallization studies. | 2010 |
Radovan Komel Igor Križaj |
| Ljerka Lah | PhD | Evolutionary and biological aspects of fungal cytochrome P450 reductases. | 2009 | Radovan Komel |
| Metka Novak | Dipl | Isolation and evaluation of cytochrome P450 reductase of the filamentous fungus Cochliobolus lunatus. | 2009 |
Radovan Komel B. Podobnik |
| Enej Kuščer | Dipl | Searching for the gene encoding steroid 11[beta]-hydroxylase of the filamentous fungus Cochliobolus lunatus. | 2002 |
Radovan Komel D. Žgur Bertok |
| Marko Vitas | PhD | Biotransformation of steroids by the fungus Cochliobolus lunatus and the yeast Saccharomyces cerevisiae. | 1999 | Radovan Komel |
| Nada Nekrep | Dipl | Rapid amplification of 5’end of complementary DNA in order to find a gene for steroid 11[beta]-hydroxylase in filamentous fungus Cochliobolus lunatus. | 1998 | Radovan Komel |
| Matjaž Barborič | Dipl | Amplification of total and subtracted complementary DNA in looking for the gene encoding steroid 11[beta]-hydroxylase of filamentous fungus Cochliobolus lunatus. | 1998 | Radovan Komel |
| Tadej Pajić | Dipl | Contribution to study of progesterone hydroxylation by filamentous fungus Cochlibolus lunatus. | 1996 |
Radovan Komel A. Pavko |
| Marko Vitas | MSc | Steroid monooxygegenases in filamentous fungi. | 1995 |
Radovan Komel Steven L. Kelly |
| Damjana Rozman | PhD | Heterologous transformation and steroid metabolism of the filamentous fungus Cochliobolus lunatus. | 1993 | Radovan Komel |
Selected publications:
- Berne s. et al. (2015): Benzoic acid derivatives with improved antifungal activity: design, synthesis, structure-activity relationship (SAR) and CYP53 docking studies. Bioorganic & Medicinal Chemistry 23(15): 4264-76.
- Novak M. et al. (2015): Oleic acid metabolism via a conserved Cytochrome P450 System-mediated ω-hydroxylation in the bark beetle-associated fungus Grosmannia clavigera. PloS One 10(3): e0120119.
- Wang Y. et al. (2014): Gene discovery for enzymes involved in limonene modification or utilization by the mountain pine beetle-associated pathogen Grosmannia clavigera. Environ. Microbiol. 80(15): 4566-4576.
- Novak M. et al. (2014): Low density Ceratocystis polonica inoclation of Norway spruce (Picea abies) triggers accumulation of monoterpenes with antifungal properties. Eur. J. Forest Res. 133(4): 573-583.
- Khajamohiddin S. et al. (2014): Genome-wide identification, annotation and characterization of novel thermostable cytochrome P450 monooxygenases from the thermophilic biomass-degrading fungi Thielavia terrestris and Myceliophthora thermophila. Genes and Genomics 36(3): 321-333.
- Korošec B. et al. (2014): Antifungal activity of cinnamic acid derivatives involves inhibition of benzoate 4-hydroxylase (CYP53). Appl. Microbiol. 4(116): 955-966.
- Jawallapersand P. et al. (2014): Comparative structural and evolutionary analysis and its role as a common alternative anti-fungal drug target. PloS One 9(9): 1-15 = e107209.
- Urbanek Krajnc A. et al. (2014): Antioxidative response patterns in Norway spruce bark to low density Ceratocystis polonica inoculation. Trees – Structure and Function 28(4): 1145-1160.
- Lah L. et al. (2013): The cytochromes P450 of Grosmannia clavigera: genome organization, phylogeny, and expression in response to pine host chemicals. Fungal Genet. Biol. 50: 72-81.
- Berne S. et al. (2012): Virtual screening yields inhibitors of novel antifungal drug target, benzoate 4-monooxygenase. Chem. Inf. Model. 52(11): 3053-3063.
- Lah L. et al. (2011): Versatility of the fungal cytochrome P450 monooxygenase system is instrumental in xenobiotic detoxification. Mol. Microbiol. 81(5): 1374-89.
- Lah L. et al. (2008): High diversity and complex evolution of fungal CPR : P450 system. Fungal Genet. & Biol. 45(4): 446-458.
- Podobnik B. et al. (2008): Benzoate para hydroxylase of Cochliobolus lunatus Curvularia lunata CYP53 – a novel antifungal drug target. J. Med. Chem. 51(12): 3480-3486.
Some relevant previous publications:
- Vitas M. et al. (1999): Induction of steroidal hydroxylase activity by plant defence compounds in the filamentous fungus Cochliobolus lunatus. Chemosphere 38(4): 853-863.
- Žnidaršič Plazl P. et al (1999): Induction of steroidal 11[alpha]-hydroxylase activity in the filamentous fungus Rhizopus nigricans by tomatidine and Primula veris root extract. Mol. Plant Pathol. 55(4): 251-254.
- Rozman D. et al. (1996): Steroid biotransforming strains designated Cohliobolus lunatus m118 and Curvularia lunata AT46 are both Curvularia lunata lunata. Mycotaxon 59: 489-498.
- Vitas M. et al. (1995): Progesterone side-chain cleavage by Paecilomyces lilacinus. Phytochemistry 40(1): 73-76.
- Rozman D. et al. (1994): Ultrastructure and genotypic characterization of the filamentous fungus Cochliobolus lunatus in comparison to the anamorphic strain Curvularia lunata. FEMS Microbiology Letters 117: 35-40.
- Hoerhold C. et al. (1986): Bioconversion of steroids by Cochliobolus lunatus. I. Transformation of REICHSTEIN’s compound S with cell-free preparation of Cochliobolus lunatus. Journal of Basic Microbiology 26(6): 335-339.
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