Tell us something about your background.
I did my bachelor’s degree in Microbiology at the University of Ljubljana, Slovenia. During this time I developed a great interest in molecular biology, which is why I decided to pursue a master’s degree in the new Molecular and Functional Biology program that my University offered. There I took an optional course in Cancer Biology which led to me working with the principal lecturer, Prof. Dr. Tamara Lah, on one of her projects. This eventually led to my first publication as a co-author in the resulting paper.
For my master’s thesis project, I joined the group of Dr. Hannes Link from the Max Planck Institute for Terrestrial Microbiology in Marburg, Germany. There I was working in the field of Protein Engineering and Metabolomics. The project entailed the creation of split metabolic enzymes, establishing a method for their re-complementation. We then measured the impact of this complementation on the Escherichia coli metabolism, using a powerful LC-MS/MS technique developed previously by Dr. Link. My time in Marburg sparked my interest in Protein Engineering, which I wanted to pursue as a Ph.D. project.
Tell us a bit about your PhD project in OXYTRAIN and your research interests.
My project in OXYTRAIN: Directed Evolution of heme-dependent monooxygenases for the production and formulation in kit applications.
Heme-dependent monooxygenases such as cytochrome P450, catalyze the incorporation of an oxygen atom, from environmental oxygen, into a substrate. These enzymes are ubiquitous in nature, found in all kingdoms of life, from archaea to humans. They catalyze a great many reactions that are mostly catabolic in nature. In humans, they are the principal family of enzymes involved in drug and hormone metabolism. Specifically, the cytochrome P450 family of enzymes is involved in 75% of the total drug metabolism in humans1. Because of their ubiquitous presence and importance, they are of great interest to the pharmaceutical industry. They are used in the synthesis of drugs, as well as predicting how drugs will metabolize in the body. For drug synthesis, oxygenases are used mostly for the catalysis of reactions that are difficult to perform chemically, as they offer great specificity and environmental friendliness.
The goal of this project will be to produce heme-dependent monooxygenases to be used in a commercial kit for a variety of industrial applications, including the pharmaceutical industry. Enzyme kits are used in the industry to screen for novel enzymes capable of catalyzing desired reactions. An ideal kit would include thousands of enzymes, each in different buffer conditions. Monooxygenase kits currently available on the market contain at most 20 different cytochrome P450 monooxygenases and thus fall short of fulfilling the promise such kits could offer.
To produce the new commercial kit, a library of monooxygenases will be created using the SeSaM method of random mutagenesis2. This method allows for a much greater variety of mutants produced compared to traditional mutagenesis methods, by inducing mutations at every site of the gene. The enzyme variants produced will be expressed using an E. coli or P. pastoris expression system, and their cell cultures optimized for enzyme production in shake flasks or fermenters. In addition to this, parameters important to monooxygenase activity, such as stability and substrate selectivity, will be assessed using TLC, GC or HPLC. Finally, as an important part of the kit production, special attention will be given to enzyme immobilization to achieve long-term enzyme stability.
- Guengerich, F. P. Cytochrome P450 and chemical toxicology. Chem. Res. Toxicol. 21, 70–83 (2008).
- Wong, T. S. Sequence saturation mutagenesis (SeSaM): a novel method for directed evolution. Nucleic Acids Res. 32, 26e–26 (2004).
My scientific interests
My scientific interests revolve around proteins. I find these little biological machines to be absolutely fascinating in all aspects, from their structure to the many functions they perform. The first time I realized the profound impact they have on life was during my time studying brain cancer. While proteins are very important in disease formation, they can do a lot of good too. This is what I am focusing on now, the creation of helpful proteins to be used in the biotech and pharmaceutical industries.
What do you expect from OXYTRAIN?
I see the primary benefit of doing my Ph.D. as a part of the OXYTRAIN project in the access to the many networking opportunities with some of the best researchers in the field. I also expect to gain an insight into the requirements of the biotech industry, as I plan to take my career in that direction after my Ph.D. Since my project is so deeply connected to the industry, this should be easily achievable.
What do you like to do in your free time, your hobbies, interests, what motivates you.
In my spare time, I like to be active outside, by cycling or running in the summer and going alpine skiing in the winter. I also really like cooking, maybe this why I also enjoy working the lab so much, the two are actually quite similar. I also like to follow the news and listen to podcasts or audio books, primarily those focusing on philosophy, economics, and politics. In general, I am motivated by my curiosity about the world we live in, and how to best act in it.