PhD FELLOW # 7

Odyssefs-Ioannis Pantelakis

Ody

#007

WP3: ENZYME ENGINEERING WITH NON-CANONICAL AMINO ACIDS

Engineering catalytic mechanisms of enzymes using non-canonical amino acids

 

Biography

As far as my background and studies are concerned, I grew up in Athens, Greece and developed an interest for the natural sciences early on, through questioning how the world around me functioned. I was lucky enough to cultivate and expand my curious nature through participation in student science clubs during my high school years. When the time came to pursue higher education, I chose the diverse field of Chemical Engineering and received my diploma from the National Technical University of Athens (NTUA) in October 2022. Apart from the basic core of natural science and engineering courses of the diploma, I settled on the direction of Biotechnology for my integrated MEng. During the last year of my studies I successfully defended my diploma thesis, titled: “Recombinant expression, isolation and biochemical characterization of an enzyme with the potential to bioremediate chlorinated aromatic pollutants”, under the supervision of professor E.Topakas. Working on this project, I got acquainted with the principles of biocatalysis and enzymology, studying the properties of the recombinant enzyme, while also gaining valuable experience handling one of the working horse host-microbes of modern biotechnology, the yeast Pichia pastoris. Every step of this process confirmed my love and affinity for research, thus giving me motivation to pursue the next appropriate step in my career. Good timing and luck struck again, as I soon took notice of the BiocatCodeExpander consortium open call, to which I immediately reached out to. One or two rounds of interviews later, the rest is history…

 

 Tell us a bit about your PhD project

This research project is set to combine the beneficial effects of non-canonical amino acid (ncAA) utilisation and the principles of enzyme engineering, while investigating an industrially relevant class of enzymes, the heme-independent iron dioxygenases. These enzymes are interesting candidates for ncAA engineering due to their ability to activate the carbon-hydrogen bond in a wide range of putative substrates, that are useful in many fields. More specifically, the main focus will rest on the subgroup of the oxoglutarate dependent amino acid hydroxylases by exchanging residues that are responsible for coordinating their metal centre with a non-canonical analogue. These alterations will be made in the facial triad of a series of AA hydroxylases, which will then be biochemically characterised to unveil changes in their activity, selectivity and promiscuity to putative substrates as well as their overall stability. In order for the incorporation to be site-specific, the stop codon suppression method will be implemented. Should the engineered variants give detailed insight into the mechanism and effect of these alterations, the same approach will be applied to the analogous enzymatic subgroup of the heme-independent halogenases. Meanwhile, the best performing variants of both groups will be structurally elucidated by x-ray crystallography. The main anticipated accomplishments include the demonstration of ncAA incorporation as an efficient enzyme engineering tool in this superfamily of non-heme iron-dependent enzymes, a fully characterised set of robust enzyme variants that can be subjected to further engineering studies and an insight into the way the proposed residue alterations with ncAAs affect both the structural and catalytic properties of the aforementioned enzymes.

Tell us about your research interests – what do you expect from the consortium

Throughout my years as a student of natural and engineering sciences I have been excited by a plethora of principles ranging from Physical Chemistry to Green Chemistry, Thermodynamics and Food Technology. However, the more I delved into Biocatalysis and found out what can be achieved through engineering enzymatic activities, all the more amazed I got. Since evolving living organisms could not utilise inorganic catalysts to achieve their purposes, nature had to find a way around this obstacle, and it did just that by combining amino acids in complex formulations to gain any activity it needed to adapt. Researchers throughout time have achieved major breakthroughs by looking up to how nature is getting the job done or just trying to mimic it to many different end goals: catalysing complex reactions, selective bioproduct formation for food or drug purposes, bioremediation or biodegradation and valorisation of waste products; the list does not end and that is particularly exciting to me. Through the wide approach of our consortium for utilising unnatural amino acids for the study, understanding, improvement and expansion of protein properties, it feels like we are doing exactly that. I am especially stoked for the project I am focusing on, since we are combining unnatural amino acid incorporation in vivo and protein engineering, two principles that could allow a new enzymatic activity to exist, that would have taken nature hundreds of years to evolve. My excitement aligns with my expectations from the consortium, as I am looking forward to collaborating with a diverse group of excellent scientists and truly making an impact on the real world.

Hobbies

Of course, cooking and baking are skills that most people develop sooner or later in life, but they feel to me like so much more than just daily chores for sustenance. Through experimenting and trying diverse recipes, both local and foreign, simple and extremely complex, with ingredients that are considered humble and others that seem more extravagant, you get to ‘travel briefly’ and understand cultural and geographical differences and similarities. Especially preparing a meal for family, friends and colleagues gives an extra sense of joy, for it allows everyone to have a bonding experience and create long lasting memories.

Also, something that I loved practising back home and that I am hoping to fit again into my weekly schedule is Taekwondo. As most martial arts, it is a great form of exercise and it can give you a strong sense of discipline and patience, both equally important for facing tough challenges.

As far as motivation is concerned, I am always in pursuit of knowledge and making sense of exactly how and why everything works the way it does, but it is equally important to me to convey that knowledge to others. I love a fun fact and if I can help someone achieve their goal through my experiences or vice-versa, that is the best feeling in the world. I feel like that is what is really motivating me to keep going and fight through the frustration of failed experiments and negative results. There is a big worldwide community of scientists that share that frustration and are overcoming one obstacle after the other together with their coworkers, which is honestly inspiring and it is exactly what I feel we are doing with my fellow PhD candidates in our consortium.

Favorite book, meal, movie and series?

Book: The Hobbit (JRR Tolkien)

This is a book that I often come back to, since it paints really vivid and imaginative scenes, while it is set apart from its siblings (Lord of The Rings) by the not so complex use of the English language. It really feels like I too am on the adventure myself!

Meal: As a cook and baker, it would be a dishonor to name out a specific meal and to that end, I wholeheartedly cherish two meals/feelings throughout the year: the dinner of Christmas Eve and the lunch of Christmas day. At that time, family and friends gather and spend time together playing games and enjoying the wonderful atmosphere. Honestly it does not really matter what the menu consists of on those occasions, I always love it.

Movie: Lord of The Rings

Even though I have seen the movies an ungodly number of times, if someone asks if we should do a LOTR marathon, I will be the first one to shape their schedule around the event. Magical, nerve-racking, inspiring, there are several reasons why they are considered unmatched masterpieces.

Series: Agatha Christie’s Hercule Poirot

Watching an iconic detective come to life, while being portrayed by equally iconic actors over the years is pure gold. I love all series-renditions of the classic books and I keep coming back to them every now and again, as they make me reflect and consider many possible outcomes for different situations.

Favorite city 

Even though it might sound cliché, I feel obligated to say Paris is one of the most amazing cities I have been to. Countless museums, iconic landmarks, good food and a certain ‘je ne sais quoi’. It felt like taking a bad photograph in this city is impossible, lots of light and just an explosion of stimuli. The locals are kind of snobbish but that is an integral part of the experience.

at what Harry Potter house do you belong?

As every other fantasy fan and Harry Potter enthusiast, I too have taken several HP House Quizzes and although nothing compared to the accursed Sorting Hat, I believe I would make an excellent addition to Ravenclaw house. Always striving to learn more and coming up with original and creative ideas are definitely worthy goals to have in life 🐦‍⬛

PyMOL or Chimera?

I tend more towards PyMOL as that is the software I learned how to use first, but after seeing the amazing work of my colleagues in recent time, I am starting to believe Chimera needs revisiting. Also it has a cooler name, PyMOL sounds like cough candy

Organization

Graz University of Technology (TUG)

Doctoral Supervisor

Dr. Anita Emmerstorfer-Augustin

Enrolment in Doctoral degree

Graz University of Technology (TUG)

Secondments

At Vrije Universiteit Amsterdam (Amsterdam, The Netherlands) with Dr. Ivana Drienovská & moloX GmbH (Berlin, Germany) with Dr. Bernhard Loll

Objectives

• • Establishment of a high-throughput system for rapid folding assessment based on split-GFP complementation
  • Biochemical characterization of biotechnologically attractive non-canonical amino acids (NCAA)-containing oxidoreductases
  • Biocatalytic and structural characterization of best variants 
  • Molecular modelling of altered reactivity

Fields related to the project

(1) Synthetic biology

(2) Biocatalysis

(3) Computational biology

(4) Structural biochemistry