PhD FELLOW # 8

Liu Yang

WP3: ENZYME ENGINEERING WITH NON-CANONICAL AMINO ACIDS

Design and evolution of 4-oxalocrotonate tautomerase for the Morita-Baylis-Hillman reaction using non-canonical amino acids

 

Biography

Liu was born in Guangyuan, Sichuan Province, located in the southwest of China. In 2020, She got her Bachelor’s degree in Food Science and Engineering at Southwest University in Chongqing. With curiosity and enthusiasm for enzymes, she moved to Guangzhou to study enzymes at South China University of Technology. During her postgraduate studies, she participated in several works on lipases, esterases and unspecific peroxygenases. In 2023, she completed the dissertation on the excavation, characterization and application of phthalate acid esters hydrolases and got the Master’s degree. Given her continuous interest in biocatalysis and enzyme engineering, in September 2023, she joined in the research group of Prof. Gerrit J. Poelarends at University of Groningen as one of the doctoral fellows of the BiocatcodeExpander doctoral network which is supported by Marie Skłodowska-Curie Actions.

Tell us a bit about your PhD project

This project focuses on the engineering of the 4-oxalocrotonate tautomerase (4-OT) enzyme by incorporating non-canonical amino acids (ncAAs). 4-OT uses its N-terminal proline as an unusual catalytic residue to perform iminium catalysis or enamine catalysis, which has shown promising capacity of forming C-C bonds as well as realizing asymmetric epoxidation reactions. The Morita–Baylis–Hillman (MBH) reaction as an important C-C bond-forming reaction draws our attention since all the steps involved in it can be realized by 4-OT. The MBH reaction provides a versatile and atom-economical approach to generate densely functionalized chiral building blocks. Therefore, this project aims to generate a set of 4-OT variants with incorporated secondary or tertiary amine-based ncAAs, having high activity and enantioselectivity for MBH and/or related reactions, as well as to explore structural and mechanistic insights into the enzyme-catalyzed MBH reaction. For this, 4-OT will be engineered by incorporating ncAAs as catalytically important residues. The synthesis and incorporation of various ncAAs will be carried out in collaboration with Dr. Ivana Drienovská at Vrije Universiteit Amsterdam. The most promising enzyme(s) will be analyzed and subjected to directed evolution to further optimize activity and selectivity, which will be performed in collaboration with Dr. Jan Vilím at Enantis.

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

Liu’s research interests include discovery and development of catalytic promiscuity of enzymes, enzyme engineering and enzymatic synthesis of pharmaceutical compounds. She is looking forward to having good communications and networking with PhD fellows and PIs within the consortium and obtaining more understanding and skills in Genetic Code Expansion.

Hobbies

Outside the lab, she enjoys sports including jogging, hiking and badminton as well as meeting with friends.

Favorite meal and movie

Favorite meals are Sichuan cuisine and Guangdong cuisine, Favorite movie is Princess Mononok

Favorite city 

Guangyuan

at what Harry Potter house do you belong?

Gryffindor 🦁

PyMOL or Chimera?

Pymol

Organization

University of Groningen (RUG)

Doctoral Supervisor

Prof. Gerrit Poelarends 

Enrolment in Doctoral degree

University of Groningen (RUG)

Secondments

At Vrije Universiteit Amsterdam (Amsterdam, The Netherlands) with Dr. Ivana Drienovská & Enantis (Brno, Czech Republic) with Dr. Jan Villim

Objectives

• Optimization of suitable assays for following the Morita-Baylis-Hillman biocatalysis
• Preparation of panel of 4-oxalocrotonate tautomerase variants with incorporated non-canonical amino acids
• Crystal structures and detailed structural and mechanistic characterization of best performing variants
• Directed evolution of best chosen variants

 

Fields related to the project

(1) Synthetic biology

(2) Biocatalysis

(3) Biochemistry

(4) Structural biochemistry