Phd on topic: Systematic investigation towards exhaustive post-translational modification of carboxylate reductases


Research interest and scientific background: The Winkler group is fascinated by enzymes and cellular systems as means to catalyze chemical transformations. The key goal is the discovery and detailed study of enzymes that are not (well) known yet, in order to understand them in depth and to determine whether and how they can become applicable for synthesis. Carboxylate reductases and other oxidoreductases are in the focus (1-6).

Affiliation: The student will work at the Institute of Molecular Biotechnology at the Graz University of Technology. This project is directly connected to the doc.fund CataLOX.


Hypothesis and objective: The project aims to gain comprehensive understanding of the activation of carboxylate reductases by phosphopantetheinylation. The working hypothesis is that balanced expression of reductase and activator will lead to exhaustive modification.


Experimental approaches: The student will follow a combinatorial approach and produce a series of CARs in the presence of a variety of phosphopantetheinyl transferases in E. coli and yeast. The student will perform all steps from designing to application of biocatalysts. These include cloning, protein expression in different host organisms, protein purification, enzyme activity assays, establishing analytics to quantify phosphopantetheinylation, whole cell biotransformations and their analysis by chromatographic methodology. The student will strongly interact with other research groups of the doc.fund CataLOX, the doc.fund Molecular Metabolism, as well as national and international training networks.

References:

  1. D Schwendenwein, G Fiume, H Weber, F Rudroff, M Winkler* Selective enzymatic transformation to aldehydes in vivo by fungal carboxylate reductase from Neurospora crassa, Adv. Syn. Catal. 2016, 358, 3414-3421.
  2. H Stolterfoht, D Schwendenwein, CW Sensen, F Rudroff, M Winkler* Four distinct types of E.C. 1.2.1.30 enzymes can catalyze the reduction of carboxylic acids to aldehydes, J. Biotechnol, 2017, 257, 222-232.
  3. M Winkler* Carboxylic acid reductase enzymes (CARs), Curr. Opin. Chem. Biol. 2018, 43, 23-29.
  4. H Stolterfoht, G Steinkellner, D Schwendenwein, T Pavkov-Keller, K Gruber, M Winkler* Identification of key residues of Carboxylate reductases, Frontiers in Microbiology, 2018, 9:250.
  5. M Winkler, M Geier, SP Hanlon, B Nidetzky, A Glieder* Human Enzymes for Organic Synthesis, Angew. Chem. Intl. Ed. 2018, 57, 13406-13423;
  6. G Strohmeier, IC Eitelj├Ârg, A Schwarz, M Winkler* Enzymatic one step reduction of carboxylate to aldehyde with cell free regeneration of ATP and NADPH, Chem. Eur J 2019, 25, DOI: 10.1002/chem.201901147 in print