8.03 Discovery, Design and Computationally Guided Evolution of Novel Oxidoreductase Bioanalytics

Research

Oxidoreductases represent the foundation of white biotechnology industry, which is forecasted to be the major driver for the global growth of chemical industry. However, development of new biotechnology based products and processes requires a substantial expansion of oxidoreductase-mediated transformations through discovery or generation of novel activities as well as expansion of substrate scope and optimization of selectivities for existing biocatalysts. The interdisciplinary project aims to identify novel oxidoreductases for industrial relevant transformations. We will use a dual focus strategy to identify new reactivities and selectivities: i) novel oxidoreductases of the Red Sea Brine Pool proteome database and from electroactive microbes will be expressed and tested and ii) unnatural amino acid incorporation methods, which were recently established at KAUST will be used to create de novo activities. Following successful expression, enzymes will be analyzed in parallel systems developed at TUM. Candidate enzymes and mutants showing novel features of interest will be structurally characterized. Based on these protein X-Ray structures or homology models, promising mutation positions will be identified. A combination of iterative saturation mutagenesis (ISM), selectivity/activity screening and mutant characterization will be used to generate new generations of optimized mutant enzymes. Such a computationally guided evolution strategy will significantly reduce the numbers of test required to identify highly selective mutants. The project team combines the complimentary expertise of four research groups at KAUST (biocatalysis and biocomputation) and TUM (structural biology and biochemical engineering).

Publications

Mader SL, Bräuer A, Groll M, Kaila VRI (2018) Catalytic mechanism and molecular engineering of quinolone biosynthesis in dioxygenase AsqJ. Nature Comm 9, 1168:1-8. doi: 10.1038/s41467-018-03442-2

Strillinger E., Grötzinger S., Allers T., Eppinger J., Weuster-Botz, D. (2016) Production of halophilic proteins using Haloferax volcanii H1895 in a stirred-tank bioreactor, Applied Microbiology and Biotechnology, p.1-13. doi. 10.1007/s00253-015-7007-1

Bräuer, A., Beck, P., Hintermann, L., Groll, M. (2016) Structure of the Dioxygenase AsqJ: Mechanistic Insights into a One-Pot Multistep Quinolone Antibiotic Biosynthesis, Angew. Chem., 55, 422-426. doi: 10.1002/anie.201507835

Weiz, A. R., Ishida, K., Quitterer, F., et al. (2014) Harnessing the evolvability of tricyclic microviridins to dissect protease-inhibitor interactions, Angewandte Chemie International Edition, Volume 53, issue 14, p. 3735-3738. doi: 10.1002/anie.201309721

Quitterer, F., et al. (2013) Structure and reaction mechanism of pyrrolysine synthase (PylD), Angewandte Chemie International Edition vol. 52 issue 27, p. 7033-7037. doi: 10.1002/anie.201301164

Quitterer, F. et al. (2012) Biosynthesis of the 22nd genetically encoded amino acid pyrrolysine: Structure and reaction mechanism of PylC at 1.5 Å resolution, Journal of Molecular Biology, Volume 424, Issue 5, Pages 270-282. doi: 10.1016/j.jmb.2012.09.007

Quitterer, F., et al. (2011) Crystal structure of methylornithine synthase (PylB): Insights into the pyrrolysine biosynthesis, Angewandte Chemie International Edition,Volume 51, Issue 6, Pages 1283–1488. doi: 10.1002/anie.201107547

Team

Project team leader

Dr. Kathrin Castiglione
Institute of Biochemical Engineering

Alumnus

Dr. rer. nat. Alois Bräuer
Chair of Biochemistry

Alumna

Dr. rer. nat. Eva Strillinger
Chair of Biochemical Engineering

Doctoral researcher / Alumnus

Dr. rer. nat. Felix Quitterer
Chair of Biochemistry

Doctoral researcher

Anastassja Leyla Akal
KAUST Catalysis Center

Principal investigator

Professor Michael Groll
Chair of Biochemistry

Principal investigator

Professor Luigi Cavallo
KAUST Catalysis Center

Principal investigator

Professor Dirk Weuster-Botz
Chair of Biochemical Engineering

Principal investigator

Professor Jörg Eppinger
KAUST Catalysis Center