Ruprecht-Karls-Universität Heidelberg
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Mack0118 - Scientist (f/m) / PhD position
Project no:
Mack0118

Project leader:

Project supervisor:
Mack, Matthias
Application deadline:
15. Dec 2018
Start of PhD project:
1. Feb 2018

Project description:

Title:
Identification and characterization of a novel enzyme cofactor in the bacterium Streptomyces davaonensis
Summary:
This project is a follow-up study of a highly successful previous work funded by the DFG (MA2510/9-1). The objective of MA2510/9-1 (previous study) was to elucidate the biosynthetic pathway for the antibiotic roseoflavin in the bacterium Streptomyces davawensis. In contrast to what we expected we found that a single enzyme is able to catalyze an impressive oxidative cascade converting riboflavin-5'-phosphate (also called flavin mononucleotide or FMN) to the key intermediate of roseoflavin biosynthesis, 8-demethyl-8-aminoriboflavin-5'-phosphate. The objective of the present project is to study the unknown role of thiamine (or a thiamine-like compound) in the RosB reaction. As a working hypothesis we suspect that a novel, thiamine-like (?) cofactor is present in S. davawensis which stimulates RosB. The identification of this novel cofactor will substantially extend our knowledge with regard to enzyme/cofactor chemistry and thus is likely to generate a “high impact” paper. The results may also challenge our current understanding of thiamine as an enzyme cofactor.
References:
1. Schwarz, J., Konjik, V., Jankowitsch, F., Sandhoff, R. and Mack, M. (2016). Identification of the Key Enzyme of Roseoflavin Biosynthesis. Angew Chem Int Ed Engl 55, 6103-6106.

2. Konjik, V., Brunle, S., Demmer, U., Vanselow, A., Sandhoff, R., Ermler, U. and Mack, M. (2017). The Crystal Structure of RosB: Insights into the Reaction Mechanism of the First Member of a Family of Flavodoxin-like Enzymes. Angew Chem Int Ed Engl 56, 1146-1151.
Methods that will be used:
Cultivation of Streptomycetes and of Escherichia coli (for cloning)
Gene deletion experiments using a CRISPR-Cas9 system adapted to Streptomycetes (from the group of Tillman Weber, Denmark)
Gene deletion experiments using PCR-targeting (from the group of Bertolt Gust, Germany)
Gene expression experiments employing different bacteria
Cloning techniques
Generation of cell-free extracts from Streptomycetes
Column chromatography
Protein purification
Enzyme assays
Fractionation of small molecules using different separation/chromatography tools
Mass spectrometry
NMR analysis of small molecules

The following working packages shall be carried out and give an idea what will happen during the experimental work in Mannheim:

Working package 1: A tagged version of the enzyme RosB is used as a bait to isolate a novel cofactor
Working package 2: Isolation and subsequent analysis of the “novel cofactor” from a concentrated cell-free extract of S. davaonensis
Working package 3: Immobilization of RosB and isolation of the unknown “novel cofactor” by using RosB as a bait on a chromatography column
Working package 4: Expression of gene clusters from S. davaonensis in Streptomyces coelicolor and identification of a stimulating cell-free extract
Working package 5: Deletion experiments using a CRISPR-Cas9 system will validate the results from working package 4
Working package 6: Expression of putative thiamine like biosynthesis genes from S. davaonensis in Streptomyces coelicolor and analysis of the stimulating effect
Cooperation partners:
Ulrich Ermler, Max Planck Institute of Biophysics, Frankfurt, Germany
Peter Macheroux, Graz Technical University, Graz, Austria
Tillman Weber, Technical University of Denmark, Lyngby, Denmark
Bertolt Gust, University of Tübingen, Germany
Wolfgang Buckel, University of Marburg, Germany
Per Bruheim, National Norwegian Technical University, Trondheim, Norway
Personal qualifications:
A person interested in bacterial enzymes and catalysis in general would be the most appropriate. Moreover, since we also will use a genetic approach to identify the novel thiamine-like cofactor, the right candidate also should be familiar with state-of-the-art cloning techniques. We will teach you to use a bacterial CRISPR-Cas9 system.
Keywords:
Microbiology, Bacteria, Vitamins, Cofactors, Streptomycetes, antibiotics