Investigation of The Antibiotic Biosynthetic
vor 5 Monaten
**Masterarbeit**
**Investigation of the antibiotic biosynthetic potential of **_Kitasatospora fiedleri _**DSM 114396**
**Background**
Actinomycetes are the origin of almost two thirds of all antibiotics in clinical use today, and still the most promising source for novel natural compounds with antimicrobial activity. While the emergence of multidrug-resistant pathogens is constantly increasing, there has been a steady decline in the discovery of new and effective antibiotics over the past decades. Thus, there is an urgent need to find new anti-infectives to combat drug resistance. _Kitasatospora fiedleri _DSM 114396 (a rare genus and novel species of the family _Streptomycetaceae_) is a strain from the DSMZ strain collection that produces one or more antimicrobials of unknown structure and biochemistry.
In actinomycetes, the genes encoding the biosynthesis of natural products are usually located in a continuous region of the genome we refer to as biosynthetic gene cluster (BGC). The _Kitasatospora _genome harbours >25 BGCs most of them cryptic (without known metabolic product). Genetic manipulation of the strains has been successfully achieved in our laboratory.
**Thesis Topic**
The ultimate goal of the project is to identify the BGCs driving the production of the different antimicrobial compounds. This will be achieved by step-wise inactivation of selected BGCs by genetic manipulation of the producer strain, using a variation of CRISPR-Cas9 technology known as CRISPR-base editing systems; establishing this technique in our lab, and testing with one BGC, will be the objective of the Master Thesis.
**_ Experiments_**_:_
- Selection of target genes from prioritised BGCs identified by antiSMASH analysis;
- Design of possible genome edits to make and required CRISPR spacers
- Construction of CRISPR delivery vectors and conjugation to _Kitasatospora_;
- Assessment of antimicrobial activity produced by mutants.
**_Methodology_**_:_
Bioinformatics (database search, analysis of DNA sequences, planning of DNA cloning). Molecular biology (DNA extraction and purification, PCR amplification, DNA cloning, _E.coli _transformation, restriction analysis). Microbiology (culture and preservation of microorganisms). Antimicrobial activity bioassays. Genetic manipulation of microorganisms (mobilisation of DNA into _Kitasatospora_, homologous recombination-based gene targeting, selection of mutants)
**Who we are**
The group _Applied Microbiology_ _(head: Prof. Yvonne Mast), located at the Leibniz-Institut DSMZ in Braunschweig-Stöckheim, investigates actinomycetes as antibiotic producers. We employ Genome Mining and Genetic Engineering techniques to fully exploit the biosynthetic potential of actinomycetes.
**Start: immediately**
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