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Exploring special antibiotics synthesis pathways

Kirromycin, an antibiotic produced by soil bacteria, reveals unusual steps for biosynthesis that might also be of interest for biotechnological applications. Dr. Ewa Maria Musiol was the first to shed light on the functions of specific acyltransferases involved in the biosynthesis of the antibiotic. She also received the 2012 DECHEMA PhD Award for Natural Products Research for these achievements.

Dr. Ewa Musiol is delighted about the DECHEMA award which she received for her excellent work on kirromycin biosynthesis. © private

In times when an increasing number of bacteria are becoming resistant to common antibiotics such as penicillin, the search for new antibiotics is an extremely urgent issue, and is receiving financial support from the German Ministry of Education and Research (BMBF). The BMBF-funded project GenBioCom, which is being coordinated by Prof. Dr. Wolfgang Wohlleben at the Interfaculty Institute of Microbiology and Infection Medicine (IMIT) at the University of Tübingen, is specifically focused on the identification of new active substances, including new antibiotics. The BMBF provides the researchers from Tübingen with around 1.2 million euros in funding. Dr. Tilmann Weber from the IMIT is coordinating a subproject that is particularly focused on substances produced by soil bacteria. The polyketide antibiotic kirromycin produced by Streptomyces collinus has been known for quite some time. Weber: “Kirromycin was discovered in Tübingen around 40 years ago by a group of researchers led by Prof. Zähner, the scientist who established the field of antibiotic research in Tübingen.” However, the biosynthesis of the antibiotic was only recently deciphered using molecular biology and bioinformatics methods that were unavailable 40 years ago. 

Dr. Ewa Maria Musiol, a 30-year-old biologist and postdoc in Weber’s group, is specifically focused on the biosynthesis of kirromycin. Her research follows her own principles: “I have always attached great importance to applied and benefit-oriented sciences. Although we focus closely on the basic research-oriented aspects of antibiotics, we do so in order to establish the basis for the development of new suitable drugs.” The biosynthesis step discovered by Ewa Maria Musiol may well become extremely important for medical applications and the biotechnological production of antibiotics. The highly specific biochemical reactions could potentially be used for controlling the specificity and toxicity of antibiotics as well as for synthesizing other polyketides. 

A biosynthesis step with broad potential

The soil bacterium Streptomyces collinum – here in the form of a laboratory smear – produces the polyketide antibiotic kirromycin. © Tilmann Weber, IMIT, University of Tübingen

The biosynthesis of polyketides takes place in the cell at a huge multifunctional enzyme complex that gradually assembles the individual building blocks of the antibiotic. “This process is similar to an industrial production line in the automotive sector, for example,” said Weber. The gene cluster encoding the enzymes involved in the biosynthesis of kirromycin was discovered several years ago. It has since become known that not all enzymes directly belong to the complex. There are two “extra” genes that code for external acyltransferases (AT), so-called trans-ATs. Musiol explains that this on its own is not that unusual. What is really essential is frequently hidden in the finer details: “It was known that trans-ATs only use malonyl-CoA as building block, and present this extender to the polyketide synthesis enzyme complex. However, we have now found that kirromycin has an unusual extender that cannot be derived from malonyl-CoA, and have examined this further."

Musiol’s results thus revealed that textbook knowledge about the synthesis of polyketides had to be expanded: she found that the trans-acyltransferase KirCII incorporates ethylmalonyl-CoA in kirromycin polyketide biosynthesis. KirCII is thus a potential tool that could be used to insert branches and change the specificity and toxicity of polyketide antibiotics. “It is also worth mentioning that subsequent synthesis modules need to be able to deal with unusual branching. And this seems to be the case here. It also means that this particular acyltransferase has huge potential to be used for biotechnological applications,” said Musiol. “It would be rather difficult and time-consuming to produce such side branches synthetically, and this would therefore not be an option for bioproduction purposes. However, our results suggest that it is possible to introduce such branches enzymatically,” said Weber highlighting the potential use of KirCII for biotechnological applications.

The long road towards kirromycin biosynthesis - the trans-acyltansferase KirCII incorporates the unusual extender unit ethylmalonyl-CoA in kirromycin polyketide biosynthesis (labelled blue). © Ewa Musiol, IMIT, University of Tübingen

DECHEMA also recognized the relevance of Musiol’s discovery and awarded her the 2012 PhD Award for Natural Products Research at the 24th Irseer Naturstofftage conference held in March 2012. This award encourages Dr. Musiol to look further into the opportunities her discovery may engender. In addition, she will also focus on the other trans-AT mentioned above. “Initial data suggest that this enzyme uses malonyl-CoA as a building block, but final evidence is still missing,” said Musiol. She still has until the end of 2012 before the cooperative project comes to an end, and she will use this time to carry out in vitro experiments involving the biochemical analysis of purified proteins.

Industry is looking forward to further results

The team also receives support from industry: the cooperative BMBF-funded project also involves Insilico Biotechnology AG, a local company that specializes in building computational models of living cells. “Insilico provides us with simulations of the bacterial metabolism. We are aiming to find conditions that will help us to increase the synthesis rate of kirromycin,” said Weber. The Göttingen-based company BioViotica Naturstoff GmbH, which is also involved in the project, produces biologically active natural products and is focused on commercialization strategies for kirromycin and other products. Kirromycin is a narrow-spectrum antibiotic; it exhibits activity against Neisseria gonorrhoeae, for example. On the other hand, hardly any toxic effect can be observed in cell culture, something that makes the antibiotic rather interesting for medical applications. If the new biosynthesis step leads to interesting modifications in other bacterial strains and as a result to interesting products, it can be expected that industrial interest in the Tübingen researchers’ results will increase still further.

At present, Dr. Musiol is deciding whether she will still be in Tübingen when this happens. She is currently considering spending some time abroad. She has also become involved in teaching at the University of Tübingen. She is lecturing on protein expression and also supervises master’s students. She also enjoys organizing and carrying out practical courses for schoolchildren, including half-day hands-on experiments which give the children insights into practical aspects of microbiology and antibiotics in particular.

Further information:
University of Tübingen
Interfaculty Institute of Microbiology and Infection Medicine (IMIT)
Dr. Ewa Maria Musiol
Auf der Morgenstelle 28
72076 Tübingen
Tel.: +49 (0)7071/ 29-74628
E-mail: ewa.musiol(at)biotech.uni-tuebingen.de
Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/exploring-special-antibiotics-synthesis-pathways