Molecular and genetic investigation of the biosynthesis of the non-ribosomal peptide cyclochlorotine

Cyclochlorotine is a potent mycotoxin of the fungus Talaromyces islandicus. Structurally, it is a cyclic and chlorinated pentapeptide containing the rare, non-proteinogenic amino acids L-β-phenyl alanine, L-α-amino butyric acid and the unique L-dichloro proline. The latter one shows a very unusual chlorination pattern.

Using genome sequencing and RNA interference studies we identified a biosynthetic gene cluster that encodes the machinery for cyclochlorotine biosynthesis. The key enzyme is a huge NRPS encoded by an almost 17 kb gene.

Interestingly, no halogenase—that would be a requisite for installing the chlorination pattern—is encoded in or nearby the cluster. Therefore, the search for a halogenase gene has been expanded to the whole genome by successively knocking-down promising candidate genes.

Production, purification and elucidating the mode of action of astin C — a pharmaceutically relevant natural product

Astin C initially has been described from the Chinese medicinal plant Aster tataricus. Its molecular structure is very similar to that of cyclochlorotine from T. islandicus. It has long been believed that astin C is a product of the host plant. However, we could show that the plant harbors an endophytic fungus, which is the actual producer of astin C. This novel fungus, Cyanodermella asteris, could be isolated and is maintained as axenic culture.

Whole genome sequencing of C. asteris led to the identification of an NRPS pathway responsible for astin C formation. That finding provides the basis for a biotechnological production of the natural product.

Astin C binds and inhibits the important regulator of the innate immunity STING, the stimulator of interferon genes. An overactivation of STING is discussed to be linked to chronic inflammations like inflammatory bowel disease and rheumatoid arthritis. Therefore, astin C as a STING-inhibitor displays an interesting, potential pharmacological drug.

In our studies, we aim to increase astin C production by heterologous expression of the biosynthesis genes in suitable hosts. On the other hand, we investigate the mode of action of astin C with respect to the ecological role for the host plant A. tataricus.

Increasing the production of the antibiotic pleuromutilin by means of metabolic engineering

The Basidiomycota fungus Clitopilus passeckerianus produces the natural product pleuromutilin, which—as a semi-synthetic derivative—is in use in veterinary and human medicine. The biosynthesis of pleuromutilin has recently been elucidated. It consists of seven enzymatic steps, that combines simple precursor building blocks to a complex, tricyclic terpenoid structure.

Here we aim at increasing the production of pleuromutilin. This is achieved, on the one hand, by manipulation of the biosynthesis machinery in the natural producer C. passeckerianus and, on the other hand, by heterologous expression of the biosynthesis genes in different microbial host strains. In both approaches, the supply of required chemical building blocks will be increased by genetic engineering.

Suggested topics for bachelor’s and master’s thesis

• Do host plant and fungal endophyte share biosynthesis? Molecular and biochemical investigations regarding a possible joint production of potent natural products in fungus and plant
• How is the chlorine attached to the molecule? Identification of components responsible for the chlorination of the natural products cyclochlorotine and astin that is unique in nature
• Heterologous expression of a synthetic fungal gene cluster in streptomycetes that have been optimised for natural product biosynthesis
• Metabolic engineering of the pleuromutilin natural producer, Clitopilus passeckerianus.

Cooperation and Funding

The research projects are conducted in close cooperation with Prof. Karl-Heinz van Pée and co-workers of the General Biochemistry department, TU Dresden, as well as with Prof. Jutta Ludwig-Müller and co-workers of the Plant Physiology department, TU Dresden. Further cooperation partners are Prof. Philippe Jacques and Prof. Luc Willems (both from Gembloux Agro-Bio Tech, University of Liège, Belgium) and Prof. Willem van Berkel (Wageningen University and Research, Netherlands).

The work is funded by ERA-IB, an ERA-NET research program of the European Community.