Approach to a enedyine biosynthesis tool box through metagenomic and targeted metabolomic analyses of actinomycetes

• Applicant: Dr. Ivana Crnovcic
• Subject Area: Biological and Biomimetic Chemistry
• Term: from 2015 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 276972917

Enediynes are a class of potent antitumor agents produced by soil and marine microorganisms. Structurally they are characterized by a core unit consisting of two acetylenic groups conjugated to a double bond or incipient double bond within a 9- or 10-membered ring. The enediynes possess anticancer and antibiotic activity based on the presence of their enediyne core whereas the peripheral chemistry found around the cores of the different enediynes plays only a role in the fine tuning of that activity. Enediynes produce in the DNA inter-strand DNA crosslinks (ICLs) and can lead to site-specific single stranded or double stranded breaks (DSBs). Comparison of the known enediyne biosynthetic gene clusters has revealed that both, 9- and 10-membered enediynes, share a conserved five-gene cassette, the enediyne PKS cassette, which encodes the biosynthesis of the pentadecaheptaene backbone of the enediyne core structure. The conservation of the central core of the pentadecaheptaene is disturbed by the peripheral parts which lead to a high structural diversity of the various enediynes. Since the enediynes have extreme cytotoxic activity as pure compounds they have only limited use as clinical drugs. However, when formulated into various polymer-based delivery systems or as anticancer antibody-drug conjugates (ADCs) they have shown great clinical success and are very promising for future developments in anticancer therapy. Therefore the biosynthetic development of new enediyne compounds with still better performance is of interest. The group of Prof. Shen has done a genome survey of 3,200 strains of their Actinomycetale collection and identified 89 strains that harbor the enediyne PKS gene cassettes. In detail, two strains, named CB02115 and CB100675 from this strain collection we will chose as models to investigate novel structural features such as e. g. specific groupings or functional groups in their biosynthesis. CB02115 encodes a gene cluster that resembles the C-1027 gene cluster from Streptomyces globisporus, while CB100675 encodes a gene cluster that shows similarity to the calicheamicin (CAL) gene cluster from Micromonospora echinospora. To address the structure and the biosynthetic features of the enediynes encoded by the enediyne gene cluster in strain CB02115 and strain CB100675 we will determine the structures of the new enediynes and analyse promising genes with apparent novel biosynthetic function in enediyne biosynthesis. Moreover, in view of the similarity of the two new enediyne gene clusters with already known clusters we will express the gene constructs in a genetic background actively synthesizing conventional enediynes. For the most successful genes in either CB02115 or CB100675 we will in vitro characterize its gene product by expressing the gene in E. coli and testing the enzyme with a suitable model substrate in vitro.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Ben Shen, Ph.D.