NATURAL PRODUCT DISCOVERY
Natural Products (NPs) originating from plants, fungi or bacteria represent a major source of medically important agents with antibacterial, antiviral, anticancer or immunosuppressive properties. They are produced as secondary metabolites and not necessarily required for the survival of the organism. However, their use for human purposes is immense. For decades these metabolites are used to develop suitable medication against human diseases or as insecticidal/herbicidal agents for agricultural purposes. Actinomycetes, Gram-positive, mostly soil dwelling, filamentous growing bacteria, is a prominent producer of such valuable NPs among bacteria. They occupy different ecological niches but are predominately distributed in soil, whereas the genus Streptomyces is one of the main representatives. Unsurprisingly, members of this genus were one of the first found producers of medically useful NPs (e.g. Streptomycin isolated from Streptomyces griseus in 1944 used for tuberculosis treatment) and provide to date more than half of all clinical used antibiotics. However, the consistent development of resistance against the currently available antibiotics requires an ongoing search for new active natural products not only to serve as antibiotics but to support development of effective agents against various diseases.
Our group investigates new actinomycetes strains collections, obtained from different ecological niches, in order to identify potentially new compounds. The new strains are cultivated, produced metabolites extracted and samples analyzed on LC-(HR)MS. A process called ‘dereplication’ is carried out to evaluate the novelty of the produced metabolites visible in the LC-MS chromatogram. This means, the exact mass of each compound is compared to the masses of known compounds by means of natural product databases (e.g. Dictionary of Natural Products (DNP, Chapman & Hall/CRC). Unknown masses and thus potentially new compounds are considered further in the discovery process. To isolate the potentially new metabolites, the corresponding strain is cultivated on a large scale. The culture broth is extracted and the resulting crude extract harboring a large number of chemically diverse substances is purified with a suitable chromatographic method or a combination of them (solid phase extraction (SPE), flash chromatography (FC), size exclusion chromatography (SEC), high-performance liquid chromatography (HPLC)) to obtain the targeted compound. The chemical structure of this new compound is elucidated by NMR spectroscopy and if necessary further experiments are carried out to determine stereogenic centers.
The option of sequencing bacterial genomes gives us additional possibility to get information about the new NPs from the side of the producer strain. Therefore, genomic DNA is subjected to genome sequencing and the obtained data is analyzed by bioinformatics software like ‘Geneious’ or ‘antiSMASH’. In bacteria, the genes for secondary metabolites are organized in groups, so called biosynthetic gene cluster (BGC), whereas one BGC is responsible for production of one metabolite and its derivatives. Thus, antiSMASH for instance can identify, annotate and compare secondary metabolite BGCs giving hints about type of BGC and possible function of each gene. This genetic information about strains obtained from such analyses gives us confirmation if known or new compounds are indeed produced by the respective strain and shows the potential of each strain to produce new interesting metabolites. Moreover, in depth analysis of specific genes gives hints about biosynthetic steps. Such a parallel pre-analysis prevents needless isolation of non-related compounds and helps us to prioritize NPs that are produced indeed by the strain in hand.
The procedure described here lead to the discovery of various new natural products and/or derivatives in our group (see below a selection of NPs and their corresponding BGCs). The putative new compounds initially identified in the crude extract of a new actinomycetes strain via HRMS, were isolated and purified and their chemical structure uncovered. In each case, the biosynthetic gene cluster responsible for production of the NP was identified in the genome of the respective strain and clearly connected to the compound by means of molecular biological experiments (e.g. cloning, heterologous expression, knock-out experiments). Further investigations are carried out to understand the exact biosynthetic machinery behind the NP and finding out functions for specific genes. Also, new compounds are usually tested to determine their biological activities (antibacterial, antifungal, herbicidal or anticancer).
