Novel metabolites hidden in the cyclodipeptide-related biosynthetic gene clusters and intriguing reactions catalyzed by cytochrome P450 enzymes

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Natural products (NPs) produced by microorganisms, especially by actinobacteria, are valuable sources of drugs and candidates. Mining the bacterial genomes revealed the presence of a large number of silent biosynthetic gene clusters (BGCs) for NPs. Activation of such BGCs in an appropriate host is a promising strategy to access these unknown NPs and to meet the need of new drugs, especially of novel antibiotics to tackle the resistance crisis. Cyclodipeptide (CDP)-derived NPs have gained increasing attention not only due to their interesting biological and pharmacological activities but also because of their forming and modification enzymes. In bacteria, CDPs are biosynthesized mainly by cyclodipeptide synthases (CDPSs) with 200–300 amino acid residues. The CDPSs are genetically associated with a variety of tailoring enzymes including cytochrome P450 enzymes (P450s). Interestingly, the small cdps-associated BGCs code often for complex and fascinating chemical structures. The P450s from these BGCs, as responsible enzymes for the novel NPs, catalyze unusual reactions such as aryl-aryl coupling, N-oxide formation and coupling of indole with nucleobases. Nine such BGCs have been successfully identified by the applicant in the last two years. It can be expected that more novel NPs and intriguing P450s are encoded by unknown cdps-associated BGCs.The main goal of this proposal is finding novel metabolites hidden in the cdps- and P450-associated BGCs and identification of new cytochrome p450 enzymes, which catalyze fascinating modifications of CDPs and derivatives thereof. Therefore, 15 candidate BGCs containing at least one CDPS and one P450 gene from different clades of a phylogenetical tree will be investigated, which was obtained by involvement of 291 bacterial P450s. To overcome the disadvantages of low or non-expression of the genes in the native hosts and often difficult genetic manipulation of the strains, we plan to express the genuine or artificial BGCs in the genetically established Streptomyces coelicolor. The newly accumulated NPs will be isolated and identified by spectroscopic methods. Detection of the identified metabolites in the native producer afterward can give indications for the expression level of the BGCs. Feeding these products into the native producer and monitoring their metabolism will provide evidence for the integrity of the identified BGCs. The obtained new products are subjected to diverse biological tests. Expression of different combinations of the genes from the BGCs and identification of the metabolites will help to elucidate the biosynthetic pathways. P450s as the key biosynthetic enzymes will be overproduced in E. coli or S. coelicolor. In vitro reconstruction of their activities will provide important insights into the catalytic activities of these intriguing enzymes.