Elucidation of novel biosynthetic pathways for the discovery of cyclodipeptide derivatives from Streptomyces species

Cyclodipeptides (CDPs) with a 2,5-diketopiperazine (DKP) as central core occur ubiquitously in living organisms, from simple bacteria and fungi to more complex ones like plants and animals. They display various biological and pharmacological effects, including antibiotic, antifungal, and antiproliferative activities. In microorganisms, CDPs are usually synthesized by one of the two distinct enzyme families, nonribosomal peptide synthetases (NRPSs), mainly occurring in fungi, or cyclodipeptide synthases (CDPSs), commonly found in bacteria. NRPS for CDP formation are comparably large (~2500 amino acids), bi-modular enzymes, using free amino acids as substrates. CDPSs on the other hand are smaller (200 – 300 amino acids) and require activated amino acyl tRNAs for peptide bond formation. In general, the formation of the DKP ring increases the stability of CDPs against proteolysis compared to their acyclic counterparts. This enables a variety of intriguing modifications carried out by tailoring enzymes. Their genetic information often lies in direct neighborhood to that of backbone enzymes, like CDPSs, arranged in biosynthetic gene clusters (BGCs). In CDPS-associated pathways, tailoring enzymes comprise cyclodipeptide oxidases (CDOs), cytochrome P450 (P450) enzymes, FeII/2-oxoglutarate dependent (FeII/2-OG) oxidases, as well as methyl- (MTs) and prenyltransferases (PTs). In this thesis, eight of such BGCs from Streptomyces species were identified using genome mining and elucidated by a combination of heterologous expression and biochemical analyses. In the first project, a BGC from Streptomyces cinnamoneus consisting of five genes was chosen for detailed investigation and termed gtm gene cluster. It codes for four enzymes, i.e. a CDPS (GtmA), a CDO (GtmBC), a P450 enzyme (GtmD), and a FeII/2-OG oxidase (GtmE). The genes were cloned in different combinations into the replicative pPWW50A vector for heterologous expression in Streptomyces albus J1074 (S. albus). Investigation using LC-MS and NMR spectroscopy revealed that GtmA synthesizes cyclo-L-Trp-L-Met, GtmBC installs a double bond at the methionine residue of the DKP, GtmD transfers a guanine onto the tryptophan moiety, and GtmE forms a second double bond at another side of the DKP. Together, this cascade results in the formation of the novel secondary metabolite guatrypmethine C. As the second dehydrogenation by GtmE displayed a novel reaction for FeII/2-OG oxidases in CDPS-dependent pathways, it was further characterized biochemically using the recombinant protein. It was proven that GtmE indeed catalyzes the conversion of the precursor guatrypmethine A to the pathway end product guatrypmethine C. No efficient conversion of the stable isomer guatrypmethine B was observed by GtmE. This experimental finding was further supported by quantum chemical calculations using density functional theory. In the second project, in cooperation with Dr. Jing Liu, a widely distributed two-gene locus, gymAB, was identified in 47 different actinobacteria. It comprises the genes gymA and gymB, coding for a CDPS and a P450 oxidase, respectively. The latter is closely related to CYP121, an essential enzyme for the viability of Mycobacterium tuberculosis. Six representative Streptomyces species were selected for functional elucidation of these BGCs. In analogy to the first project, their genes were cloned into pPWW50A and overexpressed in S. albus. Analyses of the cultural extracts by LC-MS in combination with NMR spectroscopy of the purified compounds showed that all six CDPSs produce cyclo-L-Tyr-L-Tyr (cYY) as major product. Subsequently, the P450 oxidases catalyze two different kind of reactions – either the formation of an intramolecular C-C bond within cYY resulting in mycocyclosin, or the intermolecular transfer of the nucleobases guanine or hypoxanthine, leading to the formation of the novel secondary metabolites guatyromycine A and B, respectively. The reactions catalyzed by GymBs were confirmed with biochemical assays using recombinant proteins of all six candidates. As the intramolecular coupling is the same reaction performed by CYP121 from Mycobacterium tuberculosis, the corresponding gene cluster was also expressed heterologously in the same manner. However, CYP121 merely catalyzes the formation of mycocyclosin, indicating that GymBs might have evolved from CYP121 and slightly changed during evolution. In the third project, I contributed to the elucidation of a BGC from Streptomyces aurantiacus, coding for the CDPS SasA, the PT SasB, and the MT SasC. It was proven that the sasABC gene cluster is responsible for the formation of streptoazine C. The involved PT SasB catalyzes two regular prenylations at both tryptophan residues within cyclo-L-Trp-L-Trp. By incubation with other CDPs and dehydrogenated CDPs, it was shown that SasB possesses a broad substrate flexibility and can convert at least eight other CDP derivatives efficiently