Genome mining-directed discovery of novel 2,5-diketopiperazines from actinobacteria
Natural products derived from cyclodipeptides (CDPs) with a 2,5-diketopiperazine (DKP) skeleton comprise an important class of secondary metabolites, especially indole alkaloids derived from tryptophan-containing CDPs, which are widespread in fungi, bacteria, and plants. They play vital roles in dru...
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|Natural products derived from cyclodipeptides (CDPs) with a 2,5-diketopiperazine (DKP) skeleton comprise an important class of secondary metabolites, especially indole alkaloids derived from tryptophan-containing CDPs, which are widespread in fungi, bacteria, and plants. They play vital roles in drug discovery and development owing to their significant biological and pharmacological activities. In nature, the DKP cores can be generated by two distinct enzyme groups, that is, the nonribosomal peptide synthetases (NRPSs) and the aminoacyl tRNA-dependent cyclodipeptide synthases (CDPSs). Afterwards, different types of tailoring enzymes, such as cytochrome P450s, FAD-dependent oxidoreductases, cyclodipeptide oxidases (CDOs), prenyltransferases (PTs), and methyltransferases (MTs) are involved in installing a number of functional groups to the DKP scaffolds, thus generating various chemical structures. Although CDPSs belong to a newly defined family of enzymes, a large set of CDPSs have been identified. Among them, only several CDPS-associated biosynthetic pathways have been functionally characterized. In recent years, huge amounts of microbial genome sequences have been released in public databases and revealed numerous silent or cryptic gene clusters hiding in their genomes, including those for 2,5-DKPs, indicating great potential for discovery of novel metabolites. Therefore, full exploration of these untapped gene clusters could be a promising way to expand the chemical range of 2,5-DKPs accessible to the medical industry in the future.
In the first project, in cooperation with Dr. Huili Yu, eleven CDPSs from Streptomyces strains were selected for investigation based on phylogenetic analysis. Their functions were characterized via heterologous expression in Escherichia coli. The coding sequences of these CDPSs were individually cloned into pET28a (+) vector and overexpressed in soluBL21 host. The fermentation cultures of generated transformants were then analyzed by LC-MS. Combined with structural elucidation of accumulated products by NMR analysis, nine CDPSs for the assembly of tryptophan-containing cyclodipeptides (cWXs) were identified. Therefore, these nine CDP synthases represented new members of the CDPS family that are responsible for cWX biosynthesis. Among them, there is one cyclo-L-Trp-L-Leu synthase, two cyclo-L-Trp-L-Pro synthases, and three cyclo-L-Trp-L-Trp synthases, as well as three unspecific CDPSs producing up to seven products with cyclo-L-Trp-L-Ala or cyclo-L-Trp-L-Tyr as the major product. Under optimized cultivation conditions, total product yields of generated CDPs in the E. coli supernatants reached 46 to 211 mg/L. In recent years, tryptophan-containing DKPs have received increasing attention due to their promising scaffolds for structural modification. Therefore, our study provides a valid experimental basis for further combination of these CDPSs with other tailoring enzymes to generate more interesting chemical entities in the field of synthetic biology.
Afterwards, sequence analysis revealed that eight of nine cWX synthase genes identified in the first project are surrounded by a putative cytochrome P450 gene. Among them, two CDPS genes, gutA24309 from Streptomyces monomycini NRRL B-24309 and gutA3589 from Streptomyces varsoviensis NRRL B-3589, are located in the similar gene loci containing four additional genes coding for three modification enzymes, i.e., CDO, cytochrome P450, and MT. Heterologous expression of these two p450-associated cdps-containing gene clusters in Streptomyces coelicolor led to the identification of eight rare and novel C3-guaninyl indole alkaloids, named guanitrypmycins. Expression of different gene combinations and precursor feeding experiments proved the biosynthetic steps of guanitrypmycins. The CDP skeletons, cyclo-L-Trp-L-Phe and cyclo-L-Trp-L-Tyr assembled by the CDPS GutA, will be dehydrogenated merely at the phenylalanyl/tyrosyl side by the CDO Gut(BC) and subsequently connected with a guanine moiety by the P450 GutD. Furthermore, the MT GutE governs the last modification step to transfer a methyl group to N9′ of the guaninyl residue. Moreover, the non-enzymatic epimerization of the enzymatic pathway products via keto–enol tautomerism increases the structural diversity of guanitrypmycins. In addition, biochemical characterization further confirmed that the P450 enzyme GutD functions as the key biocatalyst and catalyzes the unprecedented regio- and stereospecific 3-guaninylation at the indole ring of the tryptophanyl moiety. Therefore, this study highlights the promise of CDPS-containing pathways as sources of novel biosynthetic transformations and natural products.
In analogy, two cdps-p450-containing operons were identified in Saccharopolyspora antimicrobica via genome mining. Heterologous expression, biochemical characterization, together with structural elucidation proved that the two P450 enzymes TtpB1 and TtpB2 catalyze distinct regio- and stereospecific dimerizations of cyclo-L-Trp-L-Trp, which are differing from those previously reported in bacteria. TtpB1 represents the first bacterial P450 that catalyzes the stereospecific C3 (sp3)–C3′ (sp3) bond formation between two monomers, both from the opposite side of H-11/H-11′, while TtpB2 is characterized as the first P450 to mainly catalyze the unusual linkage between C3 (sp3) of a hexahydropyrroloindole unit and N1′ of the tryptophanyl moiety of the second monomer from the H-11 side. Thus, our finding significantly increases the repertoire of DKP-tailoring enzymes. Additionally, in comparison with chemical synthesis, this study provides a simple, direct, and efficient approach for enzymatic one-step preparation of structurally complex DKP dimers.