New secondary metabolites produced by an engineered strain Streptomyces sp. XZQH13OEΔastC.

Two previously undescribed alkaloids (1-2), five known alkaloids (3-7) and five cyclodipeptides (8-12) were obtained from an ansatrienin-producing mutant strain Streptomyces sp. XZQH13OEΔ astC. Their structures were elucidated by analysis of the 1D, 2D NMR and ESI HRMS data and by comparison with the reported data. The antibacterial activities of compounds 1-12 were evaluated.

[Advances in the biosynthesis of cyclodipeptide type natural products derived from actinomycetes].

Cyclodipeptide (CDP) composed of two amino acids is the simplest cyclic peptide. These two amino acids form a typical diketopiperazine (DKP) ring by linking each other with peptide bonds. This characteristic stable ring skeleton is the foundation of CDP to display extensive and excellent bioactivities, which is beneficial for CDPs' pharmaceutical research and development. The natural CDP products are well isolated from actinomycetes. These bacteria can synthesize DKP backbones with nonribosomal peptide synthetase (NRPS) or cyclodipeptide synthase (CDPS). Moreover, actinomycetes could produce a variety of CDPs through different enzymatic modification. The presence of these abundant and diversified catalysis indicates that actinomycetes are promising microbial resource for exploring CDPs. This review summarized the pathways for DKP backbones biosynthesis and their post-modification mechanism in actinomycetes. The aim of this review was to accelerate the genome mining of CDPs and their isolation, purification and structure identification, and to facilitate revealing the biosynthesis mechanism of novel CDPs as well as their synthetic biology design.

Total synthesis of complex 2,5-diketopiperazine alkaloids.

The 2,5-diketopiperazine (DKP) motif is present in many biologically relevant, complex natural products. The cyclodipeptide substructure offers structural rigidity and stability to proteolysis that makes these compounds promising candidates for medical applications. Due to their fascinating molecular architecture, synthetic organic chemists have focused significant effort on the total synthesis of these compounds. This review covers many such efforts on the total synthesis of DKP containing complex alkaloid natural products.

A new cyclodipeptide from Tetragonisca angustula honey active against Neospora caninum and in silico study.

A new cyclic natural compound formed by succinic acid and two alanine amino acid units was isolated from the Tetragonisca angustula honey extract. The chemical structure of 1 was established based on spectroscopic data analysis, including one- (1H and 13C NMR) and two-dimensional NMR techniques (1H-1H-COSY, HSQC and HMBC). A primary culture model previously infected with Neospora caninum was used to evaluate 1 for two time intervals (24 and 72 h), showing a reduction (40-56%) of the number of tachyzoites in the first 24 h and until 72 h, a dose-dependent reduction in parasite proliferation (25-50%). Glial cells treated with 1 did not demonstrate toxicity at concentrations up to 25 ug/mL. Treated and infected cultures showed an increase in NO when compared to control cells in 24 h and 72 h. In silico studies suggest that the new compound may affect DNA synthesis and impair -protein production.

Nanostructured Electrospun Fibers with Self-Assembled Cyclo-L-Tryptophan-L-Tyrosine Dipeptide as Piezoelectric Materials and Optical Second Harmonic Generators.

The potential use of nanostructured dipeptide self-assemblies in materials science for energy harvesting devices is a highly sought-after area of research. Specifically, aromatic cyclo-dipeptides containing tryptophan have garnered attention due to their wide-bandgap semiconductor properties, high mechanical rigidity, photoluminescence, and nonlinear optical behavior. In this study, we present the development of a hybrid system comprising biopolymer electrospun fibers incorporated with the chiral cyclo-dipeptide L-Tryptophan-L-Tyrosine. The resulting nanofibers are wide-bandgap semiconductors (bandgap energy 4.0 eV) consisting of self-assembled nanotubes embedded within a polymer matrix, exhibiting intense blue photoluminescence. Moreover, the cyclo-dipeptide L-Tryptophan-L-Tyrosine incorporated into polycaprolactone nanofibers displays a strong effective second harmonic generation signal of 0.36 pm/V and shows notable piezoelectric properties with a high effective coefficient of 22 pCN-1, a piezoelectric voltage coefficient of geff=1.2 VmN-1 and a peak power density delivered by the nanofiber mat of 0.16µWcm-2. These hybrid systems hold great promise for applications in the field of nanoenergy harvesting and nanophotonics.

Production of Diketopiperazine Derivatives by Pathway Engineering with Different Cyclodipeptide Synthases from Various Streptomyces Strains.

Cyclodipeptides from fungi and bacteria are often modified by different tailoring enzymes. They display various biological and pharmacological activities, and some derivatives are used as drugs. In a previous study, we elucidated the function of the silent guatrypmethine gene cluster from Streptomyces cinnamoneus containing a cyclodipeptide synthase (CDPS) core gene gtmA and four genes gtmB-gtmE for tailoring enzymes. The latter are used in this study for the design of modified cyclodipeptides by genetic engineering. Addition of six different cyclodipeptides to the Streptomyces albus transformant harboring gtmB-gtmE led to the detection of different pathway products. Coexpression of five CDPS genes from four Streptomyces strains with gtmB-gtmE resulted in the formation of diketopiperazine derivatives, differing in their modification stages. Our results demonstrate the potential of rational gene combination to increase structural diversity.

Advances in the biosynthesis of cyclodipeptide type natural products derived from actinomycetes / 生物工程学报

Cyclodipeptide (CDP) composed of two amino acids is the simplest cyclic peptide. These two amino acids form a typical diketopiperazine (DKP) ring by linking each other with peptide bonds. This characteristic stable ring skeleton is the foundation of CDP to display extensive and excellent bioactivities, which is beneficial for CDPs' pharmaceutical research and development. The natural CDP products are well isolated from actinomycetes. These bacteria can synthesize DKP backbones with nonribosomal peptide synthetase (NRPS) or cyclodipeptide synthase (CDPS). Moreover, actinomycetes could produce a variety of CDPs through different enzymatic modification. The presence of these abundant and diversified catalysis indicates that actinomycetes are promising microbial resource for exploring CDPs. This review summarized the pathways for DKP backbones biosynthesis and their post-modification mechanism in actinomycetes. The aim of this review was to accelerate the genome mining of CDPs and their isolation, purification and structure identification, and to facilitate revealing the biosynthesis mechanism of novel CDPs as well as their synthetic biology design.

Genome Mining and Metabolic Profiling Reveal Cytotoxic Cyclodipeptides in Streptomyces hygrospinosus var. Beijingensis.

Two new cyclodipeptide (CDP) derivatives (1-2) and another seven known cyclodipeptides (3-9) were isolated from Streptomyces 26D9-414 by the genome mining approach combined with genetic dereplication and the "one strain many compounds" (OSMAC) strategy. The structures of the new CDPs were established on the basis of 1D- and 2D-NMR and comparative electronic circular dichroism (ECD) spectra analysis. The biosynthetic gene clusters (BGCs) for these CDPs were identified through antiSMASH analysis. The relevance between this cdp cluster and the identified nine CDPs was established by genetic interruption manipulation. The newly discovered natural compound 2 displayed comparable cytotoxicity against MDA-MB-231 and SW480 with that of cisplatin, a widely used chemotherapeutic agent for the treatment of various cancers.

[Chemical constituents with α-glucosidase inhibitory activities from seeds of Morinda citrifolia (Noni)].

The present study investigated the chemical constituents and inhibitory activities against α-glucosidase from the seeds of Morinda citrifolia(Noni) by the chromatographic technique and semi-preparative HPLC.Fifteen compounds were isolated from the ethyl acetate extract of the seeds, and their structures were identified on the basis of physiochemical characteristics and spectroscopic data as(9S,2E,4Z,7Z)-9-hydroxydeca-2,4,7-trienoic acid(1), azelaic acid(2), scopoletin(3), ursolic acid(4), quercetin(5), cyclo-(L-Leu-L-Ile)(6), cyclo-(L-Phe-L-Ile)(7), cyclo-(L-Phe-L-Val)(8), cyclo-(L-Leu-L-Val)(9), cyclo-(L-Phe-L-Leu)(10), caffeic acid(11), 3,4-dihydroxycinnamaldehyde(12), p-hydroxybenzoic acid(13), p-hydroxy-cinnamic acid(14), and p-hydroxyphenethyl alcohol(15).Among them, compound 1 was a new fatty acid and compounds 7-10 and 12 were isolated from Morinda plant in the Rubiaceae family for the first time.Compounds 1, 2 and 4-15 were isolated from the seeds of M.citrifolia(Noni) for the first time.All isolated compounds were evaluated for the inhibitory activities against α-glucosidase and compounds 3-5 showed potential inhibitory activity with IC_(50) values of 160, 133, and 120 µmol·L~(-1), respectively.

Widely Distributed Bifunctional Bacterial Cytochrome P450 Enzymes Catalyze both Intramolecular C-C Bond Formation in cyclo-l-Tyr-l-Tyr and Its Coupling with Nucleobases.

Tailoring enzymes are important modification biocatalysts in natural product biosynthesis. We report herein six orthologous two-gene clusters for mycocyclosin and guatyromycine biosynthesis. Expression of the cyclodipeptide synthase genes gymA1 -gymA6 in Escherichia coli resulted in the formation of cyclo-l-Tyr-l-Tyr as the major product. Reconstruction of the biosynthetic pathways in Streptomyces albus and biochemical investigation proved that the cytochrome P450 enzymes GymB1 -GymB6 act as both intramolecular oxidases and intermolecular nucleobase transferases. They catalyze not only the oxidative C-C coupling within cyclo-l-Tyr-l-Tyr, leading to mycocyclosin, but also its connection with guanine and hypoxanthine, and are thus responsible for the formation of tyrosine-containing guatyromycines, instead of the reported tryptophan-nucleobase adducts. Phylogenetic data suggest the presence of at least 47 GymB orthologues, indicating the occurrence of a widely distributed enzyme class.

Deciphering a Cyclodipeptide Synthase Pathway Encoding Prenylated Indole Alkaloids in Streptomyces leeuwenhoekii.

Cyclodipeptide synthases (CDPSs) catalyze the formation of cyclodipeptides using aminoacylated tRNAs as the substrates and have great potential in the production of diverse 2,5-diketopiperazines (2,5-DKPs). Genome mining of Streptomyces leeuwenhoekii NRRL B-24963 revealed a two-gene locus, saz, encoding CDPS SazA and a unique fused enzyme (SazB) harboring two domains: phytoene synthase-like prenyltransferase (PT) and methyltransferase (MT). Heterologous expression of the saz gene(s) in Streptomyces albus J1074 led to the production of four prenylated indole alkaloids, among which streptoazines A to C (compounds 3 to 5) are new compounds. Expression of different gene combinations showed that the SazA catalyzes the formation of cyclo(l-Trp-l-Trp) (cWW; compound 1), followed by consecutive prenylation and methylation by SazB. Biochemical assays demonstrated that SazB is a bifunctional enzyme, catalyzing sequential C-3/C-3' prenylation(s) by SazB-PT and N-1/N-1' methylation(s) by SazB-MT. Of note, the substrate selectivity of SazB-PT and SazB-MT was probed, revealing the stringent specificity of SazB-PT but relative flexibility of SazB-MT.IMPORTANCE Natural products with a 2,5-diketopiperazine (2,5-DKP) skeleton have long sparked interest in drug discovery and development. Recent advances in microbial genome sequencing have revealed that the potential of cyclodipeptide synthase (CDPS)-dependent pathways encoding new 2,5-DKPs are underexplored. In this study, we report the genome mining of a new CDPS-encoding two-gene operon and activation of this cryptic gene cluster through heterologous expression, leading to the discovery of four indole 2,5-DKP alkaloids. The cyclo(l-Trp-l-Trp) (cWW)-synthesizing CDPS SazA and the unusual prenyltransferase (PT)-methyltransferase (MT) fused enzyme SazB were characterized. Our results expand the repertoire of CDPSs and associated tailoring enzymes, setting the stage for accessing diverse prenylated alkaloids using synthetic biology strategies.

In vivo characterization of the activities of novel cyclodipeptide oxidases: new tools for increasing chemical diversity of bioproduced 2,5-diketopiperazines in Escherichia coli.

BACKGROUND: Cyclodipeptide oxidases (CDOs) are enzymes involved in the biosynthesis of 2,5-diketopiperazines, a class of naturally occurring compounds with a large range of pharmaceutical activities. CDOs belong to cyclodipeptide synthase (CDPS)-dependent pathways, in which they play an early role in the chemical diversification of cyclodipeptides by introducing Cα-Cß dehydrogenations. Although the activities of more than 100 CDPSs have been determined, the activities of only a few CDOs have been characterized. Furthermore, the assessment of the CDO activities on chemically-synthesized cyclodipeptides has shown these enzymes to be relatively promiscuous, making them interesting tools for cyclodipeptide chemical diversification. The purpose of this study is to provide the first completely microbial toolkit for the efficient bioproduction of a variety of dehydrogenated 2,5-diketopiperazines. RESULTS: We mined genomes for CDOs encoded in biosynthetic gene clusters of CDPS-dependent pathways and selected several for characterization. We co-expressed each with their associated CDPS in the pathway using Escherichia coli as a chassis and showed that the cyclodipeptides and the dehydrogenated derivatives were produced in the culture supernatants. We determined the biological activities of the six novel CDOs by solving the chemical structures of the biologically produced dehydrogenated cyclodipeptides. Then, we assessed the six novel CDOs plus two previously characterized CDOs in combinatorial engineering experiments in E. coli. We co-expressed each of the eight CDOs with each of 18 CDPSs selected for the diversity of cyclodipeptides they synthesize. We detected more than 50 dehydrogenated cyclodipeptides and determined the best CDPS/CDO combinations to optimize the production of 23. CONCLUSIONS: Our study establishes the usefulness of CDPS and CDO for the bioproduction of dehydrogenated cyclodipeptides. It constitutes the first step toward the bioproduction of more complex and diverse 2,5-diketopiperazines.

Comparative studies on similarities and differences of cyclodipeptide oxidases for installation of C-C double bonds at the diketopiperazine ring.

Cyclodipeptide oxidases (CDOs) perform dehydrogenations on diketopiperazines and play an important role in the cyclodipeptide diversification. In this study, we investigated the two known CDOs AlbA/B and Ndas_1146/7 and one new member, CDO-Np. LC-MS monitoring of 32 cyclodipeptide biotransformations in E. coli revealed good consumption of cyclodipeptides containing aromatic amino acids. Cyclodipeptides consisting solely of aliphatic amino acids were poor substrates. In vitro assays of 34 substrates with crude enzyme extracts and product identification proved that the CDO-Np-containing extract catalyzes the formation of two C-C double bonds in many cases. The extracts containing the two other enzymes had lower activities and catalyzed mainly didehydrogenations. For didehydrogenation, the phenylalanyl or tyrosyl site was usually preferred. No or very low acceptance of benzodiazepinediones and a 2,6-diketopiperazine proved the importance of the 2,5-diketopiperazine ring. N-Methylation at the diketopiperazine ring or prenylation of the tryptophan-containing cyclodipeptides influences the enzyme activity and product spectrum. KEY POINTS: • Comparison of catalytic activities of three enzymes; Diverse cyclodipeptides and derivatives as substrates; Determination of double bond formation using2H-labeled substrates; Product identification also by interpretation of MS2fragmentation pattern.

Diketopiperazines Synthesis Gene in Shewanella baltica and Roles of Diketopiperazines and Resveratrol in Quorum Sensing.

The synthesis pathways of quorum sensing (QS) signal molecules and the mechanism of action of quorum sensing inhibitors (QSIs) have gained considerable attention as research topics in the field of food preservation. Here, Shewanella baltica was detected as the specific spoilage organism in large yellow croaker during 4 °C storage, and it produced the QS signal molecules autoinducer-2 (AI-2) and diketopiperazines (DKPs). Then, a cyclodipeptide synthase (CDPS) homologous gene, sb1370, was screened, and knockout and rescue results revealed that this gene was involved in DKP synthesis but not in AI-2 synthesis, and it also played an important role in QS. Furthermore, fish fillets and mutant strains were treated with resveratrol, and the results suggested that resveratrol was an ideal QSI for inhibition of DKPs production via the sb1370 gene and reduced QS in S. baltica, thus delaying the process of fish spoilage during chilling storage.

Functional and evolutionary characterization of a secondary metabolite gene cluster in budding yeasts.

Secondary metabolites are key in how organisms from all domains of life interact with their environment and each other. The iron-binding molecule pulcherrimin was described a century ago, but the genes responsible for its production in budding yeasts have remained uncharacterized. Here, we used phylogenomic footprinting on 90 genomes across the budding yeast subphylum Saccharomycotina to identify the gene cluster associated with pulcherrimin production. Using targeted gene replacements in Kluyveromyces lactis, we characterized the four genes that make up the cluster, which likely encode two pulcherriminic acid biosynthesis enzymes, a pulcherrimin transporter, and a transcription factor involved in both biosynthesis and transport. The requirement of a functional putative transporter to utilize extracellular pulcherrimin-complexed iron demonstrates that pulcherriminic acid is a siderophore, a chelator that binds iron outside the cell for subsequent uptake. Surprisingly, we identified homologs of the putative transporter and transcription factor genes in multiple yeast genera that lacked the biosynthesis genes and could not make pulcherrimin, including the model yeast Saccharomyces cerevisiae We deleted these previously uncharacterized genes and showed they are also required for pulcherrimin utilization in S. cerevisiae, raising the possibility that other genes of unknown function are linked to secondary metabolism. Phylogenetic analyses of this gene cluster suggest that pulcherrimin biosynthesis and utilization were ancestral to budding yeasts, but the biosynthesis genes and, subsequently, the utilization genes, were lost in many lineages, mirroring other microbial public goods systems that lead to the rise of cheater organisms.

Petroleum degradation by Pseudomonas sp. ZS1 is impeded in the presence of antagonist Alcaligenes sp. CT10.

Enhanced bioremediation is a favorable approach for petroleum pollutant cleanup, which depends on the growth of oil-eating microorganisms. In this study, we show that, by using the modified T-RFLP (mT-RFLP) methodology, one of the four major microbial populations derived from oil sludge has failed to propagate in MS medium supplemented with 2% yeast extract (YE). rDNA sequence-based analysis indicated that the four populations were Donghicola sp. CT5, Bacillus sp. CT6, Alcaligenes sp. CT10, and Pseudomonas sp. ZS1. Four purified strains grow well individually in MS medium supplemented with 2% YE, suggesting that ZS1 growth is antagonized by other strains. Co-growth analysis using mT-RFLP methodology and plate inhibitory assay indicated that ZS1 exhibited antagonistic effect against CT5 and CT6. On the other hand, co-growth analysis and plate inhibition assay showed that CT10 antagonized against ZS1. To investigate the potential compounds responsible for the antagonism, supernatant of CT10 culture was subjected to GC-MS analysis. Analysis indicated that CT10 produced a number of antimicrobial compounds including cyclodipeptide c-(L-Pro-L-Phe), which was known to inhibit the growth of Pseudomonas sp. Growth test using the purified c-(L-Pro-L-Phe) from CT10 confirmed its inhibitory activity. We further showed that, using both gravimetric and GC analysis, CT10 antagonism against the oil-eating ZS1 led to the diminishing of crude oil degradation. Together, our results indicate that bioremediation can be affected by environmental antagonists.

Regulation of the Synthesis and Secretion of the Iron Chelator Cyclodipeptide Pulcherriminic Acid in Bacillus licheniformis.

The cyclodipeptide pulcherriminic acid synthesized by Bacillus licheniformis is an iron chelator that antagonizes certain pathogens by removing iron from the environment. But since the insoluble iron-pulcherriminic acid complex cannot act as an iron carrier as siderophores do, excessive synthesized pulcherriminic acid causes iron starvation for the producer cells. At present, the regulation of pulcherriminic acid synthesis and the mechanism by which B. licheniformis strikes a balance between biocontrol and self-protection from excessive iron removal remain unclear. This study provides insights into the regulatory network and explains the mechanism of pulcherriminic acid biosynthesis. The yvmC-cypX synthetic gene cluster was directly negatively regulated by three regulators: AbrB, YvnA, and YvmB. Within the regulatory network, YvnA expression was repressed not only by AbrB but also by iron-limiting environments, while YvmB expression was repressed by YvnA. The transporter gene yvmA is repressed by YvmB and is required for pulcherriminic acid secretion. The biosynthesis window is determined by the combined concentration of the three regulators in an iron-rich environment. Under iron-limiting conditions, cells close the pulcherriminic acid synthesis pathway by downregulating YvnA expression.IMPORTANCE The cyclodipeptides are widespread in nature and exhibit a broad variety of biological and pharmacological activities. The cyclodipeptide scaffold is synthesized by nonribosomal peptide synthetases (NRPSs) and cyclodipeptide synthases (CDPSs). At present, it is clear that CDPSs use aminoacyl tRNAs as substrates to synthesize the two peptide bonds, and the pulcherriminic acid synthase YvmC is a member of the eight identified CDPSs. However, little is known about the regulation of cyclodipeptide synthesis and secretion. In this study, we show that AbrB, which is considered to be the main regulator of NRPS-dependent pathways, is also involved in the regulation of CDPS genes. However, AbrB is not the decisive factor for pulcherriminic acid synthesis, as the expression of YvnA determines the fate of pulcherriminic acid synthesis. With this information on how CDPS gene transcription is regulated, a clearer understanding of cyclodipeptide synthesis can be developed for B. licheniformis Similar approaches may be used to augment our knowledge on CDPSs in other bacteria.

Expanding tryptophan-containing cyclodipeptide synthase spectrum by identification of nine members from Streptomyces strains.

Cyclodipeptide synthases (CDPSs) comprise normally 200-300 amino acid residues and are mainly found in bacteria. They hijack aminoacyl-tRNAs from the ribosomal machinery for cyclodipeptide formation. In this study, nine new CDPS genes from eight Streptomyces strains were cloned into pET28a vector and expressed in Escherichia coli. Structural elucidation of the isolated products led to the identification of one cyclo-L-Trp-L-Leu, two cyclo-L-Trp-L-Pro, and three cyclo-L-Trp-L-Trp synthases. Other three CDPSs produce cyclo-L-Trp-L-Ala or cyclo-L-Trp-L-Tyr as the major cyclodipeptide. Total product yields of 46 to 211 mg/L E. coli culture were obtained. Our findings represent rare examples of CDPS family derived from actinobacteria that form various tryptophan-containing cyclodipeptides. Furthermore, this study highlights the potential of the microbial machinery for tryptophan-containing cyclodipeptide biosynthesis and provides valid experimental basis for further combination of these CDPS genes with other modification genes in synthetic biology.

A Comprehensive Overview of the Cyclodipeptide Synthase Family Enriched with the Characterization of 32 New Enzymes.

Cyclodipeptide synthases (CDPSs) use as substrates two amino acids activated as aminoacyl-tRNAs to synthesize cyclodipeptides in secondary metabolites biosynthetic pathways. Since the first description of a CDPS in 2002, the number of putative CDPSs in databases has increased exponentially, reaching around 800 in June 2017. They are likely to be involved in numerous biosynthetic pathways but the diversity of their products is still under-explored. Here, we describe the activity of 32 new CDPSs, bringing the number of experimentally characterized CDPSs to about 100. We detect 16 new cyclodipeptides, one of which containing an arginine which has never been observed previously. This brings to 75 the number of cyclodipeptides formed by CDPSs out of the possible 210 natural ones. We also identify several consensus sequences related to the synthesis of a specific cyclodipeptide, improving the predictive model of CDPS specificity. The improved prediction method enables to propose the main product synthesized for about 80% of the CDPS sequences available in databases and opens the way for the deciphering of CDPS-dependent pathways. Analysis of phylum distribution and predicted activity for all CDPSs identified in databases shows that the experimentally characterized set is representative of the whole family. Our work also demonstrates that some cyclodipeptides, precursors of diketopiperazines with interesting pharmacological properties and previously described as being synthesized by fungal non-ribosomal peptide synthetases, can also be produced by CDPSs in bacteria.

Global analysis of prokaryotic tRNA-derived cyclodipeptide biosynthesis.

BACKGROUND: Among naturally occurring small molecules, tRNA-derived cyclodipeptides are a class that have attracted attention for their diverse and desirable biological activities. However, no tools are available to link cyclodipeptide synthases identified within prokaryotic genome sequences to their chemical products. Consequently, it is unclear how many genetically encoded cyclodipeptides represent novel products, and which producing organisms should be targeted for discovery. RESULTS: We developed a pipeline for identification and classification of cyclodipeptide biosynthetic gene clusters and prediction of aminoacyl-tRNA substrates and complete chemical structures. We leveraged this tool to conduct a global analysis of tRNA-derived cyclodipeptide biosynthesis in 93,107 prokaryotic genomes, and compared predicted cyclodipeptides to known cyclodipeptide synthase products and all known chemically characterized cyclodipeptides. By integrating predicted chemical structures and gene cluster architectures, we created a unified map of known and unknown genetically encoded cyclodipeptides. CONCLUSIONS: Our analysis suggests that sizeable regions of the chemical space encoded within sequenced prokaryotic genomes remain unexplored. Our map of the landscape of genetically encoded cyclodipeptides provides candidates for targeted discovery of novel compounds. The integration of our pipeline into a user-friendly web application provides a resource for further discovery of cyclodipeptides in newly sequenced prokaryotic genomes.