Genome mining of biosynthetic pathways with no identifiable core enzymes can lead to discovery of the so-called unknown (biosynthetic route)–unknown (molecular structure) natural products. Here we focused on a conserved fungal biosynthetic pathway that lacks a canonical core enzyme and used heterologous expression to identify the associated natural product, a highly modified cyclo-arginine-tyrosine dipeptide. Biochemical characterization of the pathway led to identification of a new arginine-containing cyclodipeptide synthase (RCDPS), which was previously annotated as a hypothetical protein and has no sequence homology to non-ribosomal peptide synthetase or bacterial cyclodipeptide synthase. RCDPS homologs are widely encoded in fungal genomes; other members of this family can synthesize diverse cyclo-arginine-Xaa dipeptides, and characterization of a cyclo-arginine-tryptophan RCDPS showed that the enzyme is aminoacyl-tRNA dependent. Further characterization of the biosynthetic pathway led to discovery of new compounds whose structures would not have been predicted without knowledge of RCDPS function.
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All data supporting the findings of this study are available within the article and its Supplementary Information files. All unique biological materials, such as plasmids, generated in the study are available from the authors upon reasonable request. The DNA sequences of the ank cluster (accession: NKHU02000029.1), ava cluster (accession: OP596311), eshA (accession: OP596310), amaA (accession: OP622864), ateA (accession: OP622863) and pthA (accession: OP622862) are available from the National Center for Biotechnology Information. The protein sequence of AlbC (accession: Q8GED7) is available from UniProt, and the structure of AlbC (Protein Data Bank: 4Q24) is available from Research Collaboratory for Structural Bioinformatics.
Harvey, A. L., Edrada-Ebel, R. & Quinn, R. J. The re-emergence of natural products for drug discovery in the genomics era. Nat. Rev. Drug Discov. 14, 111–129 (2015).
Blin, K. et al. antiSMASH 5.0: updates to the secondary metabolite genome mining pipeline. Nucleic Acids Res. 47, W81–W87 (2019).
Kautsar, S. A., Blin, K., Shaw, S., Weber, T. & Medema, M. H. BiG-FAM: the biosynthetic gene cluster families database. Nucleic Acids Res. 49, D490–D497 (2021).
Gilchrist, C. L. M., Li, H. & Chooi, Y.-H. Panning for gold in mould: can we increase the odds for fungal genome mining? Org. Biomol. Chem. 16, 1620–1626 (2018).
Ziemert, N., Alanjary, M. & Weber, T. The evolution of genome mining in microbes—a review. Nat. Prod. Rep. 33, 988–1005 (2016).
Barra, L. et al. β-NAD as a building block in natural product biosynthesis. Nature 600, 754–758 (2021).
Patteson, J. B. et al. Biosynthesis of fluopsin C, a copper-containing antibiotic from Pseudomonas aeruginosa. Science 374, 1005–1009 (2021).
Lima, S. T. et al. Biosynthesis of guanitoxin enables global environmental detection in freshwater cyanobacteria. J. Am. Chem. Soc. 144, 9372–9379 (2022).
Tang, M.-C., Zou, Y., Watanabe, K., Walsh, C. T. & Tang, Y. Oxidative cyclization in natural product biosynthesis. Chem. Rev. 117, 5226–5333 (2017).
Rix, U., Fischer, C., Remsing, L. L. & Rohr, J. Modification of post-PKS tailoring steps through combinatorial biosynthesis. Nat. Prod. Rep. 19, 542–580 (2002).
Du, Y.-L. & Ryan, K. S. Pyridoxal phosphate-dependent reactions in the biosynthesis of natural products. Nat. Prod. Rep. 36, 430–457 (2019).
Zhao, G. et al. Structural basis for a dual function ATP grasp ligase that installs single and bicyclic ω-ester macrocycles in a new multicore RiPP natural product. J. Am. Chem. Soc. 143, 8056–8068 (2021).
Canu, N., Moutiez, M., Belin, P. & Gondry, M. Cyclodipeptide synthases: a promising biotechnological tool for the synthesis of diverse 2,5-diketopiperazines. Nat. Prod. Rep. 37, 312–321 (2020).
Chen, M., Liu, C.-T. & Tang, Y. Discovery and biocatalytic application of a PLP-dependent amino acid γ-substitution enzyme that catalyzes C–C bond formation. J. Am. Chem. Soc. 142, 10506–10515 (2020).
Faulkner, J. R. et al. On the sequence of bond formation in loline alkaloid biosynthesis. ChemBioChem 7, 1078–1088 (2006).
Hai, Y., Chen, M., Huang, A. & Tang, Y. Biosynthesis of mycotoxin fusaric acid and application of a PLP-dependent enzyme for chemoenzymatic synthesis of substituted l-pipecolic acids. J. Am. Chem. Soc. 142, 19668–19677 (2020).
Liu, N. et al. Targeted genome mining reveals the biosynthetic gene clusters of natural product CYP51 inhibitors. J. Am. Chem. Soc. 143, 6043–6047 (2021).
Yee, D. A. et al. Genome mining of alkaloidal terpenoids from a hybrid terpene and nonribosomal peptide biosynthetic pathway. J. Am. Chem. Soc. 142, 710–714 (2020).
Liu, N. et al. Identification and heterologous production of a benzoyl-primed tricarboxylic acid polyketide intermediate from the zaragozic acid a biosynthetic pathway. Org. Lett. 19, 3560–3563 (2017).
Tohyama, S. et al. Discovery and characterization of NK13650s, naturally occurring p300-selective histone acetyltransferase inhibitors. J. Org. Chem. 77, 9044–9052 (2012).
Bartnik, E. & Weglenski, P. Regulation of arginine catabolism in Aspergillus nidulans. Nature 250, 590–592 (1974).
Tsukamoto, S., Kato, H., Hirota, H. & Fusetani, N. Pipecolate derivatives, anthosamines A and B, inducers of larval metamorphosis in ascidians, from a marine sponge Anthosigmella aff. raromicrosclera. Tetrahedron 51, 6687–6694 (1995).
Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. E. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015).
Meister, A. Glutamine synthetase of mammals. in: The Enzymes Vol. 10 (ed. Boyer, P. D.) 699–754 (Academic Press, 1974).
Cotton, J. L., Tao, J. & Balibar, C. J. Identification and characterization of the Staphylococcus aureus gene cluster coding for staphyloferrin A. Biochemistry 48, 1025–1035 (2009).
Mydy, L. S., Bailey, D. C., Patel, K. D., Rice, M. R. & Gulick, A. M. The siderophore synthetase IucA of the aerobactin biosynthetic pathway uses an ordered mechanism. Biochemistry 59, 2143–2153 (2020).
Sasaki, Y., Akutsu, Y., Suzuki, K., Sakurada, S. & Kisara, K. Structure and analgesic activity relationship of cyclo-tyrosyl-arginyl and its three stereoisomers. Chem. Pharm. Bull. (Tokyo) 29, 3403–3406 (1981).
Harvey, C. J. B. et al. HEx: a heterologous expression platform for the discovery of fungal natural products. Sci. Adv. 4, eaar5459 (2018).
Liao, H.-J. et al. Insights into the desaturation of cyclopeptin and its C3 epimer catalyzed by a non-heme iron enzyme: structural characterization and mechanism elucidation. Angew. Chem. Int. Ed. 57, 1831–1835 (2018).
Walsh, C. T. & Wencewicz, T. A. Flavoenzymes: versatile catalysts in biosynthetic pathways. Nat. Prod. Rep. 30, 175–200 (2013).
Healy, F. G., Krasnoff, S. B., Wach, M., Gibson, D. M. & Loria, R. Involvement of a cytochrome P450 monooxygenase in thaxtomin a biosynthesis by Streptomyces acidiscabies. J. Bacteriol. 184, 2019–2029 (2002).
Maiya, S., Grundmann, A., Li, S.-M. & Turner, G. The fumitremorgin gene cluster of Aspergillus fumigatus: identification of a gene encoding brevianamide F synthetase. ChemBioChem 7, 1062–1069 (2006).
Katoh, K., Rozewicki, J. & Yamada, K. D. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief. Bioinform 20, 1160–1166 (2019).
Izumida, H., Imamura, N. & Sano, H. A novel chitinase inhibitor from a marine bacterium, Pseudomonas sp. J. Antibiot. (Tokyo) 49, 76–80 (1996).
Furukawa, T. et al. Cyclic dipeptides exhibit potency for scavenging radicals. Bioorg. Med. Chem. 20, 2002–2009 (2012).
Li, X. et al. Determination of absolute configuration and conformation of a cyclic dipeptide by NMR and chiral spectroscopic methods. J. Phys. Chem. A 117, 1721–1736 (2013).
Gondry, M. et al. A comprehensive overview of the cyclodipeptide synthase family enriched with the characterization of 32 new enzymes. Front. Microbiol. 9, 46 (2018).
Cusack, S. Aminoacyl-tRNA synthetases. Curr. Opin. Struct. Biol. 7, 881–889 (1997).
Gondry, M. et al. Cyclodipeptide synthases are a family of tRNA-dependent peptide bond-forming enzymes. Nat. Chem. Biol. 5, 414–420 (2009).
Sissler, M., Eriani, G., Martin, F., Giegé, R. & Florentz, C. Mirror image alternative interaction patterns of the same tRNA with either class I arginyl-tRNA synthetase or class II aspartyl-tRNA synthetase. Nucleic Acids Res. 25, 4899–4906 (1997).
John, T. R., Ghosh, M. & Johnson, J. D. Identification and expression of the Saccharomyces cerevisiae cytoplasmic tryptophanyl-tRNA synthetase gene. Yeast 13, 37–41 (1997).
Moutiez, M. et al. Unravelling the mechanism of non-ribosomal peptide synthesis by cyclodipeptide synthases. Nat. Commun. 5, 5141 (2014).
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
Moutiez, M., Belin, P. & Gondry, M. Aminoacyl-tRNA-utilizing enzymes in natural product biosynthesis. Chem. Rev. 117, 5578–5618 (2017).
Ye, Y. et al. Fungal-derived brevianamide assembly by a stereoselective semipinacolase. Nat. Catal. 3, 497–506 (2020).
Li, S. et al. Biochemical characterization of NotB as an FAD-dependent oxidase in the biosynthesis of notoamide indole alkaloids. J. Am. Chem. Soc. 134, 788–791 (2012).
Zou, L.-H. et al. Copper-catalyzed ring-opening/reconstruction of anthranils with oxo-compounds: synthesis of quinoline derivatives. J. Org. Chem. 84, 12301–12313 (2019).
Arai, N. et al. Argadin, a new chitinase inhibitor, produced by Clonostachys sp.FO-7314. Chem. Pharm. Bull. (Tokyo) 48, 1442–1446 (2000).
Garg, R. P., Qian, X. L., Alemany, L. B., Moran, S. & Parry, R. J. Investigations of valanimycin biosynthesis: elucidation of the role of seryl-tRNA. Proc. Natl Acad. Sci. USA 105, 6543–6547 (2008).
Sato, M. et al. Involvement of lipocalin-like CghA in decalin-forming stereoselective intramolecular [4+2] cycloaddition. ChemBioChem 16, 2294–2298 (2015).
Yan, Y. et al. Resistance-gene-directed discovery of a natural-product herbicide with a new mode of action. Nature 559, 415–418 (2018).
Ng, L. T., Pascaud, A. & Pascaud, M. Hydrochloric acid hydrolysis of proteins and determination of tryptophan by reversed-phase high-performance liquid chromatography. Anal. Biochem. 167, 47–52 (1987).
Ekanayake, D. I. et al. Broomeanamides: cyclic octapeptides from an isolate of the fungicolous ascomycete Sphaerostilbella broomeana from India. J. Nat. Prod. 84, 2028–2034 (2021).
Cacho, R. A. & Tang, Y. Reconstitution of fungal nonribosomal peptide synthetases in yeast and in vitro. in: Nonribosomal Peptide and Polyketide Biosynthesis: Methods and Protocols (ed Evans, B. S.) 103–119 (Springer, 2016).
Hang, L. et al. Reversible product release and recapture by a fungal polyketide synthase using a carnitine acyltransferase domain. Angew. Chem. 129, 9684–9688 (2017).
Collart, M. A. & Oliviero, S. Preparation of yeast RNA. Curr. Protoc. Mol. Biol. 23, 13.12.1–13.12.5 (1993).
Ohashi, M. et al. SAM-dependent enzyme-catalysed pericyclic reactions in natural product biosynthesis. Nature 549, 502–506 (2017).
Janssen, B. D., Diner, E. J. & Hayes, C. S. Analysis of aminoacyl- and peptidyl-tRNAs by gel electrophoresis. Methods Mol. Biol. 905, 291–309 (2012).
Petrov, A., Tsa, A. & Puglisi, J. D. Analysis of RNA by analytical polyacrylamide gel electrophoresis. in: Methods in Enzymology (ed Lorsch, J.) 301–313 (Academic Press, 2013).
This work was supported by National Institutes of Health grant R35GM118056 to Y.T. D.A.Y. is supported by the UCLA Dissertation Year Fellowship. K.N. is supported by an overseas postdoctoral fellowship from the Uehara Memorial Foundation in Japan.
Y.T. is shareholder of Hexagon Bio.
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Extended Data Fig. 1 QTOF analysis of extracts from the expression of ankA-D in A. nidulans.
Traces include ankABCD (ii), ankABC (iii), ankAB (iv), ankA (v), and the empty vector control (i), retention time 4.5–5.5 min. Ion-extracted traces correspond to the [M + H]+ for 5 ([M + H]+ = 435), 6 ([M + H]+ = 334), and 7 ([M + H]+ = 318).
Extended Data Fig. 2 QTOF analysis of extracts from heterologous expression of RCDPS from different fungal species.
Traces include expression of ankA, pthA, ateA, amaA, avaA, anoA, and eshA in A. nidulans (ii-viii) compared to the empty vector control (i). Ion-extracted traces correspond to the [M + H]+ for 8 ([M + H]+ = 320), 9 ([M + H]+ = 272), 10 ([M + H]+ = 286), 11 ([M + H]+ = 254), 12 ([M + H]+ = 270), and 13 ([M + H]+ = 343).
Extended Data Fig. 3 RCSB PDB pairwise structural alignment of the predicted structure of AvaA to the bacterial CDPS AlbC S37C mutant.
The AlphaFold59 structural prediction of AvaA is colored blue and AlbC S37C mutant (4Q24)56 is colored brown. The dark blue and dark brown portions correspond to regions with similar structure, whereas the gray (AvaA) and beige (AlbC) correspond to regions with low similarity. (A) Orientation that highlights the similar core Rossmann fold. (B) Orientation that that highlights the additional folds in AvaA not present in AlbC. (C) Sequence alignment from the RCSB PDB pairwise structural alignment between AlbC and AvaA. Aligned active site residues are colored red. (D) Overlay of AlbC S37C mutant (gray) and AvaA model (purple) generated by AlphaFold. C193 in AvaA aligned to S37C in AlbC, while D428 aligned to Y202 in AlbC. S37 and Y202 are the catalytic residues in AlbC. In the structure of AlbC S37C (4Q24), the cysteine is covalently modified with a substrate analogue which can be seen in the enzyme active site. (E) Comparison of active site architecture of AlbC and AvaA. Using cysteine rather than serine for acyl intermediate formation in AvaA is likely to provide advantages such as kinetic enhancement, as well as reducing competing solvolysis by bulk solvent. This has been demonstrated in thioesterases of polyketide synthases and NRPSs, exemplifying a strategy of convergent evolution across different enzymes. Y392 in AvaA, equivalent to E182 in AlbC, may deprotonate the positively charged amino group of the acyl acceptor in preparation for formation of the first amide bond. Given the aligned position of D428 and Y202, it is likely that once the dipeptidyl intermediate forms, D428 is responsible for deprotonating the positively charged amino group of the acyl donor to attack the C193-thioester intermediate and form the diketopiperazine ring. The exact mechanism of RCDPS requires X-ray crystal structure validation.
Extended DataFig. 4 QTOF analysis of extracts from expression of ava pathway genes in A. nidulans.
Ion-extracted traces correspond to the [M + H]+ for 13 ([M + H]+ = 343), 14 ([M + H]+ = 359), 15 ([M + H]+ = 317), 16 ([M + H]+ = 347), 17 ([M + H]+ = 345), 18 ([M + H]+ = 387), and 24 ([M + H]+ = 389). Structures are shown in Supplementary Fig. 7.
Supplementary Tables 1–20 and Supplementary Figs. 1–93
Supplementary Data 1
Table of oligo sequences
Supplementary Data 2
Unmodified gels for Supplementary Figs. 8 and 18
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Yee, D.A., Niwa, K., Perlatti, B. et al. Genome mining for unknown–unknown natural products. Nat Chem Biol (2023). https://doi.org/10.1038/s41589-022-01246-6