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Genome mining for unknown–unknown natural products


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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Fig. 1: Reconstitution of the ank pathway from A. thermomutatus leads to new natural products.
Fig. 2: AnkA and homologs from fungi are RCDPSs.
Fig. 3: Biochemical characterization of AvaA.
Fig. 4: Heterologous expression of ava-tailoring enzymes from A. versicolor.

Data availability

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.


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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.

Author information

Authors and Affiliations



D.A.Y., M.C. and Y.T. developed the hypothesis and designed the study. D.A.Y. and M.C. performed bioinformatics analysis of the ank and ava gene clusters and additional RCDPS candidates. D.A.Y. and Y.L. constructed the heterologous expression plasmids and performed in vivo and in vitro studies. M.C. analyzed the structural predictions by AlphaFold and designed the mutagenesis experiment of AvaA. D.A.Y., B.P. and K.N. purified the compounds and performed NMR spectroscopy for structural elucidation. All authors analyzed and discussed the results and prepared the manuscript.

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Correspondence to Yi Tang.

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Y.T. is shareholder of Hexagon Bio.

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Nature Chemical Biology thanks Yasushi Ogasawara and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

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 information

Supplementary Information

Supplementary Tables 1–20 and Supplementary Figs. 1–93

Reporting Summary

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).

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