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A genomics-guided approach for discovering and expressing cryptic metabolic pathways

Nature Biotechnology volume 21, pages 187–190 (2003)Cite this article

Abstract

Genome analysis of actinomycetes has revealed the presence of numerous cryptic gene clusters encoding putative natural products1,2. These loci remain dormant until appropriate chemical or physical signals induce their expression. Here we demonstrate the use of a high-throughput genome scanning method to detect and analyze gene clusters involved in natural-product biosynthesis. This method was applied to uncover biosynthetic pathways encoding enediyne antitumor antibiotics in a variety of actinomycetes. Comparative analysis of five biosynthetic loci representative of the major structural classes of enediynes reveals the presence of a conserved cassette of five genes that includes a novel family of polyketide synthase (PKS)3,4. The enediyne PKS (PKSE) is proposed to be involved in the formation of the highly reactive chromophore ring structure (or “warhead”) found in all enediynes3,4. Genome scanning analysis indicates that the enediyne warhead cassette is widely dispersed among actinomycetes. We show that selective growth conditions can induce the expression of these loci, suggesting that the range of enediyne natural products may be much greater than previously thought. This technology can be used to increase the scope and diversity of natural-product discovery.

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Figure 1: A diagrammatic view of the genome scanning method for high-throughput discovery of natural-product biosynthetic gene clusters.
Figure 2: Chemical structures of enediynes and genes involved in warhead formation.
Figure 3: Enediyne production measured by the BIA.

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Acknowledgements

We thank S. Mercure, V. Dodelet, and M. Piraee for helpful discussions and J. McAlpine for critical reading of the manuscript. B.S. is a recipient of a NSF CAREER Award (MCB9733938) and a NIH Independent Scientist Award (AI51689). Enediyne studies in the Shen lab are supported in part by NIH grant CA78747. Research in the Thorson lab is supported in part by NIH grants CA84347, GM58196, and AI52218. J.S.T. is an Alfred P. Sloan Fellow.

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Authors and Affiliations

  1. Ecopia BioSciences, Inc., 7290 Frederick Banting, Montreal, H4S 2A1, Quebec, Canada

    Emmanuel Zazopoulos, Kexue Huang, Alfredo Staffa, Brian O. Bachmann & Chris M. Farnet

  2. Division of Pharmaceutical Sciences, University of Wisconsin, Madison, 53706, WI

    Wen Liu, Koichi Nonaka, Joachim Ahlert, Jon S. Thorson & Ben Shen

  3. Laboratory for Biosynthetic Chemistry, University of Wisconsin, Madison, 53706, WI

    Joachim Ahlert & Jon S. Thorson

  4. Department of Chemistry, University of Wisconsin, Madison, 53706, WI

    Ben Shen

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  1. Emmanuel Zazopoulos
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Supplementary information

41587_2003_BFnbt784_MOESM1_ESM.pdf

Supplementary Fig. 1. Amino acid alignment of the PKSE family of proteins. Approximate boundaries of domains are indicated above the alignment; KS, ketosynthase domain; AT, acyl transferase domain; ACP, acyl carrier protein domain; KR, ketoreductase domain; DH, dehydratase domain; PPTE, 4'-phosphopantetheinyl transferase domain (described further in Fig. F); domain ?, possible domain. Key residues involved in cofactor binding or catalysis are highlighted in black (Kakavas et al. Identification and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis. J Bacteriol. 179, 7515-7522 (1997); Fisher et al. The X-ray structure of Brassica napus beta-keto acyl carrier protein reductase and its implications for substrate binding and catalysis. StructureFold Des. 8, 339-347 (2000)). (PDF 51 kb)

Supplementary Fig. 2. Amino acid alignment of the TEBC family of proteins. (PDF 21 kb)

41587_2003_BFnbt784_MOESM3_ESM.pdf

Supplementary Fig. 3. Amino acid alignment of the UNBL family of proteins. PSORT (Nakai K, and Horton P. PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci. 24, 34-36 (1999)) analysis predicts this family to be cytosolic with high confidence. (PDF 26 kb)

41587_2003_BFnbt784_MOESM4_ESM.pdf

Supplementary Fig. 4. Amino acid alignment of the UNBU family of proteins. PSORT analysis predicts this family to be integral membrane proteins with high confidence. The predicted transmembrane-spanning regions are highlighted. (PDF 31 kb)

41587_2003_BFnbt784_MOESM5_ESM.pdf

Supplementary Fig. 5. Amino acid alignment of the UNBV family of proteins. PSORT analysis predicts this family to be secreted proteins with high confidence. Fifteen of the sixteen UNBV members are predicted to have an N-terminal signal sequence and of these, only that from the 059A cassette is not predicted to be cleavable. (PDF 31 kb)

41587_2003_BFnbt784_MOESM6_ESM.pdf

Supplementary Fig. 6. Alignment of the PPTE domains of the warhead polyketide synthase with the Sfp 4'-phosphopantetheinyl transferase of B.subtilis. For the purpose of comparison, the amino acid numbering scheme of the Sfp protein is used to refer to corresponding residues in the PPTE domains. Predicted secondary structure elements found in the PPTE domains are shown above the alignments, while structural elements found in the X-ray crystal structure of the Sfp protein are shown below the alignments (Reuter et al. Crystal structure of the surfactin synthetase-activating enzyme Sfp: a prototype of the 4'-phosphopantetheinyl transferase superfamily. EMBO 18, 6823-6831 (1999). (PDF 182 kb)

Supplementary Methods. (PDF 91 kb)

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Zazopoulos, E., Huang, K., Staffa, A. et al. A genomics-guided approach for discovering and expressing cryptic metabolic pathways. Nat Biotechnol 21, 187–190 (2003). https://doi.org/10.1038/nbt784

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