1. Greg Challis Department of Chemistry Lecture 2: Methods for experimental identification of cryptic biosynthetic gene cluster products Microbial Genomics and Secondary Metabolites Summer School, MedILS, Split, Croatia, 25-29 June 2007

2. Overview Overview of available approaches Identification of a S. coelicolor cryptic NRPS product prediction of properties, gene KO / metabolic profiling Identification of S. coelicolor cryptic type III PKS products gene KO / metabolic profiling Identification of a S. coelicolor cryptic terpene synthase product in vitro reconstiution

3. Overview of approaches Corre and Challis, Chem. Biol. (2007) 14, 7-9

4. Gene knockout / comparative metabolic profiling X wild type mutant Lautru, Deeth, Bailey and Challis, Nat. Chem. Biol. (2005) 1, 265-269 Song et al., J. Am. Chem. Soc. (2006), 128, 14754

5. Expression of pathway specific activator / comparative metabolic profiling host + activator host - activator Bergman et al., Nat. Chem. Biol. (2007) 3, 213-217

6. Heterologous gene expression / comparative metabolic profiling host host + genes host - genes Hornung et al., ChemBioChem (2007) 8, 757-766

7. Prediction of physicochemical properties predicted precursors hh Banskota et al., J. Antibiot., (2006) 59, 533-542

8. “Genomisotopic” approach labelled predicted precursor * * * * Gross et al. Chem. Biol. (2007) 14, 53-63

9. In vitro pathway reconstitution predicted precursors purified enzymes Lin, Hopson and Cane, J. Am. Chem. Soc. (2006) 128, 6022-6023

10. H. Vlamakis, P. Straight, M. Fischbach Addition of a soil metabolite to Streptomyces avermitilis induces it to produce a cryptic metabolite Ueda et al., J. Antibiotics, 2000 Diffusible compound from a soil organism induces another organism to generate a new antibiotic activity Supernatant of soil organism A (stimulating compound) Soil organism B (antibiotic producer) Paper discs containing extracts from the culture broth of organism A were placed adjacent to inoculated spots of organism B Organism B grew for 1 – 3 days Soft agar containing spores of Bacillus subtilis was overlain to indicate antibiotic production

11. Example 1: isolation of a novel cryptic NRPS product

12. A new S. coelicolor NRPS gene cluster cchAcchBcchH Flavin-dependent monooxygenase (cchB) Non-ribosomal peptide synthetase (cchH) Formyl-tetrahydrofolate-dependent formyl transferase (cchA) MbtH-like protein (cchK) Esterase (cchJ) Challis and Ravel FEMS Microbiol. Lett. (2000) 187, 111-114 Export functions Ferric-siderophore import cchJcchI

13. Prediction of substrates and possible products for the S. coelicolor cryptic NRPS Challis and Ravel FEMS Microbiol. Lett. (2000) 187, 111-114

14. Gene KO / comparative metabolic profiling targeting predicted properties cchH X Retention time / min Mutant Wild type A 435 / nm

15. Mass spectrometric analysis of coelichelin ESI-FTICR-MS ESI-MS-MS C 21 H 39 N 7 O 11

16. NMR analysis of Ga-coelichelin complex

17. 2D-NMR analysis of Ga-coelichelin complex HMBC ROESY

18. Molecular modelling of Ga-coelichelin

19. Structure of coelichelin

20. Assembly of a tetrapeptide by a trimodular NRPS cchHcchJ

21. Heterologous expression of the cch cluster in Streptomyces fungicidicus S. fungicidicus S. fungicidicus + cch cluster S.coelicolor M145 Lautru, Deeth, Bailey and Challis, Nat. Chem. Biol. (2005) 1, 265-269

22. Example 2: isolation of novel products of a cryptic iterative PKS

23. Archetypal type III PKS products from bacteria

24. Mechanism of 3,5-DHPA-CoA assembly by DpgA 3,5-DHPA-CoA Tseng, McLoughlin, Kelleher and Walsh Biochemistry (2004) 43, 970-980

25. Type III polyketide synthases

26. In vitro investigation of the products formed by Sco7221 from acyl thioesters + malonyl CoA Moore, Noel and coworkers, unpublished

27. Identification of a new S. coelicolor type III PKS products by genome mining sco7221 EIC 197  sco7221 EIC 183  sco7221 EIC 197 M145 EIC 183 M145 X

28. Structures of the products Germicidin A (MW = 196) Germicidin B (MW = 182) Isogermicidin B* (MW = 182) Isogermicidin A* (MW = 196) Germicidin C (MW = 182) Song, Barona-Gomez, Corre, Xiang, Udwary, Austin, Noel, Moore and Challis, J. Am. Chem. Soc. (2006), 128, 14754 Petersen, Zahner, Metzger, Freund and Hummel, J. Antibiot. (1993) 46, 1126-1138 Isogermicidin C* (MW = 182)

29. Biosynthetic origins of germicidin A in S. coelicolor EIC 202EIC 197

30. Proposed mechanism 1 for germicidin assembly

31. Proposed mechanism 2 for germicidin assembly

32. Heterologous expression of sco7221 in Streptomyces venezualae ISP5230 EIC 197 ISP5230 + sco7221 EIC 183 ISP5230 + sco7221 EIC 197 ISP5230 EIC 183 ISP5230 gcs (sco7221) P ermE*

33. Fatty acid biosynthesis in Streptomyces coelicolor and E. coli compared

34. Analysis of germicidin production in S. coelicolor YL/ecFabH EIC 197 YL/ecFabH EIC 183 YL/ecFabH EIC 197 M511 EIC 183 M511 fabD aac(3)IVfabCfabBoriTecfabH + P ermE*

35. Proposed mechanism for germicidin assembly

36. X-ray structure of germicidin synthase Ser Cys Gcs AcpP

37. Example 3: a novel product of a cryptic sesquiterpene synthase

38. Cryptic sesquiterpene synthases of S. coelicolor

39. In vitro investigation of the product formed by Sco5222 from farensyl pyrophosphate Lin, Hopson and Cane, J. Am. Chem. Soc. (2006) 128, 6022-6023 sco5222 overexpressed in E. coli with N-terminal His 6 and purified from CFE sco5222sco5223 Sesquiterpene synthase Cytochrome P-450

40. Conclusions Several different approaches for the identification of the products of cryptic biosynthetic gene clusters have been developed in recent years Several novel bioactive metabolites have been discovered from well- studied microbes by these approaches Activation of silent cryptic gene clusters is a challenge that awaits generic solutions Genome mining is a promising approach for new bioactive metabolite discovery