Greg Challis Department of Chemistry

1. 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 Greg Challis Department of Chemistry

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

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

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

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

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

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

10. Supernatant of soil organism A (stimulating compound) Soil organism B (antibiotic producer) Diffusible compound from a soil organism induces another organism to generate a new antibiotic activity • 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 Ueda et al., J. Antibiotics, 2000 Addition of a soil metabolite to Streptomyces avermitilis induces it to produce a cryptic metabolite H. Vlamakis, P. Straight, M. Fischbach

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

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

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

14. Mutant A435 / nm Wild type Retention time / min Gene KO / comparative metabolic profiling targeting predicted properties cchH X

15. Mass spectrometric analysis of coelichelin ESI-FTICR-MS C21H39N7O11 ESI-MS-MS

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 cchJ cchH

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

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. X EIC 197 M145 EIC 183 M145 EIC 197 Dsco7221 EIC 183 Dsco7221 Identification of a new S. coelicolor type III PKS products by genome mining sco7221

28. 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 Structures of the products Germicidin B (MW = 182) Germicidin C (MW = 182) Germicidin A (MW = 196) Isogermicidin B* (MW = 182) Isogermicidin C* (MW = 182) Isogermicidin A* (MW = 196)

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

30. Proposed mechanism 1 for germicidin assembly

31. Proposed mechanism 2 for germicidin assembly

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

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

34. EIC 197 M511 EIC 183 M511 EIC 197 YL/ecFabH EIC 183 YL/ecFabH Analysis of germicidin production in S. coelicolor YL/ecFabH PermE* + fabD oriT aac(3)IV fabC fabB ecfabH

35. Proposed mechanism for germicidin assembly

36. Ser Cys X-ray structure of germicidin synthase 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 sco5222 sco5223 Sesquiterpene synthase Cytochrome P-450 sco5222 overexpressed in E. coli with N-terminal His6 and purified from CFE Lin, Hopson and Cane, J. Am. Chem. Soc. (2006) 128, 6022-6023

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