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Greg Challis Department of Chemistry

Lecture 1: Methods for in silico analysis of cryptic natural product biosynthetic gene clusters. Microbial Genomics and Secondary Metabolites Summer School, MedILS, Split, Croatia, 25-29 June 2007. Greg Challis Department of Chemistry. Overview. Introduction

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Greg Challis Department of Chemistry

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  1. Lecture 1: Methods for in silico analysis of cryptic natural product biosynthetic gene clusters Microbial Genomics and Secondary Metabolites Summer School, MedILS, Split, Croatia, 25-29 June 2007 Greg Challis Department of Chemistry

  2. Overview • Introduction • cryptic (orphan) gene clusters in microbial genomes • Clusters encoding nonribosomal peptide synthetases (NRPSs) • domains, modules,substrate specificity, predicting products • Clusters encoding modular polyketide synthases (PKSs) • domains, modules, substrate specificity, predicting products • Clusters encoding other biosynthetic systems • terpene synthases, iterative PKSs

  3. Introduction

  4. ‘Cryptic’ (orphan) biosynthetic gene clusters • Present in many of the 300 or so sequenced microbial genomes • e.g. Streptomyces avermitilis • Streptomyces coelicolor • Bacillus subtilis • Pseudomonas fluorescens • Pseudomonas syringae • Nostoc punctiforme • Aspergillus nidulans • Polyketide synthases • Nonribosomal peptide • synthetases • Terpene synthases • May prove a valuable new source of bioactive metabolites

  5. Genome sequence of the model antibiotic-producer Streptomyces coelicolor M145

  6. Gene clusters directing complex metabolite biosynthesis in the S. coelicolor genome Bentley et al. Nature (2002) 417, 141-147

  7. Part 1: Nonribosomal peptide synthetase analysis

  8. Recap of NRPS organisation and function: the gramicidin S synthetase as an example grsT grsA grsB synthetase 1 synthetase 2 module 2 module 4 module 1 module 3 module 5 A E C A C A C A C A TE PCP PCP PCP PCP PCP A = Adenylation PCP = peptidyl carrier protein C = Condensation E = Epimerisation TE = Thioesterase

  9. Recap of NRPS organisation and function: the gramicidin S synthetase as an example TE PCP TE For further information see Lars Robbel’s poster

  10. Nonribosomal peptide synthetases encoded by the S. coelicolor genome

  11. 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

  12. Deduced domain and module organization Prediction of domain and module structure Conserved Domain (CD) search (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)

  13. GrsADASVWEMFMALLTGASLYIILKDTINDFVKFEQYINQKEITVITLPPTYVVHL-----DPERILSIQTLITAGSATSPSLVNKWKEK--VTYINAYGPTETTIGrsADASVWEMFMALLTGASLYIILKDTINDFVKFEQYINQKEITVITLPPTYVVHL-----DPERILSIQTLITAGSATSPSLVNKWKEK--VTYINAYGPTETTI Ncs1-M1DIAVWELLAAFVGGARLVIAEHRLRGVVPHLPELMTDHRVTVAHFVPSVLEELLGWMADGGRVG-LRLVVCGGEAVPPSQRDRLLALSGARMVHAYGPTETTI GrsA D A W T I A A I Ncs1-M1 D I W H V G A I Prediction of A-domain selectivity pocket residues Stachelhaus, Mootz and Marahiel Chem. Biol. (1999) 6, 493-505 Challis, Ravel and Townsend Chem. Biol. (2000) 7, 211-224

  14. Empirical correlation between specificity pocket residues and substrate Challis, Ravel and Townsend Chem. Biol. (2000) 7, 211-224

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

  16. Part 2: Modular polyketide synthase analysis

  17. Recap of modular PKS organisation and function: the erythromycin synthase as an example • Three large modular enzymes (DEBS 1-3), encoded by eryAI, eryAII, and eryAIII, assemble 6-DEB • Each module performs one chain extension

  18. -CO2 Recap of modular PKS organisation and function: the erythromycin synthase as an example

  19. Recap of modular PKS organisation and function: the erythromycin synthase as an example • Three large modular enzymes (DEBS 1-3), encoded by eryAI, eryAII, and eryAIII, assemble 6-DEB • Each module performs one chain extension

  20. Gene clusters directing complex metabolite biosynthesis in the S. coelicolor genome Bentley et al. Nature (2002) 417, 141-147

  21. A new S. coelicolor modular PKS cluster Genes encoding a modular PKS

  22. Prediction of domain and modules in CpkA Conserved Domain (CD) search (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)

  23. Prediction of domain and modules in CpkB

  24. Prediction of domain and modules in CpkC

  25. Prediction of domains and modules in CpkABC Pawlik, Kotowska, Chater, Kuczek and Takano Arch. Microbiol. (2007) 187, 87-99

  26. Prediction of AT domain substrate selectivity Haydock et al.FEBS Lett. (1995) 374, 246-248 Banskota et al. J. Antibiot. (2006) 59, 168-176

  27. Prediction of KR domain stereoselectivity

  28. Prediction of KR domain stereoselectivity Caffrey ChemBioChem (2003) 4, 654-657 Reid et al. Biochemistry (2003) 42, 72-79

  29. Prediction of substrates and possible products for the S. coelicolor cryptic PKS

  30. Non-linear enzymatic logic can complicate things! Haynes and Challis, Curr. Op. Drug Discov. Develop. (2007) 10, 203-218

  31. Non-linear enzymatic logic can complicate things! Haynes and Challis, Curr. Op. Drug Discov. Develop. (2007) 10, 203-218

  32. Part 3: Analysis of other biosynthetic systems

  33. Terpene synthases

  34. Iterative polyketide synthases – type III PKSs

  35. Conclusions • Reasonably confident in silico predictions of domain / module organisation and substrate specificity of modular PKS / NRPS can be made • Non-linear enzymatic logic can complicate the reliable prediction of product structure(s) • For other types of biosynthetic system, reasonably confident predictions of substrate specificity can sometimes be made • Prediction of chain length and substrate specificity in some iterative PKS systems, especially type III and fungal type I, remains difficult

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