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David Hopwood Lecture 2 (DH2)

David Hopwood Lecture 2 (DH2). Part 1 Aspects of the programming of Type II PKSs Chain length control Tang, Tsai & Khosla (2003) JACS 125: 12708 Keatings-Clay, A. T. et al . (2004) Nature Struct. Biol. 11: 888. Chain length control by Type II ketosynthases. ACP. tcm KS -act CLF. ACP.

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David Hopwood Lecture 2 (DH2)

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  1. David Hopwood Lecture 2 (DH2)

  2. Part 1 • Aspects of the programming of • Type II PKSs • Chain length control • Tang, Tsai & Khosla (2003) JACS 125: 12708 • Keatings-Clay, A. T. et al. (2004) Nature Struct. Biol. 11: 888

  3. Chain length control by Type II ketosynthases ACP tcm KS-act CLF ACP act KS-tcm CLF no product!

  4. McDaniel, Ebert-Khosla, Hopwood, Khosla (1993) Science 262: 1546 “Engineered Biosynthesis of Novel Polyketides” “The CLF (perhaps in conjunction with the KS) could provide a water-excluding pocket with appropriate molecular dimensions … for the nascent polyketide chain”

  5. Role of the chain length factor CLF KS 18 Å channel lid

  6. Role of the chain length factor

  7. Act (C16) DYDMGVVTANACGGFDFTHREFRKLWSEGPKSVSVYESFAWFYAVNTGQI 144 Fren (C16/18) EYGASAVTSNATGGFEFTHREIRKLWTEGPARVSVYESFAWFYAVNTGQI 161 Tcm (C20) EYGLGVLTAAGAGGFEFGQREMQKLWGTGPERVSAYQSFAWFYAVNTGQI 148 Dps (C20) PLEAGVITASASGGFAFGQRELQNLWSKGPAHVSAYMSFAWFYAVNTGQI 166 R1128 (C20) DYSMGVVTSSAIGGFEFTHGEVHKLWTKGPQHVSVYESFAWFYAVNTGQL 152 Gris (C20) ANGMGVVTAAGSGGFEFGERELRKLWSLGANHVSAYQSFAWFPTANTGQI 153 WhiE (C24) PFGIGVVTAAGSGGGEFGQRELQRLWGQGPRFVGPYQSIAWFYAASTGQI 152 Role of the chain length factorin chain length control Critical residues in the channel are smaller for longer carbon chains

  8. (b) Unimodular and bimodular Type II PKSs Tang, Y. et al. (2003) Biochemistry 42: 6588 Tang, Y. et al. (2004) Public Library of Science Biology 2:227 Tang, Y. et al. (2004) Biochemistry 43: 9546

  9. ER ER DH DH KR KR ACP ACP AT AT Unimodular and bimodular PKSs KS KS Initiation module Elongation module

  10. ER ER DH DH KR KR ACP ACP AT AT Unimodular and bimodular PKSs KS KS X Elongation module X Initiation module Initiation module

  11. O O 8 X - O O C S - C o A O H ARO KR CYC CYC O H O O O O O O O O O ACP O O O O O O O O O O O O O O O O H O O S - E S - E H O S - E S - E O H O H O O H O O C O O H AT O O O H O H O Actinorhodin biosynthesis by a unimodular polyketide synthase KS DMAC Actinorhodin

  12. R1128 biosynthesis by a bimodular PKS Initiation module 1 1 1 1 1 Elongation module 2 1 2 2 2 2

  13. Initiation ketosynthases prefer initiation ACPs

  14. Elongation ketosynthases prefer elongation ACPs

  15. Recombining initiation and elongation modules R1128 initiation module + octaketide synthase 2 1

  16. ER DH KR Recombining initiation and elongation modules R1128 initiation module + decaketide synthase

  17. Part 2 PKS gene synthesis and morphing of modular Type I PKSs KOSAN Biosciences

  18. Requirements for PKS gene synthesis and morphing • E. coli as expression host • Pfeifer, B. A. et al. (2001) Microbiol. Mol. Biol. Rev. 65: 106 • Rapid gene synthesis, e.g. ~32 kb DEBS cluster • Kodumal, S. J. (2004) PNAS 101: 15573 • Synthetic PKS building blocks • Combinatorial biosynthesis of novel polyketides • Menzella, H. G. et al. (2005) Nat. Biotech. 23: 1171

  19. PK ~1g/L of 6dEB! E. coli as host for polyketide biosynthesis

  20. One letter code for 2-C extensions added by modules in database

  21. AD G JDD 6dEB The ‘code’ for erythromycin

  22. N J D D J G B N Target polyketide:dissect structure to define necessary modules

  23. Obtain functional hybrid interfaces to connect modules

  24. GEMS software • Input: PKS module sequence • Optimize and randomize codon usage • Automated restriction site assignment • Avoid secondary structures in RNA • Optimized oligo overlap specificity • Output: overlapping oligos 40mers Jayaraj, S. et al. (2005) Nucleic Acids Res. 33: 3011

  25. 40mer oligos Assemble, amplify ~500-800 bp Synthon Error rate ~2 per 1,000 bp Synthon stitching ~5,000 bp DNA SynX Syn3 Syn1 Syn2 Completely automated Fast and accurate gene synthesis

  26. Generic module design Generic module design Alignment of 150 modules revealed conserved sequences at borders

  27. Synthetic PKS building blocks Current collection LM = loading module 4 LI = intrapeptide linker 40 LN = N-terminal linker module 40 LC = C-terminal linker 40 TE = thioesterase 3

  28. Bimodular test system 17 donor X 17 acceptor modules = 289 bimodules 47% gave TKL product

  29. Bimodular test system LI: Intrapeptide linker LC: C- terminal Interpeptide linker LN: N- terminal interpeptide linker

  30. TKLs from bimodular tests 6x6=36 polyketides expected from the 289 bimodular PKSs

  31. TKLs from bimodular tests 264 unnatural PKSs tested, 118 active (45%)

  32. Rescuing inactive bimodules Chandran, S. S. et al. (2006) Chemistry & Biology 13:469

  33. Rescuing inactive bimodules

  34. LD LD LD KS KS KS AT AT AT KR KR KR ACP ACP ACP KS AT KR ACP TE KS AT KR ACP eryM2 eryM3 20 mg/L KS AT KR ACP TE eryM2 sorM6 gelM3 rifM5 0 mg/L KS AT KR ACP TE eryM2 (KSeryM3)Sor6 (KSeryM3)Gel3 (KSeryM3)Rif5 10 mg/L 5 mg/L 3 mg/L Rescuing inactive bimodules

  35. Rational design and assembly of synthetic trimodular PKSs Menzella, H. G. et al. (2007) Chemistry & Biology 14: 143

  36. TE LM TE LM LM TE Rational assembly of trimodular PKSs If modA - modB makes a product, and modB - modC makes a product, will modA - modB - modC make a product?

  37. Rational assembly of trimodular PKSs

  38. Rational assembly of trimodular PKSs 54 A-B-C trimodular PKSs assembled , with A-B and B-C active as bimodules e.g.: pairs sor6-ery5 and ery5-rap3 are active, so sor6-ery5-rap3 is tested

  39. O O O H O H O O O O O O O H O H O O O O Rational assembly of trimodular PKSs Expected tetraketide products from 54 trimodular PKSs assembled

  40. Rational assembly of trimodular PKSs 52 out of 54 trimodular PKSs active (96%)

  41. Searching for the discodermolide PKS genes Schirmer, A. et al. (2005) Appl. Env. Microbiol. 71: 4840

  42. Discodermia dissoluta Discodermolide

  43. A multimodular PKS KS probe pool An abundant, simple PKS

  44. A multimodular PKS gene cluster from Discodermia

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