1. David Hopwood Lecture 2 (DH2)

2. Part 1 Aspects of the programming of Type II PKSs (a) 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 act KS-tcm CLFno 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) DYDMGVVTANACGG F DF T HRE F RKLWSEGPKSVSVYES F AWFYAVNTGQI 144 Fren (C16/18) EYGASAVTSNATGG F EF T HRE I RKLWTEGPARVSVYES F AWFYAVNTGQI 161 Tcm (C20) EYGLGVLTAAGAGG F EF G QRE M QKLWGTGPERVSAYQS F AWFYAVNTGQI 148 Dps (C20) PLEAGVITASASGG F AF G QRE L QNLWSKGPAHVSAYMS F AWFYAVNTGQI 166 R1128 (C20) DYSMGVVTSSAIGG F EF T HGE V HKLWTKGPQHVSVYES F AWFYAVNTGQL 152 Gris (C20) ANGMGVVTAAGSGG F EF G ERE L RKLWSLGANHVSAYQS F AWFPTANTGQI 153 WhiE (C24) PFGIGVVTAAGSGG G EF G QRE L QRLWGQGPRFVGPYQS I AWFYAASTGQI 152 Role of the chain length factor in 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. Unimodular and bimodular PKSs ER KR DH ACP KS AT ER KR DH ACP KS AT Elongation moduleInitiation module

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

11. KR ACP KS AT Actinorhodin biosynthesis by a unimodular polyketide synthase ARO CYC DMAC Actinorhodin

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

13. Initiation ketosynthases prefer initiation ACPs

14. Elongation ketosynthases prefer elongation ACPs

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

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

25. ~500-800 bp Synthon 40mer oligos Assemble, amplify Error rate ~2 per 1,000 bp Synthon stitching ~5,000 bp DNA Syn1Syn2 Syn3 SynX Completely automated Fast and accurate gene synthesis

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

27. Synthetic PKS building blocks Current collection LM = loading module 4 LI = intrapeptide linker40 LN = N-terminal linker module40 LC = C-terminal linker40 TE = thioesterase 3

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

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

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

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

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

33. Rescuing inactive bimodules

34. ACPTEKRATKS TEACPKRATKS TEACPKRATKS ACPKRATKS ACPKRATKS LD ACPKRATKS LD eryM2eryM3 eryM2sorM6 gelM3 rifM5 eryM2 20 mg/L (KS eryM3 )Sor6 (KS eryM3 )Gel3 (KS eryM3 )Rif5 10 mg/L 5 mg/L 3 mg/L 0 mg/L ACPKRATKS LD Rescuing inactive bimodules

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

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

38. 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 Rational assembly of trimodular PKSs

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

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

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

42. Discodermia dissoluta Discodermolide

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

44. A multimodular PKS gene cluster from Discodermia