David Hopwood Lecture 2 DH 2 Part

David Hopwood Lecture 2 (DH 2)

Part 1 Aspects of the programming of Type II PKSs (a) Chain length control (b) Tang, Tsai & Khosla (2003) JACS 125: 12708 (c) 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 act KS-tcm CLF no product!

Mc. Daniel, 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”

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

Role of the chain length factor

Role of the chain length factor in chain length control Act Fren Tcm Dps R 1128 Gris Whi. E (C 16) (C 16/18) (C 20) (C 24) DYDMGVVTANACGG FDFTHREFRKLWSEGPKSVSVYES FAWFYAVNTGQI EYGASAVTSNATGG FEFTHREIRKLWTEGPARVSVYES FAWFYAVNTGQI EYGLGVLTAAGAGG FEFGQREMQKLWGTGPERVSAYQS FAWFYAVNTGQI PLEAGVITASASGG FAFGQRELQNLWSKGPAHVSAYMS FAWFYAVNTGQI DYSMGVVTSSAIGG FEFTHGEVHKLWTKGPQHVSVYES FAWFYAVNTGQL ANGMGVVTAAGSGG FEFGERELRKLWSLGANHVSAYQS FAWFPTANTGQI PFGIGVVTAAGSGG GEFGQRELQRLWGQGPRFVGPYQS IAWFYAASTGQI 144 161 148 166 152 153 152 Critical residues in the channel are smaller for longer carbon chains

(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

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

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

Actinorhodin biosynthesis by a unimodular polyketide synthase O 8 X -OOC S-Co. A KS OH ACP O O O O O AT KR S-E HO O O O O CYC S-E O O O HO O ARO O OH O O O S-E OH S-E CYC OH O COOH OH O O OH DMAC Actinorhodin

R 1128 biosynthesis by a bimodular PKS Initiation module 1 1 1 Elongation module 1 2 2 2

Initiation ketosynthases prefer initiation ACPs

Elongation ketosynthases prefer elongation ACPs

Recombining initiation and elongation modules R 1128 initiation module + octaketide synthase 2 1

Recombining initiation and elongation modules R 1128 initiation module + decaketide synthase KR DH ER

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

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

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

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

A D G J 6 d. EB The ‘code’ for erythromycin D D

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

Obtain functional hybrid interfaces to connect modules

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

Fast and accurate gene synthesis 40 mer oligos Assemble, amplify ~500 -800 bp Synthon Error rate ~2 per 1, 000 bp Synthon stitching Syn 1 Syn 2 Syn 3 Syn. X Completely automated ~5, 000 bp DNA

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

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

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

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

TKLs from bimodular tests 6 x 6=36 polyketides expected from the 289 bimodular PKSs

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

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

Rescuing inactive bimodules

Rescuing inactive bimodules LD KS AT KR ACP ery. M 2 LD KS AT KR ery. M 3 KR ACP ery. M 2 KS ACP TE 20 mg/L AT KR ACP TE sor. M 6 gel. M 3 0 mg/L rif. M 5 LD KS AT ery. M 2 KR ACP KS AT KR ACP TE (KSery. M 3)Sor 6 10 mg/L (KSery. M 3)Gel 3 5 mg/L (KSery. M 3)Rif 5 3 mg/L

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

Rational assembly of trimodular PKSs TE LM LM LM TE TE If mod. A - mod. B makes a product, and mod. B - mod. C makes a product, will mod. A - mod. B - mod. C make a product ?

Rational assembly of trimodular PKSs

Rational assembly of trimodular PKSs 54 A-B-C trimodular PKSs assembled , with A-B and B-C active as bimodules e. g. : pairs sor 6 -ery 5 and ery 5 -rap 3 are active, so sor 6 -ery 5 -rap 3 is tested

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

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

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

Discodermia dissoluta Discodermolide

An abundant, simple PKS A multimodular PKS KS probe pool

A multimodular PKS gene cluster from Discodermia