Download presentation
Presentation is loading. Please wait.
Published byImogene Sims Modified over 9 years ago
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
Similar presentations
© 2025 SlidePlayer.com. Inc.
All rights reserved.