Post on 12-Jan-2016
description
David Hopwood
Lecture 2
(DH2)
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
Chain length control by Type II ketosynthases
R S
O
KS
act KS-CLF(C16)
HO
O
S
O
ACP
S
OOOO
O R R
O ACP
tcm KS-CLF(C20)
S
OOOO
O O O
O ACP
R R
ACP
ACPtcm KS-act CLF
act KS-tcm CLF no product!
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”
Role of the chain length factor
CLFKS
18 Åchannel
lid
Role of the chain length factor
Act (C16) DYDMGVVTANACGGFDFTHREFRKLWSEGPKSVSVYESFAWFYAVNTGQI 144Fren (C16/18) EYGASAVTSNATGGFEFTHREIRKLWTEGPARVSVYESFAWFYAVNTGQI 161Tcm (C20) EYGLGVLTAAGAGGFEFGQREMQKLWGTGPERVSAYQSFAWFYAVNTGQI 148Dps (C20) PLEAGVITASASGGFAFGQRELQNLWSKGPAHVSAYMSFAWFYAVNTGQI 166R1128 (C20) DYSMGVVTSSAIGGFEFTHGEVHKLWTKGPQHVSVYESFAWFYAVNTGQL 152Gris (C20) ANGMGVVTAAGSGGFEFGERELRKLWSLGANHVSAYQSFAWFPTANTGQI 153WhiE (C24) PFGIGVVTAAGSGGGEFGQRELQRLWGQGPRFVGPYQSIAWFYAASTGQI 152
Role of the chain length factorin chain length control
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
ER
KR DH
ACPKS
AT
ER
KR DH
ACPKS
AT
Elongation moduleInitiation module
Unimodular and bimodular PKSs
ER
KR DH
ACPKS
AT
ER
KR DH
ACPKS
AT
Elongation module
Initiation moduleXInitiation module
X
KR
ACPKS AT
Actinorhodin biosynthesis bya unimodular polyketide synthase
AROCYC
CYC
DMAC
Actinorhodin
O
S-CoA-OOC
S-E
OOO
O O OO
O
S-E
OOO
O OHO
OO
S-EHO
OOO
O O
OH
O
S-E
OO
O O
OH
O
8 X
OH
OO
O
OH
O
O
OH
O
O
COOH
OH
OH
ACP
KS
ACP
KR DH ER
ACP
ACP KS
ACP ACP
ACP
AT
AT
O
S-CoA
O
-O
O
S
O
-O
O
S
O
R
O
S-CoA
O
-O7 X
O O O
O
O O O
S
R
O
Tailoring Steps OOH
HO
O
OH
R
R1128R=Me, Et, Pr, iPr
O
SR
O
SR
O
SR
O
S
O
-O SH
R
O
S-CoA
11 1
1
1
2
2 2
2
1
2
R1128 biosynthesis bya bimodular PKS
Initiation module
Elongation module
Initiation ketosynthases prefer initiation ACPs
0
200
400
600
800
0 2 4 6 8 10
Time (min)
Tu
rno
ver
of
[1-1
4C
]b-k
eto
pe
nta
no
yl-A
CP
ZhuG: 69 min-1
FrenJ: 7.6 min-1
GraACP: 4.0 min-1
Elongation ketosynthases prefer elongation ACPsAct KS/CLF:ACP titration
0
0.05
0.1
0.15
0.2
0.25
0.3
0 10 20 30 40 50
ACP Concentration (mM)
SE
K4/
SE
K4B
Tu
rno
ver
per
min
ute
FrenN
ZhuN
ZhuG, FrenJ
DpsG, Gra
ACP
AT KS
KR
ACP
ACP
ACPACP
AT KS
DHKR ER
O
S-CoA-OOC
S-
O
O O OO
O5 X
2
2
2
2
R S
O
CoA
O
S-R1
R
S
O
O O OHO
O
R
HO
O
O
OH
O
R
1
11
2
Recombining initiation and elongation modulesR1128 initiation module + octaketide synthase
ACP
AT KS
KR
ACP
ACP
ACPACP
AT KS
O
S-CoA-OOC7 X
2
2
2
2
R S
O
CoA
O
S-R1
1
1
S-
OOO
O O OO
O
R
S
OOO
O O OHO
O
R
O
OHO
O
HO
OH
R
2
1
ERKR DH
Recombining initiation and elongation modulesR1128 initiation module + decaketide synthase
Part 2
PKS gene synthesis and morphingof modular Type I PKSs
KOSANKOSAN BiosciencesBiosciences
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
PK
MM-CoA
PKSsfp
PKSSH
Prop-CoA
accA2 pccB
Propionate +CoA +ATP
PK
Propionate
prpE
NADPH NADP
MM-CoA
PKSsfp
PKSSH
Prop-CoA
accA2 pccB
Propionate +CoA +ATP
PK
Propionate
prpE
NADPH NADP
~1g/L of 6dEB!
E. coli as host for polyketide biosynthesis
One letter code for 2-C extensions added by modules in database
I GHO
O O
OH
OH
OH OH
OH OH
E B A
F D C
N
M
L K J
O
O
O
OH
OH
OH
OH OH O OH
OH
OOH
1
A D G J D D
6dEB
The ‘code’ for erythromycin
OH OH O OH
N J D D J G B N
Target polyketide: dissect structure to define necessary modules
Target sequence: N J D D J G B N
Pikromycin A N G J D H
Erythromycin A D G J D D
Erythromycin A D G J D D
Pikromycin A N G J D H
Rifamycin B I U M’ D A G B N
Target sequence: N J D D J G B N
Pikromycin A N G J D H
Erythromycin A D G J D D
Erythromycin A D G J D D
Pikromycin A N G J D H
Rifamycin B I U M’ D A G B N
Target sequence: N J D D J G B N
Pikromycin A N G J D H
Erythromycin A D G J D D
Erythromycin A D G J D D
Pikromycin A N G J D H
Rifamycin B I U M’ D A G B N
Target sequence:Target sequence: NN JJ DD DD JJ GG BB NN
PikromycinPikromycin AA NN GG JJ DD HH
ErythromycinErythromycin AA DD GG JJ DD DD
ErythromycinErythromycin AA DD GG JJ DD DD
PikromycinPikromycin AA NN GG JJ DD HH
RifamycinRifamycin BB II UU M’M’ DD AA GG BB NN
OH OH O OH
Obtain functional hybrid interfaces to connect modules
Input:Input: PKS module sequence PKS module sequence
•Optimize and randomize codon usageOptimize and randomize codon usage
•Automated restriction site assignmentAutomated restriction site assignment
•Avoid secondary structures in RNAAvoid secondary structures in RNA
•Optimized oligo overlap specificityOptimized oligo overlap specificity
Output:Output: overlapping oligos 40mers overlapping oligos 40mers
GEMS software
Jayaraj, S. et al. (2005) Nucleic Acids Res. 33: 3011
~500-800 bp
Synthon
40mer oligos
Assemble, amplify
Error rate ~2 per 1,000 bp
Synthon stitching
~5,000 bp DNASyn1 Syn2 Syn3 SynX
Completely automated
Fast and accurate gene synthesis
Generic module design
KS DH DH
AT KR ER ER
ACP LI
eryM1 PVAeryM2 PIAeryM6 PIAsorM6 PIAepoM7 PIAeryM5 PIAgelM3 PVAlepM10 PIArifM5 PIArapM2 PLAepoM3 PIAeryM3 PIArapM3 PLApikM6 PMADesign PIA
SpeI MfeI
GTGTGTGTGTGTGTGTGTGTGTGTGTGTGT
KpnI
RVWLERFWLLRYWLARFWLDRCWIERYWLPHYWLTRFWLEHFWLSRYWLERYWIERYWLQRYWLRSYWIS
LQ
PstI
TGTGTGTGTGTGTGTGTGTGTGTGTG**TG
AgeI
LAAELGGLDAWLGTIGQQIDSLRDSLAHLLTDVLKYLERLVGIRTELGHIDGELFALRDELGGLGELIFTLLSQALELRARLVGL-DELFTISDELAE
SS
XbaI/SpeI
Alignment of 150 modules revealed conserved sequences at borders
Generic module design
Synthetic PKS building blocks
Current collectionLM = loading module 4LI = intrapeptide linker 40LN = N-terminal linker module 40LC = C-terminal linker 40TE = thioesterase 3
Bimodular test system
17 donor X 17 acceptor modules = 289 bimodules
47% gave TKL product
AT
AT KR AT KR
Donor AcceptorLMery
SO
SO
HO
HO
SO
HO
TE
TEery
ACPACP ACPKS KS
O
OH
OSO
HO
SO
TKL
LC LNLI
LI: Intrapeptide linker
LC: C- terminal Interpeptide linker
LN: N- terminal interpeptide linker
Bimodular test system
6x6=36 polyketides expected from the 289 bimodular PKSs
O
R1
R2
R4 R3
R5
R6
O O
R1
R2
R4 R3
R5
R6
O
A-A: R1, R5=Me; R2, R3, R6=H; R4=OHA-D: R2, R5=Me; R1, R4, R6=H; R3=OHA-E: R5=Me; R1, R2, R3, R6=H; R4=OHE-A: R1=Me; R2, R3, R5, R6=H; R4=OHE-D: R2=Me; R1, R4, R5, R6=H; R3=OHF-D: R2=Me; R1, R4, R5, R6=H; R3=OHE-E: R1, R2, R3, R5, R6=H; R4=OHF-E: R1, R2, R3, R5, R6=H; R4=OH
D-A: R1, R6=Me; R2, R3, R5=H; R4=OHD-D: R2, R6=Me; R1, R4, R5=H; R3=OHD-E: R6=Me; R1, R2, R3, R5=H; R4=OHD-F: R6=Me; R1, R2, R3, R5=H; R4=OHA-F: R6=Me; R1, R2, R3, R5=H; R4=OHF-A: R2=Me; R1, R4, R5, R6=H; R3=OHE-F: R6=Me; R1, R2, R3, R5=H; R4=OHF-F: R6=Me; R1, R2, R3, R5=H; R4=OH
O
R1
R2R3
R4
O
O
A-H: R2, R3=Me; R1, R4=HA-G: R1, R3=Me; R2, R4=HE-G: R1=Me; R2, R3, R4=HE-H: R2=Me; R1, R3, R4=H
O
R1
R2R3
R4
O
O
D-G: R1, R4=Me; R2, R3=HD-H: R2, R4=Me; R1, R3=HF-G: R2, R4=Me; R1, R3=HF-H: R2, R4=Me; R1, R3=H
COOH
O
R1 R2 R3 R4
R5 R6
COOH
O
R1 R2 R3 R4
O
G-A: R2, R3=Me; R1, R4, R5=H , R6=OH G-D: R1, R3=Me; R2, R4, R5=H, R6=OH G-E: R1=Me; R2, R3, R4, R5=H, R6=OH G-F: R2=Me; R1, R3, R4, R5=H, R6=OH H-A: R2, R3=Me; R1, R4, R5=H , R6=OH H-D: R1, R3=Me; R2, R4, R5=H, R6=OH H-E: R1=Me; R2, R3, R4, R5=H, R6=OH H-F: R2=Me; R1, R3, R4, R5=H, R6=OH
G-G: R2, R3=Me; R1, R4=H G-H: R1, R3=Me; R2, R4=HH-G: R1=Me; R2, R3, R4=H H-H: R2=Me; R1, R3, R4=H
TKLs from bimodular tests
264 unnatural PKSs tested, 118 active (45%)
LN
-ep
oM
3-T
E
LN
-fo
sM
4-T
E
LN
-pik
M6
-TE
LN
-ery
M3
-TE
LN
-ra
pM
3-T
E
LN
-le
pM
4-T
E
LN
-ra
pM
6-T
E
LM-eryM1-LC (A)
LM-eryM2-LC (D)
LM-eryM6-LC (D)
LM-sorM6-LC (D)
LM-epoM7-LC (D)
LM-eryM5-LC (D)
LM-gelM3-LC (D)
LM-lepM10-LC (D)
LM-rifM5-LC (D)
LM-rapM2-LC (E)
LM-epoM3-LC (E)
LM-fosM4-LC (F)
LM-pikM6-LC (H)
LM-eryM3-LC (G)
LM-rapM3-LC (G)
LM-lepM4-LC (G)
LM-rapM6-LC (G)
LN
-ery
M2
-TE
LN
-ery
M1
-TE
LN
-ery
M6
-TE
LN
-so
rM6
-TE
LN
-ep
oM
7-T
E
LN
-ery
M5
-TE
LN
-ge
lM3
-TE
LN
-le
pM
10
-TE
LN
-ra
pM
2-T
E
LN
-rif
M5
-TE
TKLs from bimodular tests
Rescuing inactive bimodules
Chandran, S. S. et al. (2006) Chemistry & Biology 13:469
LN
-ep
oM
3-T
E
LN
-fo
sM
4-T
E
LN
-pik
M6
-TE
LN
-ery
M3
-TE
LN
-ra
pM
3-T
E
LN
-le
pM
4-T
E
LN
-ra
pM
6-T
E
LM-eryM1-LC (A)
LM-eryM2-LC (D)
LM-eryM6-LC (D)
LM-sorM6-LC (D)
LM-epoM7-LC (D)
LM-eryM5-LC (D)
LM-gelM3-LC (D)
LM-lepM10-LC (D)
LM-rifM5-LC (D)
LM-rapM2-LC (E)
LM-epoM3-LC (E)
LM-fosM4-LC (F)
LM-pikM6-LC (H)
LM-eryM3-LC (G)
LM-rapM3-LC (G)
LM-lepM4-LC (G)
LM-rapM6-LC (G)
LN
-ery
M2
-TE
LN
-ery
M1
-TE
LN
-ery
M6
-TE
LN
-so
rM6
-TE
LN
-ep
oM
7-T
E
LN
-ery
M5
-TE
LN
-ge
lM3
-TE
LN
-le
pM
10
-TE
LN
-ra
pM
2-T
E
LN
-rif
M5
-TE
Rescuing inactive bimodules
ACP TEKRATKS
TEACPKRATKS
TEACPKRATKSACPKRATKS
ACPKRATKSLD
ACPKRATKSLD
eryM2 eryM3
eryM2 sorM6
gelM3
rifM5
eryM2
20 mg/L
(KSeryM3)Sor6
(KSeryM3)Gel3
(KSeryM3)Rif5
10 mg/L
5 mg/L
3 mg/L
0 mg/L
ACPKRATKSLD
O O
OH
O O
OH
O O
OH
Rescuing inactive bimodules
Rational design and assemblyof synthetic trimodular PKSs
Menzella, H. G. et al. (2007) Chemistry & Biology 14: 143
If If modAmodA - - modBmodB makes a product, makes a product,
and and modBmodB - - modCmodC makes a product, makes a product,
will will modAmodA - - modBmodB - - modCmodC make a product make a product ??
LM
LM
LM
TE
TE
TE
Rational assembly of trimodular PKSs
LN
-ep
oM
3-T
E
LN
-fo
sM
4-T
E
LN
-pik
M6
-TE
LN
-ery
M3
-TE
LN
-ra
pM
3-T
E
LN
-le
pM
4-T
E
LN
-ra
pM
6-T
E
LM-eryM1-LC (A)
LM-eryM2-LC (D)
LM-eryM6-LC (D)
LM-sorM6-LC (D)
LM-epoM7-LC (D)
LM-eryM5-LC (D)
LM-gelM3-LC (D)
LM-lepM10-LC (D)
LM-rifM5-LC (D)
LM-rapM2-LC (E)
LM-epoM3-LC (E)
LM-fosM4-LC (F)
LM-pikM6-LC (H)
LM-eryM3-LC (G)
LM-rapM3-LC (G)
LM-lepM4-LC (G)
LM-rapM6-LC (G)
LN
-ery
M2
-TE
LN
-ery
M1
-TE
LN
-ery
M6
-TE
LN
-so
rM6
-TE
LN
-ep
oM
7-T
E
LN
-ery
M5
-TE
LN
-ge
lM3
-TE
LN
-le
pM
10
-TE
LN
-ra
pM
2-T
E
LN
-rif
M5
-TE
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
Rational assembly of trimodular PKSs
O O
O
O H
O O
O
O H
O O
O
O H
O O
O
O H
Expected tetraketide products from 54 trimodular PKSs assembled
Rational assembly of trimodular PKSs
52 out of 54 trimodular PKSs active (96%)
LN
-ery
M2-
eryM
3-T
E
(D-G
)
LN
-ery
M2-
pik
M6-
TE
(D
-H)
LN
-ery
M2-
rap
M3-
TE
(D
-G)
LN
-ery
M2-
rap
M6-
TE
(D
-H)
LN
-ery
M5-
eryM
3-T
E
(D-G
)
LN
-ery
M5-
pik
M6-
TE
(D
-G)
LN
-ery
M6-
eryM
3-T
E
(D-G
)
LN
-ery
M6-
pik
M6-
TE
(D
-G)
LN
-ery
M6-
rap
M3-
TE
(D
-G)
LN
-ery
M6-
rap
M6-
TE
(D
-G)
+++ ++ ++ ++ + + + + + +
+ + + + + + + + + +
+ + + + nd + ++ ++ ++ ++
+ + + + + + + + + +
nd + + + + +
+ + + + + + + +
LM-eryM1-LC (A)
LM-eryM2-LC (D)
LM-eryM5-LC (D)
LM-eryM6-LC (D)
LM-sorM6-LC (D)
LM-gdmM3-LC (D)
Rational assembly of trimodular PKSs
Searching for the discodermolidePKS genes
Schirmer, A. et al. (2005) Appl. Env. Microbiol. 71: 4840
Discodermiadissoluta
Discodermolide
KS probe pool
A multimodular PKS
An abundant, simple PKS
A multimodular PKS gene cluster from Discodermia