David Hopwood Lecture 2 (DH2)

44
David Hopwood Lecture 2 (DH2)

description

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. - PowerPoint PPT Presentation

Transcript of David Hopwood Lecture 2 (DH2)

Page 1: David Hopwood Lecture 2 (DH2)

David Hopwood

Lecture 2

(DH2)

Page 2: 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

Page 3: David Hopwood Lecture 2 (DH2)

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!

Page 4: David Hopwood Lecture 2 (DH2)

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”

Page 5: David Hopwood Lecture 2 (DH2)

Role of the chain length factor

CLFKS

18 Åchannel

lid

Page 6: David Hopwood Lecture 2 (DH2)

Role of the chain length factor

Page 7: David Hopwood Lecture 2 (DH2)

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

Page 8: David Hopwood Lecture 2 (DH2)

(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

Page 9: David Hopwood Lecture 2 (DH2)

Unimodular and bimodular PKSs

ER

KR DH

ACPKS

AT

ER

KR DH

ACPKS

AT

Elongation moduleInitiation module

Page 10: David Hopwood Lecture 2 (DH2)

Unimodular and bimodular PKSs

ER

KR DH

ACPKS

AT

ER

KR DH

ACPKS

AT

Elongation module

Initiation moduleXInitiation module

X

Page 11: David Hopwood Lecture 2 (DH2)

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

Page 12: David Hopwood Lecture 2 (DH2)

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

Page 13: David Hopwood Lecture 2 (DH2)

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

Page 14: David Hopwood Lecture 2 (DH2)

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

Page 15: David Hopwood Lecture 2 (DH2)

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

Page 16: David Hopwood Lecture 2 (DH2)

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

Page 17: David Hopwood Lecture 2 (DH2)

Part 2

PKS gene synthesis and morphingof modular Type I PKSs

KOSANKOSAN BiosciencesBiosciences

Page 18: David Hopwood Lecture 2 (DH2)

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

Page 19: David Hopwood Lecture 2 (DH2)

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

Page 20: David Hopwood Lecture 2 (DH2)

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

Page 21: David Hopwood Lecture 2 (DH2)

O

O

O

OH

OH

OH

OH OH O OH

OH

OOH

1

A D G J D D

6dEB

The ‘code’ for erythromycin

Page 22: David Hopwood Lecture 2 (DH2)

OH OH O OH

N J D D J G B N

Target polyketide: dissect structure to define necessary modules

Page 23: David Hopwood Lecture 2 (DH2)

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

Page 24: David Hopwood Lecture 2 (DH2)

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

Page 25: David Hopwood Lecture 2 (DH2)

~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

Page 26: David Hopwood Lecture 2 (DH2)

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

Page 27: David Hopwood Lecture 2 (DH2)

Synthetic PKS building blocks

Current collectionLM = loading module 4LI = intrapeptide linker 40LN = N-terminal linker module 40LC = C-terminal linker 40TE = thioesterase 3

Page 28: David Hopwood Lecture 2 (DH2)

Bimodular test system

17 donor X 17 acceptor modules = 289 bimodules

47% gave TKL product

Page 29: David Hopwood Lecture 2 (DH2)

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

Page 30: David Hopwood Lecture 2 (DH2)

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

Page 31: David Hopwood Lecture 2 (DH2)

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

Page 32: David Hopwood Lecture 2 (DH2)

Rescuing inactive bimodules

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

Page 33: David Hopwood Lecture 2 (DH2)

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

Page 34: David Hopwood Lecture 2 (DH2)

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

Page 35: David Hopwood Lecture 2 (DH2)

Rational design and assemblyof synthetic trimodular PKSs

Menzella, H. G. et al. (2007) Chemistry & Biology 14: 143

Page 36: David Hopwood Lecture 2 (DH2)

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

Page 37: David Hopwood Lecture 2 (DH2)

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

Page 38: David Hopwood Lecture 2 (DH2)

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

Page 39: David Hopwood Lecture 2 (DH2)

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

Page 40: David Hopwood Lecture 2 (DH2)

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

Page 41: David Hopwood Lecture 2 (DH2)

Searching for the discodermolidePKS genes

Schirmer, A. et al. (2005) Appl. Env. Microbiol. 71: 4840

Page 42: David Hopwood Lecture 2 (DH2)

Discodermiadissoluta

Discodermolide

Page 43: David Hopwood Lecture 2 (DH2)

KS probe pool

A multimodular PKS

An abundant, simple PKS

Page 44: David Hopwood Lecture 2 (DH2)

A multimodular PKS gene cluster from Discodermia