Engineering biosynthetic

pathways to generate Universidad de Oviedo

antitumor compounds

César Sánchez
Departamento de Biología Funcional
e Instituto Universitario de Oncología del
Principado de Asturias (IUOPA)

University of Oviedo
Spain
 Antitumor natural products from actinomycetes
H
H3C H3C H3CO OH O N O
O HO O
HO O CH3
HO O
H
O OH OH
H3C O
O
OH OH O O N N
HO H
CH3 H3C Cl Cl
H3C O O
O HO O O
HO O
OH
H3C
O HO

OH
OH
MITHRAMYCIN OH O OH
OCH3
(inhibition of DNA replication/transcription)
Streptomyces argillaceus OVIEDOMYCIN REBECCAMYCIN
(induction of apoptosis) (inhibition of DNA topoisomerase I)
Streptomyces antibioticus Lechevalieria aerocolonigenes
OCH3
CH3 OOCCH3
O
HO CH3 OCH3 OH H
O H N O
O O CH3 CH3 CH3 CH3

O OH OH
H3C O
OH OH O OH O
H3C N N
H3C
H3C O
O HO
O O
NC H
HO O
O
CH3 COOH MeO
H3C
O
H3CCOO
NHCH3
HO

CHROMOMYCIN BORRELIDIN STAUROSPORINE

(inhibition of DNA replication/transcription) (inhibition of angiogenesis) (inhibition of protein kinases)
Streptomyces griseus Streptomyces parvulus Streptomyces longisporoflavus
 Indolocarbazoles

N N
H H

INDOLO[2,3-a]CARBAZOLE
 Indolocarbazoles as antitumor drug leads

H
N H
O O N O

N N
H N N
Cl Cl
O
O OH H3C H
HO H3CO
OH NHCH3
OCH3
STAUROSPORINE
REBECCAMYCIN (inhibition of protein kinases)
Produced by Streptomyces longisporoflavus
(inhibition of DNA topoisomerase I)
and other actinomycetes
Produced by Lechevalieria aerocolonigenes
 Indolocarbazole derivatives in clinical trials
OH H
Cancer N O
Parkinson’s disease
OH
HN H
N
N O
O O
N N S S
O
H3C H
HO N N OH N N
H H3CO O
H3C H
OH NCH3
O O HO
HO COOCH3
N(CH2CH3)2 OH
CEP-1347 (KT-7515)
OH
Midostaurin (PKC-412,
O N O Edotecarin 4’-N-benzoyl-staurosporine)
(J-107088) Diabetic retinopathy
H H
HO N O N H
O
N N O N O
H
Cl Cl
O OH
N N N N
HO N N
O O
OH H3C H H3C H
OCH3 H3CO HO
NHCH3 CH2OH O
Becatecarin N(CH3)2
(DEAE-rebeccamycin) UCN-01 CEP-701
(7-hydroxystaurosporine) (KT-5555) Ruboxistaurin (LY333531)

Butler (2005) Nat. Prod. Rep. 22, 162-195

 Building blocks for rebeccamycin and staurosporine

H2N COOH H
H
N N O
O O

N
H
Tryptophan N N
N N
H (2x)
Cl Cl O
O OH -
H3C H
Cl
O
HO Glucose NH
OH CH3
O CH3
H 3C Methionine

Meksuriyen & Cordell (1988) J. Nat. Prod. 51, 893-899

Pearce et al. (1988) J. Nat. Prod. 51, 937-940
 Structural differences = Opportunities for further diversity

REBECCAMYCIN STAUROSPORINE

7-oxo 7-deoxo
H H
O N O N O

Halogenation
N N N N
Cl H Cl Additional
linkage O
O OH H3C H
D-Glucose O 2,3,6-trideoxy-
HO NH L-sugar
OH CH3
O CH3
H3C
Methylation Amination
Methylations
 Gene cluster for rebeccamycin biosynthesis

1 kb From Lechevalieria aerocolonigenes ATCC39243

G O D C P M R F U H T

Indolocarbazole formation Sugar methylation

Halogenation H Resistance / Secretion
O N O
Sugar attachment Regulation

N N
Cl H Cl
O OH

HO
OH
O
H3C

C. Sánchez et al. (2002) Chem. Biol. 9, 519-531

Onaka et al. (2003) Biosci. Biotechnol. Biochem. 67, 127-138
Hyun et al. (2003) ChemBioChem 1, 114-117 ngt: Ohuchi et al. (2000) J. Antibiot. 53, 393-403
 Heterologous expression of reb genes in Streptomyces albus

G O D C P M R F U H T cosmid 14E8

G O D C P M R F U pREB7

H
p O D C P M pREB6 O N O

N N
Cl H Cl
O OH

HO
OH
O
H3C
rebeccamycin

C. Sánchez et al. (2002) Chem. Biol. 9, 519-531

 Heterologous expression of reb genes in Streptomyces albus

G O D C P M R F U H T cosmid 14E8

G O D C P M R F U pREB7

H
p O D C P M pREB6 O N O

H
O N O N N
Cl H Cl
H
O N O O OH

N N HO
H OH
N N O OH O
H H H3C
dideschloro-rebeccamycin HO rebeccamycin
OH
aglycone O
H 3C
dideschloro-
rebeccamycin
C. Sánchez et al. (2002) Chem. Biol. 9, 519-531
 Rebeccamycin biosynthesis: proposed pathway (I-III)

I. Tryptophan modification

NH2 NH2
HOOC HOOC
RebH
RebH

N N
H Cl H
L-tryptophan
 Rebeccamycin biosynthesis: proposed pathway (I-III)

II. Dimerization
I. Tryptophan modification
III. Ring closure

RebO H
NH2 NH2 N
HOOC HOOC RebD O O
RebH
RebH
x2
N N
H Cl H RebC N N
H H
L-tryptophan RebP Cl Cl
aglycone
 Rebeccamycin biosynthesis: proposed pathway (IV-V)

IV. Glycosylation V. Sugar modification

H
H N
N O O
O O

H
O N O N N
RebG N N RebM
RebG RebM Cl H Cl
Cl H Cl
OH
OH O
N N O
Cl H H Cl HO HO

OH OH
aglycone
OH O
CH3
rebeccamycin
Dissection and reconstitution of the rebeccamycin pathway

rebO

rebD

rebO rebD
- Multiple combinations of reb genes
rebO rebD rebC

rebO rebD rebP - Under the control of the ermE*p promoter

rebO rebD rebC rebP

rebD rebC rebP

- In shuttle vectors (pWHM3, pKC796)
rebO rebC rebP
- Expression in Streptomyces albus
rebO rebD rebC rebP rebG

rebO rebD rebC rebP rebG rebM - Identification of metabolites (HPLC-MS, NMR)
rebO rebD rebH

rebO rebD rebC rebP rebH

rebO rebD rebC rebP rebT rebG rebH

rebO rebD rebC rebP rebT rebG rebM rebH

C. Sánchez et al. (2005) PNAS 102, 461-466
 Dissection and reconstitution of the rebeccamycin pathway

Biosynthesis of indolocarbazole core and non-chlorinated rebeccamycin

H
HOOC NH2 RebO HOOC N COOH
RebD

(2 x)
N N N
H H H
L-tryptophan 1
chromopyrrolic
acid

C. Sánchez et al. (2005) PNAS 102, 461-466

 Dissection and reconstitution of the rebeccamycin pathway

Biosynthesis of indolocarbazole core and non-chlorinated rebeccamycin

H
HOOC NH2 RebO HOOC N COOH
RebD

(2 x)
N N N
H H H RebP
L-tryptophan 1
chromopyrrolic H H H
acid O N O N O HO N O

N
H
N
H
+ N
H
N
H
+ N
H
N
H
2 3 4
dideschloro- staurosporine 7-OH-staurosporine
rebeccamycin aglycone aglycone
aglycone

C. Sánchez et al. (2005) PNAS 102, 461-466

 Dissection and reconstitution of the rebeccamycin pathway

Biosynthesis of indolocarbazole core and non-chlorinated rebeccamycin

H
O N O
RebC
RebP N N
H H
2
H dideschloro-
HOOC NH2 RebO HOOC N COOH
RebD rebeccamycin
aglycone
(2 x)
N N N
H H H RebP
L-tryptophan 1
chromopyrrolic H H H
acid O N O N O HO N O

N
H
N
H
+ N
H
N
H
+ N
H
N
H
2 3 4
dideschloro- staurosporine 7-OH-staurosporine
rebeccamycin aglycone aglycone
aglycone

C. Sánchez et al. (2005) PNAS 102, 461-466

 Dissection and reconstitution of the rebeccamycin pathway

Biosynthesis of indolocarbazole core and non-chlorinated rebeccamycin

H H
O N O O N O
H
O N O
RebG RebM
N N N N
RebC H H
O OH O OH
RebP N N
H H HO HO
2 OH OH
OH OCH3
H dideschloro-
HOOC NH2 RebO HOOC N COOH 5 6
RebD rebeccamycin
aglycone dideschloro-
(2 x) rebeccamycin
N N N
H H H
RebP
L-tryptophan 1 H H H
O N O N O HO N O
chromopyrrolic
acid
+ +
N N N N N N
H H H H H H
2 3 4
dideschloro- staurosporine 7-OH-staurosporine
rebeccamycin aglycone aglycone
aglycone

C. Sánchez et al. (2005) PNAS 102, 461-466

 Dissection and reconstitution of the rebeccamycin pathway

Biosynthesis of rebeccamycin
H H
O N O O N O
H H
HOOC NH2 HOOC N COOH RebC O N O
RebO
RebD RebP RebG RebM
(x 2) N N N N
H Cl H Cl
N N N N N Cl Cl O OH
H H H H H O OH
Cl Cl R Cl R
HO HO
7-chlorotryptophan OH OH
7 R=H 9 R=H OH OCH3
8 R=Cl 10 R=Cl
11 12
RebH
demethyl- REBECCAMYCIN
rebeccamycin
HOOC NH2

N
H Here, co-expression of rebT
L-tryptophan was needed to obtain transformants

C. Sánchez et al. (2005) PNAS 102, 461-466

 Production of brominated derivatives

By replacing chloride with bromide in the fermentation medium

H H
O N O O N O
H H
HOOC NH2 HOOC N COOH RebC O N O
RebO
RebD RebP RebG RebM
(x 2) N N N N
H Br H Br
N N N N N Br Br O OH
H H H H H O OH
Br Br R Br R
HO HO
7-bromotryptophan OH OH
26 R=H 28 R=H OH OCH3
27 R=Br 29 R=Br
31 32
RebH 30 R=Cl
bromorebeccamycin
HOOC NH2

N
H
L-tryptophan

C. Sánchez et al. (2005) PNAS 102, 461-466

 Production of derivatives chlorinated at novel positions

PyrH, a tryptophan 5-halogenase from Streptomyces rugosporus LL-42D005

S. Zehner et al. (2005) Chem. Biol. 12, 445-452 OH
O O
O O

H2N O
HOOC NH2 HOOC NH2 O O
O
OH
PyrH Cl NH

OH
NH
N N O
H H N
COOH
O
H
L-tryptophan Cl pyrroindomycin B
5-chlorotryptophan

H
HOOC NH2 HOOC NH2 O N O
RebH

N N
N N Cl H Cl
H H O OH
Cl
HO
L-tryptophan OH
OCH3
7-chlorotryptophan
REBECCAMYCIN
 Production of derivatives chlorinated at novel positions

PyrH, a tryptophan 5-halogenase from Streptomyces rugosporus LL-42D005

S. Zehner et al. (2005) Chem. Biol. 12, 445-452 OH
O O
O O

NH2 NH2 H2N O

HOOC HOOC O
O
PyrH O
Cl OH

NH
N N OH
H H NH
O
COOH
L-tryptophan O N
5-chlorotryptophan Cl pyrroindomycin B H

Co-expression of reb genes and pyrH in Streptomyces albus

H H
HOOC NH2 HOOC NH2 HOOC N COOH RebC O N O
RebO
PyrH Cl RebD Cl R RebP Cl
(x 2)
N N N N N N
H H H H H H
L-tryptophan
5-chlorotryptophan 21 R=H 23
22 R=Cl
C. Sánchez et al. (2005) PNAS 102, 461-466
Gene clusters for biosynthesis of rebeccamycin and staurosporine

1 kb
reb G O D C P M R F U H T

sta R B A N G O D P MA J K I E MB C

REB O
H
N O
Indolocarbazole formation STA H
N O
Sugar attachment
Halogenation
N N N N
Cl H Cl Sugar biosynthesis O
O OH H 3C H
Resistance / Secretion
O
HO Regulation NH
OH CH3
O CH3
H3C

Identification of sta genes: From Streptomyces longisporoflavus DSM10189:

From Streptomyces sp. TP-A0274: - Schupp et al. (1997) WIPO Patent WO9708323
Onaka et al. (2002) J. Antibiot. 55, 1063-1071 - C. Sánchez et al. (2005) PNAS 102, 461-466
- A.P. Salas et al. (2005) Mol. Microbiol. in press
 C-7 oxidation state in indolocarbazoles
1 kb
reb G O D C P M R F U H T

sta R B A N G O D P MA J K I E MB C

Indolocarbazole formation
7-oxo Sugar attachment 7-deoxo H
H N
N O
O O Halogenation
Sugar biosynthesis
Resistance / Secretion
N N
N N Regulation
H O
Cl Cl
H3C H
O OH
H3CO
HO
NHCH3
OH
OCH3
 Dissection and reconstitution of the rebeccamycin pathway

Biosynthesis of indolocarbazole core and non-chlorinated rebeccamycin

H
O N O
RebC
RebP N N
H H
2
H dideschloro-
HOOC NH2 RebO HOOC N COOH
RebD rebeccamycin
aglycone
(2 x)
N N N
H H H RebP
L-tryptophan 1
chromopyrrolic H H H
acid O N O N O HO N O

N
H
N
H
+ N
H
N
H
+ N
H
N
H
2 3 4
dideschloro- staurosporine 7-OH-staurosporine
rebeccamycin aglycone aglycone
aglycone

C. Sánchez et al. (2005) PNAS 102, 461-466

 C-7 oxidation state is determined by RebC/StaC
Co-expression of reb genes and staCP genes in Streptomyces albus

rebO rebD H
HOOC N COOH
rebO rebD rebC = chromopyrrolic acid
rebD N N
rebO staC H H

rebO rebD rebP

rebO rebD staP

rebO rebD rebC rebP

rebO rebD rebC staP

rebO rebD staC rebP

rebO rebD staC staP

C. Sánchez et al. (2005) PNAS 102, 461-466

 C-7 oxidation state is determined by RebC/StaC
Co-expression of reb genes and staCP genes in Streptomyces albus

rebO rebD H
HOOC N COOH
rebO rebD rebC = chromopyrrolic acid
rebD N N
rebO staC H H

H H H
HO N O O N O N O
rebO rebD rebP

rebO rebD staP

N N N N N N
H H H H H H

rebO rebD rebC rebP

rebO rebD rebC staP

rebO rebD staC rebP

rebO rebD staC staP

C. Sánchez et al. (2005) PNAS 102, 461-466

 C-7 oxidation state is determined by RebC/StaC
Co-expression of reb genes and staCP genes in Streptomyces albus

rebO rebD H
HOOC N COOH
rebO rebD rebC = chromopyrrolic acid
rebD N N
rebO staC H H

H H H
HO N O O N O N O
rebO rebD rebP

rebO rebD staP

N N N N N N
H H H H H H

H
O N O
rebO rebD rebC rebP
= non-chlorinated rebeccamycin aglycone
rebO rebD rebC staP
N N
H H

H
N O
rebO rebD staC rebP
= staurosporine aglycone
rebO rebD staC staP
N N
H H

C. Sánchez et al. (2005) PNAS 102, 461-466

 Production of staurosporinone (= 7-deoxo) derivatives

Non-halogenated H
N O
H
N O
H
N O
rebO rebD staC rebP
N N N N
H H
O OH O OH
rebO rebD staC rebP rebG N N
H H HO HO
OH OH
3 OH OCH3
rebO rebD staC rebP rebG rebM
13 14

H H
N N
Halogenated O O

rebO rebD staC rebP rebH N N N N H

H H H N O
R Cl R Cl
O OH
15 R=H
rebO rebD staC rebP rebG rebH HO
16 R=Cl OH
N N
H OH H
N O R Cl
O OH
17 R=H
rebO rebD staC rebP rebG rebM rebH Cl HO
18 R=Cl OH
OCH3
N N
pyrH H H
rebO rebD staC rebP 19 R=H
24 20 R=Cl

C. Sánchez et al. (2005) PNAS 102, 461-466

 Sugar attachment and methylation in staurosporine
Using a two-plasmid system in Streptomyces albus H
N
O

Reconstitution of staurosporine biosynthesis:

N N
NHCH3
pKC39GNT rebO rebD staC rebP staG staN rebT
H3 C O
H3CO
pETAS staMA staJ staK staI staE staMB
STAUROSPORINE

A.P. Salas et al. (2005) Mol. Microbiol. in press

 Sugar attachment and methylation in staurosporine
Using a two-plasmid system in Streptomyces albus H
N
O

Reconstitution of staurosporine biosynthesis:

N N
NHCH3
pKC39GNT rebO rebD staC rebP staG staN rebT
H3 C O
H3CO
pETAS staMA staJ staK staI staE staMB
STAUROSPORINE

H
N
O

After deletion of staN:

pKC39GT rebO rebD staC rebP staG rebT N N

H

O
pETAS staMA staJ staK staI staE staMB
CH3
HO
NH2
HOLYRINE A

A.P. Salas et al. (2005) Mol. Microbiol. in press

 Sugar attachment and methylation in staurosporine
Feeding holyrine A to S. albus strains expressing sta genes

H
N
O

N N
H

CH3
HO
NH2

N N
HOLYRINE A NHCH3
H3 C O
staMA staN staMB
H3CO
STAUROSPORINE

A.P. Salas et al. (2005) Mol. Microbiol. in press

 Sugar attachment and methylation in staurosporine
Feeding holyrine A to S. albus strains expressing sta genes

N N
NH2
H3 C O
H
N HO
O

staN
N N
H

CH3
HO
NH2

N N
HOLYRINE A NHCH3
H3 C O
staMA staN staMB
H3CO
STAUROSPORINE

A.P. Salas et al. (2005) Mol. Microbiol. in press

 Sugar attachment and methylation in staurosporine
Feeding holyrine A to S. albus strains expressing sta genes

N N
staMA NO MODIFICATION
NH2
H3 C O
H
N staMB HO
O

N N
NHCH3
staN
N N H3 C O
H HO

O
staMA staN N N
CH3
NH2
HO H 3C O
NH2
H3CO
staN staMB
N N
HOLYRINE A NHCH3
H3 C O
staMA staN staMB
H3CO
STAUROSPORINE

A.P. Salas et al. (2005) Mol. Microbiol. in press

 Staurosporine biosynthesis
NH2 H
HOOC StaO HOOC
N
COOH
StaC H NDP-D-glucose
N
StaD StaP O

StaA StaI
(2x)
N N N StaB StaJ
H H H N
H
N
H
StaE StaK
L-Tryptophan Chromopyrrolic acid
K-252c
NDP-L-ristosamine
H
N
O
StaG

StaMA H
StaMB N
O
N N
H H
N NHCH3 N
O O
H 3C O
HO
N N
H
H
N N N N N
NHCH3
O
NH2
StaN O
H 3C O H3 C O
StaMA CH3
H3CO HO
HO
Staurosporine N N StaMB NH2
NH2
H3 C O
Holyrine A
H3CO
 Sugar substrate flexibility of StaG and StaN
H
Using a two-plasmid system in Streptomyces albus N
O

pKC39GNT rebO rebD staC rebP staG staN rebT

N N
NHCH3
pETAS staMA staJ staK staI staE staMB
H3C O
H3CO
STAUROSPORINE

Plasmids expressing genes for biosynthesis of different deoxysugars:

Rodríguez et al. (2000) J. Mol. Microbiol. Biotechnol. 2, 271-6; Fischer et al. (2002) J. Nat. Prod. 65, 1685-9
H3C O
pRHAM oleL oleS oleE oleU HO
L-rhamnose
HO
OH
pLN2 oleV oleW oleU oleY oleL oleS oleE
H3C O
L-olivose HO
pLNR oleV oleW urdR oleY oleL oleS oleE HO
OH

pLNBIV
L-digitoxose H3C O
oleV oleW eryBIV oleY oleL oleS oleE HO
CH3
O
D-olivose HO
ole urdR eryBIV HO

oleandomycin urdamycin erythromycin

S. antibioticus S. fradiae Saccharopolyspora erythraea
 Sugar substrate flexibility of StaG and StaN
Using a two-plasmid system in Streptomyces albus H
N H

α-L-rhamnose
O N
O

N N
pKC39GNT rebO rebD staC rebP staG staN rebT H N N

O OH
H 3C O
CH3 HO
pRHAM oleL oleS oleE oleU OH HO
OH
HO

pKC39GNT rebO rebD staC rebP staG staN rebT N

H
N α-L-olivose N N

O
H3 C O
CH3 HO
pLN2 oleV oleW oleU oleY oleL oleS oleE OH
HO
HO

N N α-L-digitoxose
pKC39GNT rebO rebD staC rebP staG staN rebT H
N N
O OH
CH3 H3 C O
pLNR oleV oleW urdR oleY oleL oleS oleE HO
HO
HO

N N
H
pKC39GNT rebO rebD staC rebP staG staN rebT
O
β-D-olivose
H3 C
pLNBIV oleV oleW eryBIV oleY oleL oleS oleE
HO
OH

A.P. Salas et al. (2005) Mol. Microbiol. in press

Structural differences = Opportunities for generating diversity

RebC StaC
H H
O N O N O

N N N N
Cl H Cl StaN StaG
RebH O
H3C H
O OH
O
RebG CH3 NH StaB
HO
OH CH3 StaE
O StaI
H3C StaMB StaJ
RebM StaK
StaMA
 César Sánchez
Aaroa P. Salas
Alfredo F. Braña
Carmen Méndez
José A. Salas

University of Oviedo
(Spain)

In collaboration with:
Lili Zhu
Igor A. Butovich
Jürgen Rohr

University of Kentucky
(USA)