Indolocarbazole antitumour compounds by combinatorial biosynthesis

Available online at www.sciencedirect.com

Indolocarbazole antitumour compounds by combinatorialbiosynthesisJose A Salas and Carmen Mendez

The indolocarbazoles constitute a family of natural products

with potential therapeutic applications in the treatment of

cancer and neurodegenerative disorders. Members of this

family are either potent stabilizers of topoisomerase I-DNA

covalent complex or potent inhibitors of protein kinases.

Rebeccamycin, staurosporine, AT2433 and K252a are

members of this family, which are produced by different

actinomycete strains, and whose biosynthesis gene clusters

have been isolated and characterized. A number of

indolocarbazole derivatives have been generated by applying

combinatorial biosynthesis technologies to these clusters

either in the producer strain or in the heterologous hosts after

expression of part or the entire gene cluster. Combinatorial

biosynthesis is therefore providing a new approach for

increasing structural diversity in this family of natural products.

Addresses

Departamento de Biologıa Funcional and Instituto Universitario de

Oncologıa del Principado de Asturias (I.U.O.P.A), Universidad de

Oviedo, 33006 Oviedo, Spain

Corresponding author: Salas, Jose A ([email protected])

Current Opinion in Chemical Biology 2009, 13:152–160

This review comes from a themed issue on

Molecular Diversity

Edited by Christian Hertweck and Hiroyuki Osada

Available online 27th February 2009

1367-5931/$ – see front matter

# 2009 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.cbpa.2009.02.003

IntroductionThe indolocarbazole alkaloids constitute an important

class of natural products, which have been isolated from

actinomycetes, cyanobacteria, fungi, slime moulds and

marine invertebrates [1�]. After the isolation of the first

indolocarbazole in 1977, this family of compounds has

attracted the attention of many researchers from different

disciplines because of the variety of chemical structures

and the interesting biological activities showed by this

family of compounds. Structurally (Figure 1a), the mem-

bers of this family are characterized by a core consisting of

either an ‘open’ bisindolylmaleimide (e.g. arcyriarubin

A), or a ‘closed’ indolo[2,3-a]carbazole (e.g. rebeccamy-

cin, staurosporine). Most of the latter compounds are, in

fact, derivatives of the indolo[2,3-a]pyrrolo[3,4-c]carba-

zole ring system, to which a sugar residue is often


attached, but they differ in the way the sugar is attached

to the indolocarbazole moiety: through one (as in rebec-

camycin and AT2433) or two (as in staurosporine and

K-252a) N-glycosidic bonds.

Indolocarbazoles display a wide range of biological activi-

ties, including antibacterial, antifungal, antiviral, hypo-

tensive, antitumour or neuroprotective properties, but

their greatest interest is based on their antitumour proper-

ties [2,3], and great efforts are made to generate indolo-

carbazole derivatives with improved properties for the

treatment of cancer. Some indolocarbazole derivatives

have entered into clinical trials for cancer and also for

the treatment of neurodegenerative disorders, and dia-

betes-associated pathologies [4�]. When considering the

mode of action, indolocarbazoles can be subdivided into

two different subgroups. The first subgroup is

represented by rebeccamycin that is a potent stabilizer

of topoisomerase I-DNA covalent complex. The second

one, represented by staurosporine, includes potent inhibi-

tors of protein kinases A, C and K. This suggests that the

structural differences between these two indolocarba-

zoles are essential for target selectivity.

A number of indolocarbazole derivatives have been pro-

duced by chemical synthesis or semi-synthesis [5,6].

However, in the past few years the isolation and charac-

terization of biosynthesis gene clusters for several indo-

locarbazoles produced by actinomycetes offers a

promising alternative for increasing structural diversity

in this family of compounds through the use of combi-

natorial biosynthesis technology. This article revises the

gene clusters characterized, the enzymatic characteriz-

ation of different steps in indolocarbazole biosynthesis

and how combinatorial biosynthesis is being used as a tool

for diversifying and expanding the chemical space in this

family of natural products.

Biosynthetic origin of indolocarbazolesEarly studies on the biosynthetic origin of indolocarbazoles

shed light on the biosynthetic pathways through the identi-

fication of metabolic precursors and some biosynthesis

intermediates. Furthermore, the flexibility of the biosyn-

thetic machinery was tested for accepting alternative pre-

cursors, and several new analogues were generated.

The biosyntheses of staurosporine and rebeccamycin were

studied by feeding isotope-labeled precursors to their

producer organisms: Lentzea albida (formerly Streptomycesstaurosporeus) staurosporine producer and Lechevalieriaaerocolonigenes (formerly Saccharothrix aerocolonigenes)rebeccamycin producer [7–10]. It was established that

www.sciencedirect.com

mailto:[email protected]

http://dx.doi.org/10.1016/j.cbpa.2009.02.003

Indolocarbazole antitumour compounds by combinatorial biosynthesis Salas and Mendez 153

Figure 1

Indolocarbazoles: structures and biosynthetic origin. (a) Chemical structures of the indolocarbazoles whose gene clusters have been characterized. (b)Biosynthetic origin of indolocarbazoles. Feeding isotope-labeled precursors and analysis of the incorporation pattern showed that L-tryptophan (blue),

D-glucose (red) and methionine (green) are precursors in the biosynthesis.

the indolocarbazole core was derived from two tryptophan

units with the carbon skeleton incorporated intact, while

the sugar moiety was derived from glucose and methionine

(Figure 1b). The identity of early tryptophan-derived

intermediates was studied by feeding tryptamine, indole-

pyruvate, indoleacetaldehyde, indoleacetamide and indo-

leacetate, and showing that indolepyruvate was an

intermediate in rebeccamycin biosynthesis, while trypta-

mine was not incorporated [8,9]. On the contrary, feeding

tryptamine to the staurosoporine producer resulted in

efficient incorporation into the molecule, suggesting that

tryptamine was a precursor in staurosporine biosynthesis.

The apparently conflicting results obtained with the two


strains were perhaps due to the different experimental

conditions employed, in addition to putative metabolic

differences existing between the two organisms such as

transaminases and other enzymes not directly involved in

indolocarbazole biosynthesis.

Novel indolocarbazoles accumulated bymutants or produced by precursor feedingA number of indolocarbazole derivatives or biosynthesis

intermediates have been isolated from mutants generated

by classical mutagenesis, by changing the fermentation

conditions or by feeding precursors. After UV irradiation

of Streptomyces longisporoflavus (staurosporine producer)


154 Molecular Diversity

mutants were isolated that accumulated K-252c and 30-O-

demethyl-staurosporine [11,12]. The latter compound

was converted into staurosporine by feeding experiments

and hence it was proposed as the last intermediate in

staurosporine biosynthesis [13]. Addition of potassium

bromide (but not fluoride or iodide salts) to cultures of

L. aerocolonigenes resulted in production of bromorebec-

camycin with concomitant suppression of rebeccamycin

accumulation [14]. On the contrary, addition of 6-fluoro-

tryptophan, 5-fluoro-tryptophan, or 4-fluoro-tryptophan

produced fluoroindolocarbazoles [15,16]. Attempts to per-

form precursor-directed biosynthesis with the staurospor-

ine producer using different indole derivatives (indole-3-

acetonitrile, indole-3-acetic acid, indole-3-acetamide, 5-

fluorotryptophan and 6-fluorotryptophan, 5-hydroxytryp-

tophan, indole-3-pyruvate, indole-3-propionic acid, tryp-

tamine, 5-fluorotryptamine and 6-fluorotryptamine and 5-

hydroxytryptamine) were unsuccessful.

Isolation of indolocarbazole gene clustersThe first report on the cloning of an indolocarbazole

biosynthesis gene was based on an indirect observation.

In the course of some bioconversion studies, it was found

that the aglycone J-104303 (identical to the rebeccamycin

aglycone but possessing hydroxyl groups instead of the

chlorine atoms) was converted into its N-glucosylated

derivative by a rebeccamycin producer strain. From a

gene library of genomic DNA of this producer strain, a

clone was isolated in Streptomyces lividans capable of

carrying out this conversion and, upon sequencing of

the DNA insert, found to contain a gene (designated as

ngt gene) coding for a glycosyltransferase [17�]. These

results suggested that ngt was probably involved in rebec-

camycin biosynthesis in this organism, although no defi-

nite proof was reported at that time. Using the ngt gene as

a probe, the rebeccamycin gene cluster was identified,

characterized and heterologously expressed in Strepto-myces albus [18�]. The same cluster was later on reported

by two different laboratories [19,20�] and expressed in

Escherichia coli, although at low level [20�]. A number of

biosynthetic intermediates in rebeccamycin biosynthesis

were identified either by co-expressing different sets of

genes of the cluster [18�] or by generating insertional

inactivation mutants [19] and further analysis of the

accumulated products. Three other indolocarbazole gene

clusters have been also reported: staurosporine [21�],AT2433 [22] and K-252a [23], together with that of the

closely related violacein [24,25]. The genetic organization

of these four indolocarbazole gene clusters is quite similar

and all of them contain genes required for the biosyn-

thesis of the aglycone, biosynthesis and transfer of the

deoxysugar and regulation (Figure 2a). Interestingly, the

AT2433 cluster is unique in containing an extra gene

AtmA, absent in the other clusters, that has been pro-

posed to participate in the biosynthesis of the aglycon

(see below). Furthermore, the rebeccamycin, AT2433

and K252a contain two genes each coding for transmem-


brane proteins possibly involved in secretion, but these

genes are missing in the staurosporine cluster.

Pathways for indolocarbazole biosynthesisand characterization of enzymes involvedOn the basis of similarities of the deduced gene products

with proteins in databases, on the chemical structures of

products accumulated by different mutants or produced

by recombinant strains co-expressing different sets of

genes and on in vitro testing of enzyme activities, path-

ways for the biosynthesis of these indolocarbazoles have

been proposed (Figure 2b). Conversion of L-tryptophan to

7-chloro-L-tryptophan is the first step in rebeccamycin

and AT2433 biosynthesis and serves to supply a unique

monomer precursor. This reaction is catalyzed in the

rebeccamycin pathway by a two-component enzyme,

RebF (flavin reductase)-RebH (halogenase), enzymes

that have been characterized in vitro [26–28]. In the

case of the AT2433 cluster, the flavin reductase gene is

absent [22]. Staurosporine and K-252a lack halogen atoms

and therefore these clusters do not contain these two-

component enzymes. A second pair of enzymes is then

responsible for the formation of the chromopyrrolic acid

intermediate through the fusion of two tryptophans or

halogenated tryptophan moieties. This process is cata-

lyzed by the tandem action of two enzymes: an L-trypto-

phan oxidase (RebO, StaO and AtmO in rebeccamycin,

staurosporine and AT2433 biosyntheses, respectively)

and a heme protein dimer with chromopyrrolic acid

synthase activity (RebD, StaD and AtmD for the same

pathways) [29,30�,31,32]. Interestingly, AT2433 is the

only naturally occurring indolocarbazole to have an asym-

metrically halogenated indolocarbazole core. It has been

proposed that AtmO and AtmD might have unique sub-

strate specificities, leading to the final formation of an

asymmetrically halogenated aglycone [22]. In addition, an

extra protein AtmA has been postulated to participate in

the introduction of the nitrogen atom at the pyrrole

moiety [22].

The ‘open’ chromopyrrolic acid intermediate must be

now converted into a ‘closed’ indolocarbazole aglycone

either containing an imide (rebeccamycin-like as in arcyr-

iaflavin A) or an amide (staurosporine-like as in K252c)

function at the pyrrole moiety. This requires an enzyme

pair that is present in all four clusters (RebC/RebP, StaC/

StaP, AtmC/AtmP and InkE/InkP). RebP (or its homol-

ogue StaP) are cytochrome P450s that appear to catalyze

the oxidation of chromopyrrolic acid to a set of three

aglycones (K252c, arcyriaflavin A and 7-hydroxy-K252c),

differing in the oxidation state in the pyrrole-derived ring

[33]. A catalytic mechanism for the StaP enzyme has been

proposed on the basis of the analysis of the crystal

structure of StaP with chromopyrrolic acid [34]. The

second enzyme, RebC (or its homologue StaC) is a

flavin-dependent hydroxylase, whose structure has been

recently elucidated alone as well as in the presence of



Figure 2

Biosynthesis of indolocarbazoles. (a) Genetic organization of the gene cluster for rebecamycin (reb), staurosporine (sta), K252a (ink) and AT2433

(atm). (b) Proposed pathways for indolocarbazoles biosynthesis. R Cl in rebeccamycin and R H or Cl for intermediates during AT2433-A1

biosynthesis.

chromopyrrolic acid and K252c [35]. The P450 enzymes

(RebP or StaP), in the presence of StaC convert the three

aglycones into a predominant K252c product, while in the

presence of RebC basically arcyriaflavin A is formed [36].


It has been proposed that the role of RebC (and that

of StaC) is to sequester a reactive intermediate produced

by its partner protein (RebP or StaP) and to react with

it enzymatically, preventing its conversion to several



degradation products that includes, at low levels, the

desired product. After the formation of the aglycone,

sugar transfer occurs catalyzed by a glycosyltransferase.

Rebeccamycin and staurosporine contain an hexose sugar,

D-glucose and L-ristosamine, respectively, while K-252a

contains a dihydrostreptose and AT2433 an aminodideox-

ypentose and a D-glucose moieties. Genes for the bio-

synthesis of these sugars are part of the gene clusters, with

the exception of rebeccamycin that can incorporate D-

glucose from primary metabolism. Candidate N-glycosyl-

transferase genes for sugar transfer have been found in all

four clusters. In the case of staurosporine and K-252a, a

second C–N bond must be established between the sugar

and the aglycone, a reaction catalyzed by a P450 mono-

oxygenase (StaN for staurosporine and InkC for K-252a).

As final steps in the biosynthesis, several methylation

steps decorate the final indolocarbazole structure acting

on the sugars (in all four cases) or in the imide function (in

the case of AT2433).

Figure 3

Indolocarbazoles produced by combinatorial biosynthesis with modification


Combinatorial biosynthesis for novelindolocarbazoles: modifications in theaglycone moietyThe availability of the genes for the biosynthesis of the

four indolocarbazoles mentioned above, together with the

relative substrate flexibility of secondary metabolism

enzymes, has made possible the generation of a good

number of novel indolocarbazole derivatives (Figures 3

and 4). One of the more efficient strategies was the use of

combinatorial biosynthesis approaches to create meta-

bolic pathways in another actinomycete host such as S.albus by co-expression of different indolocarbazole bio-

synthesis genes either from a pathway or in combination

with genes from other indolocarbazole pathways, thus

rendering 24 novel indolocarbazoles (Figure 3) [37��].This allowed the dissection and reconstitution of the

rebeccamycin pathway and the understanding of how

this indolocarbazole is synthesized. These studies

showed that rebeccamycin derivatives can be produced

s in the aglycone moiety.



Figure 4

Indolocarbazoles produced by combinatorial biosynthesis with modifications in the deoxysugar moiety.

www.sciencedirect.com Current Opinion in Chemical Biology 2009, 13:152–160


in the absence of the RebH halogenase (Figure 3, com-

pounds 1, 10, 19–21), indicating that a full pathway for

dideschloro-rebeccamycin can be reconstituted and also

showing that the rest of the enzymes of the pathway were

able to use non-halogenated intermediates [37��]. Rebec-

camycin derivatives with chlorine atoms attached to

different positions of the tryptophan moiety were pro-

duced by co-expressing genes involved in the biosyn-

thesis of the rebeccamycin aglycone together with two

genes coding for halogenases acting on different positions

of the tryptophan moiety (Figure 3, compounds 2, 3, 9, 11,

18). These were the PyrH 5-chloro-tryptophan halogen-

ase involved in pyrroindomycin biosynthesis and the

ThaI 6-chloro-tryptophan halogenase involved in thieno-

lodin biosynthesis. These experiments showed that halo-

gen atoms can be positioned in different parts of the

indolocarbazole ring. Another interesting feature for

creating indolocarbazole structural diversity is the pivotal

role of RebC vs. StaC. These two enzymes are respon-

sible for the oxidation state of the indolocarbazole ring:

RebC leading to the formation of an imide function and

StaC to an amide function. This implies that when co-

expressing indolocarbazole genes, just by exchanging

these two genes in any gene construct we can produce

rebeccamycin-like (Figure 3, compounds 14–18, 20–24;

Figure 4, compounds 42, 47, 48, 52) or staurosporine-like

derivatives (Figure 3, compounds 10–13; Figure 4, com-

pounds 25–30) ([37��], AP Salas et al., unpublished data).

Combinatorial biosynthesis for novelindolocarbazoles: modifications in the sugarmoietyIncreasing evidence suggest the existence of some flexi-

bility in secondary metabolite glycosyltransferases

regarding the sugar donor [38�,39,40]. Taking advantage

of this flexibility a number of novel glycosylated deriva-

tives from staurosporine have been generated. The com-

plete staurosporine pathway was reconstituted in S. albususing a two-plasmid system [41��]. The first plasmid

(‘aglycone plasmid’) contained the genes required for

staurosporine aglycone formation together with a gene

coding for the staurosporine glycosyltransferase StaG and

a second gene coding for the cytochrome P450 enzyme

StaN. The latter has been shown to be responsible for the

establishment of the second C–N linkage between the

sugar and the indolocarbazole moiety [42,43]. The second

plasmid (‘sugar plasmid’) contained the genes necessary

for the biosynthesis of the staurosporine sugar moiety

with or without two genes coding for an O-methyltrans-

ferase and a N-methyltransferase that modify the sugar

moiety. Coexistence of these two plasmids in S. albusresulted in the production of staurosporine and several

glycosylated derivatives with differences in the sugar

moiety (Figure 4, compounds 31, 36–38). On the basis

of this two-plasmid system, the replacement of the ‘sugar

plasmid’ by a family of ‘sugar cassette plasmids’, each one

directing the biosynthesis of different L-deoxysugars and


D-deoxysugars [44] lead to the formation of novel glyco-

sylated indolocarbazoles containing L-rhamnose, L-oli-

vose, L-digitoxose or D-olivose attached either through

single (in all the cases) (Figure 4, compounds 32–35) or

double (in the case of the former three sugars but not for

D-olivose) C–N bonds (Figure 4, compounds 39–41)

[41��]. Recently, similar rebeccamycin derivatives have

been produced by replacing the staC by the rebC gene in

the ‘aglycone plasmid’ (AP Salas et al., unpublished data)

(Figure 4, compounds 43–46, 49–51). Through biocon-

version experiments it has been also shown that the RebG

rebeccamycin glycosyltransferase can glucosylate a set of

indolocarbazole surrogates, and evidence for a remarkable

lack of regioselectivity of this transferase in the presence

of asymmetric substrates has been provided (Figure 4,

compounds 53–57)[45]. Interestingly, the RebM O-meth-

yltransferase can modify in vitro many of the glycosylated

compounds produced by RebG (Figure 4) [45]. The RebM

O-methyltransferase also shows an interesting peculiarity

regarding the methyl group donor. RebM is capable of

using a non-natural cofactor analogue of S-adenosyl-L-

methionine (a novel synthetic cofactor bearing a pendant

50-amino acid N-mustard) as methyl group donor to gen-

erate a number of new rebeccamycin analogues [46��].This could be another way to increase the molecular

biodiversity in indolocarbazoles for drug discovery.

Concluding remarksAs an alternative to chemical synthesis, the introduction of

combinatorial biosynthesis technology and its application

to indolocarbazole biosynthesis gene clusters may

represent a promising way of expanding the structural

diversity of this family of biologically active natural pro-

ducts. The identification of the genes responsible for the

biosynthesis of four indolocarbazoles (rebeccamycin, staur-

osporine, AT2433 and K252a) provides genetical tools for

such purpose. More than 50 different indolocarbazole

derivatives have been produced in a rational way through

the expression of selected genes in a convenient bacterial

host. Two interesting features of their biosynthetic path-

ways make them interesting targets for combinatorial

biosynthesis. Firstly, indolocarbazole pathways are rela-

tively simple mainly in comparison to pathways for other

more complex natural products and, secondly, some of the

indolocarbazole biosynthetic enzymes have some degree

of substrate flexibility, being capable of recognizing and

processing different intermediates thus making possible

the generation of novel derivatives. Applying combinator-

ial biosynthesis to this group of compounds will contribute

to further expanding the chemical space of indolocarba-

zoles and thus to provide potentially active derivatives as

inhibitors of topoisomerases or protein kinases.

AcknowledgementsResearch in the authors’ laboratory has been supported by grants from theSpanish Ministry of Science and Innovation (BFU2006-00404 to JAS andBIO2005-04115 to CM), the Red Tematica de Investigacion Cooperativa de



Centros de Cancer to JAS (Ministry of Health, Spain; ISCIII-RETIC RD06/0020/0026) and from the UE FP6 (ActinoGen; Integrated project no005224).

References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:

� of special interest�� of outstanding interest

1.�

Sanchez C, Mendez C, Salas JA: Indolocarbazole naturalproducts: occurrence, biosynthesis, and biological activity.Nat Prod Rep 2006, 23:1007-1045.

This review provides a survey of 257 indolocarbazoles isolated fromdifferent biological sources or generated by combinatorial biosynthesisand describes recent developments in molecular genetics, enzymologyand metabolic engineering, with an emphasis on the development ofanalogues that have entered clinical trials for its future use against canceror other diseases.

2. Akinaga S, Sugiyama K, Akiyama T: UCN-01 (7-hydroxystaurosporine) and other indolocarbazolecompounds: a new generation of anti-cancer agents for thenew century? Anti-Cancer Drug Design 2000, 15:43-52.

3. Bailly C: Topoisomerase I poisons and suppressors asanticancer drugs. Curr Med Chem 2001, 7:39-58.

4.�

Butler MS: Natural products to drugs: natural product derivedcompounds in clinical trials. Nat Prod Rep 2005, 22:162-195.

A comprehensive description on natural products or derived compoundsthat are being evaluated in clinical trials or in registration.

5. Knolker HJ, Reddy KR: Isolation and synthesis of biologicallyactive carbazole alkaloids. Chem Rev 2002, 102:4303-4427.

6. Prudhomme M: Biological targets of antitumorindolocarbazoles bearing a sugar moiety. Curr Med ChemAnticancer Agents 2004, 4:509-521.

7. Pearce CJ, Doyle TW, Forenza S, Lam KS, Schroeder DR: Thebiosynthetic origins of rebeccamycin. J Nat Prod 1988,51:937-940.

8. Lam KS, Forenza S, Doyle TW, Pearce CJ: Identification ofindolepyruvic acid as an intermediate of rebeccamycinbiosynthesis. J Ind Microbiol 1990, 6:291-294.

9. Yang SW, Cordell GA: Origin of nitrogen in the indolocarbazoleunit of staurosporine. J Nat Prod 1997, 60:788-790.

10. Yang SW, Lin LJ, Cordell GA, Wang P, Corley DG: O- and N-Methylation in the biosynthesis of staurosporine. J Nat Prod1999, 62:1551-1553.

11. Goeke K, Hoehn P, Ghisalba O: Production of the staurosporineaglycone K-252c with a blocked mutant of the staurosporineproducer strain Streptomyces longisporoflavus and bybiotransformation of staurosporine with Streptomycesmediocidicus ATCC 13279. J Antibiot (Tokyo) 1995, 48:428-430.

12. Hoehn P, Ghisalba O, Moerker T, Peter HH: 30-Demethoxy-30-hydroxystaurosporine, a novel staurosporine analogueproduced by a blocked mutant. J Antibiot (Tokyo) 1995,48:300-305.

13. Weidner S, Kittelmann M, Goeke K, Ghisalba O, Zahner H: 30-Demethoxy-30-hydroxystaurosporine-O-methyltransferasefrom Streptomyces longisporoflavus catalyzing the last step inthe biosynthesis of staurosporine. J Antibiot (Tokyo) 1998,51:679-682.

14. Lam KS, Schroeder DR, Veitch JM, Matson JA, Forenza S: Isolationof a bromo analog of rebeccamycin from Saccharothrixaerocolonigenes. J Antibiot (Tokyo) 1991, 44:934-939.

15. Lam KS, Schroeder DR, Veitch JM, Colson KL, Matson JA,Rose WC, Doyle TW, Forenza S: Production, isolation andstructure determination of novel fluoroindolocarbazoles fromSaccharothrix aerocolonigenes ATCC 39243. J Antibiot (Tokyo)2001, 54:1-9.

16. Long BH, Rose WC, Vyas DM, Matson JA, Forenza S: Discoveryof antitumor indolocarbazoles: rebeccamycin, NSC 655649,


and fluoroindolocarbazoles. Curr Med Chem AntiCancer Agents2002, 2:255-266.

17.�

Ohuchi T, Ikeda-Araki A, Watanabe-Sakamoto A, Kojiri K,Nagashima M, Okanishi M, Suda H: Cloning and expression of agene encoding N-glycosyltransferase (ngt) from SaccharothrixaerocolonigenesATCC39243.JAntibiot (Tokyo)2000,53:393-403.

This paper reports the cloning of the first indolocarbazole biosynthesisgene and experimental evidence for the formation of an N-glucosylatedderivative by biotransformation.

18.�

Sanchez C, Butovich IA, Brana AF, Rohr J, Mendez C, Salas JA:The biosynthetic gene cluster for the antitumor rebeccamycin:characterization and generation of indolocarbazolederivatives. Chem Biol 2002, 9:519-531.

In this paper, the cloning, sequencing and heterologous expression of thefirst indolocarbazole cluster characterized is reported together with theformation of some biosynthesis intermediates by expression of differentsets of genes of the cluster in S. albus.

19. Onaka H, Taniguchi S, Igarashi Y, Furumai T: Characterization ofthe biosynthetic gene cluster of rebeccamycin fromLechevalieria aerocolonigenes ATCC 39243. Biosci BiotechnolBiochem 2003, 67:127-138.

20.�

Hyun CG, Bililign T, Liao J, Thorson JS: The biosynthesis ofindolocarbazoles in a heterologous Escherichia coli host.Chembiochem 2003, 4:114-117.

The first report on the heterologous expression of an indolocarbazolegene cluster in Escherichia coli.

21.�

Onaka H, Taniguchi S, Igarashi Y, Furumai T: Cloning of thestaurosporine biosynthetic gene cluster from Streptomycessp. TP-A0274 and its heterologous expression inStreptomyces lividans. J Antibiot (Tokyo) 2002, 55:1063-1071.

It describes the cloning, sequencing and heterologous expression of thestaurosporine cluster and a number of insertional inactivation mutantsaccumulating specific biosynthetic intermediates.

22. Gao Q, Zhang C, Blanchard S, Thorson JS: Decipheringindolocarbazole and enediyne aminodideoxypentosebiosynthesis through comparative genomics: insights fromthe AT2433 biosynthetic locus. Chem Biol 2006, 13:733-743.

23. Kim SY, Park JS, Chae CS, Hyun CG, Choi BW, Shin J, Oh KB:Genetic organization of the biosynthetic gene cluster for theindolocarbazole K-252a in Nonomuraea longicatena JCM11136. Appl Microbiol Biotechnol 2007, 75:1119-1126.

24. August PR, Grossman TH, Minor C, Draper MP, MacNeil IA,Pemberton JM, Call KM, Holt D, Osburne MS: Sequence analysisand functional characterization of the violacein biosyntheticpathway from Chromobacterium violaceum. J Mol MicrobiolBiotechnol 2000, 2:513-519.

25. Sanchez C, Brana AF, Mendez C, Salas JA: Reevaluation of theviolacein biosynthetic pathway and its relationship toindolocarbazole biosynthesis. Chembiochem 2006, 7:1231-1240.

26. Yeh E, Garneau S, Walsh CT: Robust in vitro activity of RebF andRebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis. Proc NatlAcad Sci U S A 2005, 102:3960-3965.

27. Yeh E, Blasiak LC, Koglin A, Drennan CL, Walsh CT: Chlorinationby a long-lived intermediate in the mechanism of flavin-dependent halogenases. Biochemistry 2007, 46:1284-1292.

28. Yeh E, Cole LJ, Barr EW, Bollinger JM Jr, Ballou DP, Walsh CT:Flavin redox chemistry precedes substrate chlorination duringthe reaction of the flavin-dependent halogenase RebH.Biochemistry 2006, 45:7904-7912.

29. Howard-Jones AR, Walsh CT: Enzymatic generation of thechromopyrrolic acid scaffold of rebeccamycin by the tandemaction of RebO and RebD. Biochemistry 2005, 44:15652-21563.

30.�

Nishizawa T, Aldrich CC, Sherman DH: Molecular analysis of therebeccamycin L-amino acid oxidase from Lechevalieriaaerocolonigenes ATCC 39243. J Bacteriol 2005, 187:2084-2092.

The last two papers report the first in vitro characterization of enzymesinvolved in early steps in the biosynthesis of rebeccamycin.

31. Nishizawa T, Gruschow S, Jayamaha DH, Nishizawa-Harada C,Sherman DH: Enzymatic assembly of the bis-indole core ofrebeccamycin. J Am Chem Soc 2006, 128:724-725.



32. Asamizu S, Kato Y, Igarashi Y, Furumai T, Onaka H: Directformation of chromopyrrolic acid from indole-3-pyruvic acidby StaD, a novel hemoprotein in indolocarbazole biosynthesis.Tetrahedron Lett 2006, 47:473-475.

33. Howard-Jones AR, Walsh CT: Nonenzymatic oxidative stepsaccompanying action of the cytochrome P450 enzymes StaPand RebP in the biosynthesis of staurosporine andrebeccamycin. J Am Chem Soc 2007, 129:11016-11107.

34. Makino M, Sugimoto H, Shiro Y, Asamizu S, Onaka H, Nagano S:Crystal structures and catalytic mechanism of cytochromeP450 StaP that produces the indolocarbazole skeleton. ProcNatl Acad Sci U S A 2007, 104:11591-11596.

35. Ryan KS, Howard-Jones AR, Hamill MJ, Elliott SJ, Walsh CT,Drennan CL: Crystallographic trapping in the rebeccamycinbiosynthetic enzyme RebC. Proc Natl Acad Sci U S A 2007,104:15311-21536.

36. Howard-Jones AR, Walsh CT: Staurosporine and rebeccamycinaglycones are assembled by the oxidative action of StaP,StaC, and RebC on chromopyrrolic acid. J Am Chem Soc 2006,128:12289-12298.

37.��

Sanchez C, Zhu L, Brana AF, Salas AP, Rohr J, Mendez C,Salas JA: Combinatorial biosynthesis of antitumorindolocarbazole compounds. Proc Natl Acad Sci U S A 2005,102:461-466.

A good example of the use of combinatorial biosynthesis to increasestructural diversity in natural products with the generation of more than 30different indolocarbazoles in the heterologous host Streptomyces albus.

38.�

Salas JA, Mendez C: Engineering the glycosylation of naturalproducts in actinomycetes. Trends Microbiol 2007, 15:219-232.

In this paper, the ‘state-of-the-art’ on the use of combinatorial biosynth-esis to alter the glycosylation pattern in natural products produced byactinomycetes is reviewed.

39. Luzhetskyy A, Mendez C, Salas JA, Bechthold A:Glycosyltransferases, important tools for drug design.Curr Top Med Chem 2008, 8:680-709.


40. Mendez C, Luzhetskyy A, Bechthold A, Salas JA: Deoxysugars inbioactive natural products: development of novel derivativesby altering the sugar pattern. Curr Top Med Chem 2008,8:710-724.

41.��

Salas AP, Zhu L, Sanchez C, Brana AF, Rohr J, Mendez C,Salas JA: Deciphering the late steps in the biosynthesis of theanti-tumour indolocarbazole staurosporine: sugar donorsubstrate flexibility of the StaG glycosyltransferase. MolMicrobiol 2005, 58:17-27.

In this paper, a number of novel glycosylated staurosporine derivativesare generated in an heterologous host by combining two plasmids eachone coding for different parts of the indolocarbazole molecule.

42. Onaka H, Asamizu S, Igarashi Y, Yoshida R, Furumai T:Cytochrome P450 homolog is responsible for C-N bondformation between aglycone and deoxysugar in thestaurosporine biosynthesis of Streptomyces sp. TP-A0274.Biosci Biotechnol Biochem 2005, 69:1753-1759.

43. Sanchez C, Mendez C, Salas JA: Engineering biosyntheticpathways to generate antitumor indolocarbazole derivatives.J Ind Microbiol Biotechnol 2006, 33:560-568.

44. Rodrıguez L, Aguirrezabalaga I, Allende N, Brana AF, Mendez C,Salas JA: Engineering deoxysugar biosynthetic pathways fromantibiotic-producing microorganisms. A tool to produce novelglycosylated bioactive compounds. Chem Biol 2002, 9:721-729.

45. Zhang C, Albermann C, Fu X, Peters NR, Chisholm JD, Zhang G,Gilbert EJ, Wang PG, Van Vranken DL, Thorson JS: RebG- andRebM-catalyzed indolocarbazole diversification.Chembiochem 2006, 7:795-804.

46.��

Zhang C, Weller RL, Thorson JS, Rajski SR: Natural productdiversification using a non-natural cofactor analogue of S-adenosyl-L-methionine. J Am Chem Soc 2006, 128:2760-2761.

Interesting description on the ability of the RebM O-methyltransferaseto use a non-natural cofactor analogue of S-adenosyl-L-methionine asmethyl group donor to generate a number of new rebeccamycinanalogues.


Indolocarbazole antitumour compounds by combinatorial biosynthesis

Documents

Transcript of Indolocarbazole antitumour compounds by combinatorial biosynthesis