Enediyne Natural Products: Biosynthesis and Prospect Towards Engineering Novel Antitumor Agents

Current Medicinal Chemistry, 2003, 10, 2317-2325 2317 Enediyne Natural Products: Biosynthesis and Prospect Towards Engineering Novel Antitumor Agents

Ben Shen1,2*, Wen Liu1 and Koichi Nonaka1

1Division of Pharmaceutical Sciences and 2Department of Chemistry, University of Wisconsin, Madison, WI 53705, USA Abstract: This review gives a brief account on the current status of enediyne biosynthesis and the prospective of applying combinatorial biosynthesis methods to the enediyne system for novel analog production. Methods for cloning enediyne biosynthetic gene clusters are first reviewed. A unified paradigm for enediyne biosynthesis, characterized with (a) the enediyne PKS, (b) the enediyne PKS accessory enzymes, and (c) tailoring enzymes, is then presented. Strategies and tools for novel enediyne analog production by combinatorial biosynthesis are finally discussed. The results set the stage to decipher the molecular mechanism for enediyne biosynthesis and lay the foundation to engineer novel enediynes by combinatorial biosynthesis for future endeavors. Keywords: Biosynthesis, C-1027, calicheamicin, combinatorial biosynthesis, enediyne, polyketide synthase.

INTRODUCTION categories according to the enediyne core structures. Members of the 9-membered enediyne core sub-category are Neocarzinostatin (NCS), the first member of the enediyne chromoproteins consisting of an apo-protein and the family of antitumor antibiotics, was originally discovered as enediyne chromophore, with N1999A2 (5 ) from a macromolecular antitumor antibiotic from the culture Streptomyces sp. AJ9493 as the only exception that was filtrates of a Streptomyces carzinostaticus strain in 1965 [1]. isolated as a chromophore alone [15]. The apo-protein acts as Although it became clear shortly after its discovery that all a stabilizer and specific carrier for the otherwise unstable biological activities of NCS resided in a nonprotein chromophore and its transport and interaction with target chromophore, the NCS chromophore structure (1) was not DNA. Due to their intrinsic reactivity, only three other elucidated until 1985 [2], revealing an unprecedented chromoprotein chromophore structures, in additional to 1 bicyclo[7,3,0]dodecadiynene system, i.e., the 9-membered and 5, are currently known—kedarcidin (6) from enediyne core, decorated with an aminosugar and a naphthoic Actinomycete L585-6 [16], C-1027 (7) from Streptomyces acid moiety. However, this seminal work was globisporus [17-20], maduropeptin (8) from Actinomadura underappreciated in the subsequent two years, and it was the madurae [21]. Members of the 10-membered enediyne core discovery of the calicheamicins (2) from a Micromonospora sub-category are in general more stable, all of which were echinospora strain [3,4] and the esperamicins (3) from an isolated as discrete small molecules. In addition to 2, 3, 4, Actinomadura verrucosospora strain [5,6] in 1987 that other members of this sub-category whose structures have grabbed the attention of chemists and biologists alike to the been elucidated include dynemicin (9 ) from enediynes. In contrast to 1, structural elucidation of 2 and 3 Micromonospora chersina sp. nov. No. M965-1 [22,23] and unveiled a novel biocyclo[7,3,1]tridecadiynene system, i.e., namenamicin (10) from a marine ascidian Polysyncraton the 10-membered enediyne core, decorated with several lithostrotum species [24] (Figure 1). deoxysugar moieties. Since then, the enediyne family of Members of both sub-categories of enediynes share a natural products has been the focus of intense research common mechanism of action, despite their structural activity in the fields of chemistry, biology, and medical difference. The enediyne core undergoes an electronic sciences because of their highly unusual molecular rearrangement (Bergman or Myers rearrangement) to form a architectures, biological activities, and modes of actions [7- transient benzenoid diradicals that damage DNA by 13]. abstracting hydrogen atoms from the deoxyribose moiety on Over twenty enediyne natural products are currently both strands. Subsequent reactions of the resultant known, and new members of the enediyne family are deoxyribose carbon-centered radicals with molecular oxygen continuously to be discovered with the newest addition, the initiate a process that leads in both single-strand and double- shishijimicins (4) from a marine ascidian Didemnum strand DNA cleavage [7-13,25-27]. This novel mechanism of proliferum species, reported in early 2003 [14]. The DNA damage has important implications for the enediyne natural products could be classified into two sub- development of the enediynes into clinical anticancer drugs [25-36]. Although the natural enediynes have seen limited use as clinical drugs mainly because of substantial toxicity, *Address correspondence to this author at the Division of Pharmaceutical various polymer-based delivery systems or enediyne- Sciences, School of Pharmacy, University of Wisconsin-Madison, 777 Highland Ave., Madison, WI 53705, USA; Tel: +1-608-263-2673; Fax: antibody conjugates have shown great clinical success and/or +1-608-262-5345; E-mail: [email protected] promise in anticancer chemotherapy [8,10,12,13,37,38]. For

0929-8673/03 $41.00+.00 © 2003 Bentham Science Publishers Ltd. 2318 Current Medicinal Chemistry, 2003, Vol. 10, No. 21 Shen et al. example, the poly(styrene-co-maleic acid)-conjugated NCS harnessed and delivered onto the target tumor cells. A great was approved in Japan in 1993 and has been marketed since challenge will be to develop ways to make new enediynes 1994 for use against hepatoma [10]. A CD33 monoclonal for mechanistic and clinical studies. antibody (mAB)-calicheamicin conjugate was approved in Access to complex natural products such as the enediynes US in 2000 and has been marked under the trade name of and their analogs by total synthesis poses a monumental Mylotarg to treat acute myeloid leukemia [37]. Several challenge to synthetic chemists, and yet tour de force effort antiheptoma mAB-C-1027 conjugates have also been by synthetic chemists has led to the total syntheses of prepared and showed to display high tumor specificity and almost every member of the enediyne family of natural to exert a strong inhibitory effect on the growth of products as well as a myriad of analogs. Significant progress established tumor xenografts [38]. These examples clearly in developing the enediynes into clinical agents has been demonstrate that the enediynes can be developed into made towards (a) improving cancer cell specificity, (b) powerful drugs when their extremely potent cytotoxicity is developing efficient delivery systems to the tumor targets,

O A H2C O OCH3 O O O N OC H3 O

O O O

H3C O H C 3 O O O (C H3 )2 N OH O R CH3 OH OH O CH3HN 22 O HO O Cl CH 3 N HO R = OH (7) NH2 (1) R = H (11) O H Cl

O OH HO O O OH H3 CO OH CH O NH 3

HO CH3 (8)

H3C

i-PrO OH OH HO O H3CO N(CH ) OC H3 O OCH 3 2 3 NH H3C Cl O O Cl N OH

O HOH2C O O O O OH

(5) O O OH H3C O HO (6) H3C Biosynthesis and Prospect Towards Engineering Novel Antitumor Agents Current Medicinal Chemistry, 2003, Vol. 10, No. 21 2319

(Fig. 1). contd..... B SSSC H3 O NHCO2CH3 OH O CH HO O 3 O O CH3 O N S O OH H

OCH3 H3COCH3 O NHEt

IOCH3

H3C O (2) HO CH O 3 OH

SSSC H3 O NHCO 2CH3 OH O CH HO O 3 O O CH3 N SCH O O OH H 3

OCH3 H3C O O NHPr-i O HO H3CO O

(3) H3CO NH

O OC H3

SSSCH3 CH3 O H NHCO2CH3 CO2H OH OH O HN O CH O HO O 3 O O CH3 O HO SC H3 OC H3 SCH3 OH OCH3 O NHPr-i OH OOH (10) (9) OH

SSSCH3 O N NHCO 2CH3 N H CH3 HO O O O HO O SCH3 OCH3 O NHPr-i (4)

Fig. (1). The chromophore structures of naturally occurring 9-membered enediyne chromoproteins: neocarzinostatin (1), C-1027 (7), maduropetin (8), kedarcidin (6), and N1999A2 (5) and as well as the engineered deshydroxy-C-1027 (11). (B) Structural I representation of naturally occurring 10-membered enediynes: calicheamicin r1 (2), esperamicin A1 (3), dynemicin A (9), namenamicin (10), and shishijimicin A (4). and (c) designing triggers and trapping the enediynes as modification of the natural products can only access to prodrugs [11-13]. However chemical total synthesis has very limited functional groups, often requiring multiple limited practical value for complex natural products such as protection and deprotection steps. the enediynes, and analog generation by chemical 2320 Current Medicinal Chemistry, 2003, Vol. 10, No. 21 Shen et al.

Complementary to organic synthesis, genetic of genome of the antibiotic-producing actinomycetes), a manipulations of genes governing secondary metabolism— daunting task that is further complicated by the fact that an emerging technology also known as combinatorial most of these organisms contain multiple secondary biosynthesis—offer a promising alternative to preparing metabolite biosynthetic gene clusters [49-51]. complex natural products and their analogs biosynthetically [39-46]. Specific structural alteration in the presence of other functional groups can often be achieved, and the target dNDP-Glucose 4,6-Dehydratase as A Probe molecules will be produced by a recombinant organism that This strategy was exemplified by the cloning of the is amenable for large-scale fermentation, thereby lowering biosynthetic gene cluster for 7 from S. globisporus [52,53] the production cost and reducing the environment concern and should be applicable to gene clusters of both the 9- and associated with conventional chemical synthesis. The 10-membered enediynes. Except for 5 and 9, enediynes with success of this approach depends on (a) the cloning and known structures all contain at least one deoxyhexose genetic and biochemical characterization of the biosynthetic moiety. The biosynthesis of various deoxyhexoses shares a pathways of the target metabolites and (b) the development common key intermediate, 4-keto-6-deoxyglucose nucleoside of strategies, methods, and expedient tools for combinatorial diphosphate, the formation of which from glucose manipulation of natural product biosynthetic gene clusters. nucleoside diphosphate is catalyzed by the dNDP-glucose The enediynes offer an outstanding opportunity to 4,6-dehydratase [54,55]. Taking advantage of the high amino investigate the genetic and biochemical basis for the acid sequence homology observed among dNDP-glucose biosynthesis of structurally complex natural products and to 4,6-dehydratases from actinomycetes, Bechthold and co- apply the combinatorial biosynthesis principle and methods workers developed a general approach for cloning dNDP- to enediyne biosynthesis for generating novel enediyne glucose 4,6-dehydratase genes by PCR with primers analogs, some of which could potentially be developed into designed according to the conserved regions [56]. We anticancer drugs. adopted this PCR method to clone the biosynthetic gene In this review, we provide a brief account on the current cluster for 7 from S. globisporus [52,53]. We first cloned a status of enediyne biosynthesis and the prospective of putative dNDP-glucose 4,6-dehydratase gene, sgcA, from S. applying combinatorial biosynthesis methods to the globisporus by PCR and confirmed its involvement in 7 enediyne system for novel analog production. We first biosynthesis by gene disruption and complementation [52]. review the strategies available for cloning enediyne We then screened the S. globisporus genomic library using biosynthetic gene clusters from various organisms. We then sgcA as a probe and localized the entire biosynthetic gene examine the genetic and biochemical data for the cluster for 7 to an 85-kb contiguous region of the S. biosynthesis of 7 and 2, as examples for the 9- and 10- globisporus chromosome [53]. We finally sequenced and membered enediynes, respectively, to illustrate a common genetically characterized the cloned sgc cluster, revealing that polyketide pathway for the enediyne family of natural sgcA is indeed clustered with the other structural genes, the products, characterized by an iterative type I polyketide previously cloned cagA gene that encodes the C-1027 apo- synthase (PKS). We finally discuss challenges and possible protein [57], as well as resistance and regulatory genes for 7 solutions for combinatorial biosynthesis of enediyne natural production [53]. products with the production of deshydroxy-C-1027 (11) to demonstrate the feasibility of this approach. Early studies on enediyne biosynthesis by isotope feeding experiments and Apo-Protein as A Probe on cloning and characterization of the apo-proteins can be This strategy depends on the availability of the apo- seen in a previous review [47]. protein, many of which have been previously cloned and characterized [47], and its applicability, therefore, is limited METHODS FOR CLONING OF ENEDIYNE only to the chromoprotein enediynes as exemplified by the BIOSYNTHETIC GENE CLUSTERS cloning of the biosynthetic gene cluster for 1 from S. carzinostaticus [48]. We initially attempted to localize the 1 Antibiotic production genes are often clustered in one biosynthetic gene cluster by the PCR method [56] to clone region of the microbial chromosome, consisting of the dNDP-glucose-4,6-dehydratase as a probe, following the structural, resistance, and regulatory genes. This clustering same strategy as we did for 7 [52]. Although we were phenomenon has become the cornerstone from which successful in amplifying a putative dNDP-glucose-4,6- strategies for cloning microbial natural product biosynthetic dehydratase gene from S. carzinostaticus, inactivation of it gene clusters were developed [39,48-51]. Practically, once failed to abolish 1 production, auguring against its one gene is cloned and confirmed to encode the production involvement in 1 biosynthesis. We then reasoned that the of the target molecule, chromosomal walking from this apo-protein gene could be used as a probe to clone the locus is virtually certain to lead to the identification of the chromoprotein enediyne gene cluster, viewing the apo- entire biosynthetic gene cluster (ranging in size from 20-200- protein as a resistance mechanism via drug sequestering. kb [48]). Recent technological advancement in DNA This hypothesis gained further support after we confirmed sequencing has dramatically reduced the time and effort in that the cagA gene, encoding the CagA aproprotein for 7, determining the DNA sequence once the cluster is cloned. was indeed clustered with the rest of its biosynthesis, However, it remains to be a great challenge to resistance, and regulatory genes [52,53]. We then took unambiguously identify, localize, and confirm the target advantage of the previously cloned ncsA gene, which gene cluster from a genome of 9 Mbp (the approximate size encodes the NcsA apoprotein for 1 [58,59], as a probe to screen the S. carzinostaticus genomic library and localized Biosynthesis and Prospect Towards Engineering Novel Antitumor Agents Current Medicinal Chemistry, 2003, Vol. 10, No. 21 2321 the biosynthetic gene cluster for 1 to a 95-kb contiguous as other natural products [48]. Farnet and co-workers region of the S. carzinostaticus chromosome. Our developed this so-called “high-throughput genome scanning confidence that the cloned gene cluster encoded 1 method to discover metabolic loci” on the basis that genes biosynthesis was further increased after identifying another encoding secondary metabolite biosynthesis in copy of the dNDP-glucose-4,6-dehydratase gene, different microorganisms are clustered [48]. Similar to the so-called from the one amplified by PCR, within this locus. The co- “perfect probes” approach to identify and clone type I PKS localization of ncsA and a dNDP-glucose-4,6-dehydratase gene clusters in a genome [63], this method consists gene, reminiscent to that for the 7 biosynthetic gene cluster essentially three steps: (a) shotgun DNA sequencing to [53], was viewed as an evidence of clustering between generate short (700 bp) random genome sequence tags biosynthesis and resistance genes. DNA sequence of the 95- (GSTs) from a genomic library, (b) sequence comparison of kb region finally unveiled the ncs cluster, consisting of the GSTs to a database of microbial gene clusters known to be ncsA gene, a dNDP-glucose-4,6-dehydratase gene, as well as involved in natural product biosynthesis to identify other biosynthesis, resistance, and regulatory genes, a candidate genes, and (c) genomic library screening with genetic organization that is highly homologous to that of 7 selected GTSs as probes for clones containing genes of [48,53]. interest as well as neighboring ones that together may constitute a biosynthetic gene cluster. This method provides an efficient way to survey the biosynthetic potential for a Resistance Gene as A Probe given microorganism and to access specific biosynthetic This strategy was exemplified by the cloning of the gene cluster of interest. Using this method, Farnet and co- biosynthetic gene cluster for 2 from M. echanospora [60,61] workers cloned and sequenced several enediyne biosynthetic and should be applicable to gene clusters of both the 9- and loci from various microorganisms, including not only these 10-membered enediynes. To clone the 2 biosynthetic gene that were known to be enediyne producers such as M. cluster, Thorson and co-workers first screened a M. cherisina strain M956-1 for 9 and Streptomyces echanospora genomic library with probes designed macromyceticus strain M480-M1 for macromomycin but according to the conserved regions of both type I and type II also ones that previously were not known as enediyne PKSs on the assumption that the enediyene core is of producers, revealing several novel enediyne biosynthetic polyketide origin. Since it is known that most gene clusters. It came as a surprise that the enediyne PKS actinomycetes contain multiple polyketide pathways [49- loci were found at a very high frequency (~15%) among the 51,62,63], the resultant clones were further screened with organisms surveyed, suggesting that the ability of these probes designed according to the conserved regions of both organisms to produce enediynes or other bioactive natural glucose-1-phosphate nucleotidyltransferase and dNDP- products might have been greatly underestimated. Guided by glucose 4,6-dehydratase targeting the biosynthesis of the these genetic data, enediyne production by these organisms, deoxyhexose moieties of 2. While these preliminary including those that were not known as enediyne producers screenings were effective in identifying putative clones for previously, were demonstrated under selective growth the 2 biosynthetic locus, it was the finding that some of the conditions and detected by the enediyne-specific biochemical clones, positive towards both the PKS and deoxyhexose induction assays, supporting the involvement of these probes, also conferred 2 resistance that ultimately convinced clusters in enediyne biosynthesis [48]. these researchers that they had localized the biosynthetic gene cluster for 2 [47]. They subsequently mapped the 2 Enediyne PKS Gene as A Probe resistance gene to calC and characterized CalC as a non- heme iron metalloprotein, although the precise mechanism This strategy was exemplified by the cloning of the of how CalC confers 2 resistance remains to be established biosynthetic gene clusters for 3 from A. verrucosopora, 8 [60]. DNA sequencing of these clones finally unveiled the from A. madurea, and 9 from M. chersina and should be entire cal gene cluster, consisting of PKS genes, applicable to gene clusters of both 9- and 10-membered deoxyhexose biosynthesis genes, the calC resistance gene, as enediynes. The cloning and characterization of 7 and 2, as well as other biosynthesis, resistance, and regulatory genes. model systems for the 9- and 10-membered enediynes, Inactivation of the calE8 enediyne PKS gene abolished 2 respectively, revealed a common polyketide pathway for production, unambiguously confirming the involvement of enediyne biosynthesis characterized by the enediyne PKS, the cloned cal gene cluster in 2 biosynthesis [61]. iterative type I PKSs that exhibited head-to-tail sequence homology and have identical domain organization (Figure 2) [53,61,64]. Taking advantage of the remarkable similarity in Genome Scanning Method both sequence and domain organization of the enediyne This strategy was exemplified by the cloning of the PKSs and their associated accessory enzymes, we developed biosynthetic gene clusters for 9 from M. chersina strain a PCP-based approach to access the enediyne PKS loci using M956-1, macromomycin (the precise structure remains a set of primers designed according to various conserved unknown) from Streptomyces macromyceticus strain M480- region of these enzymes. The effectiveness of this approach M1, as well as several putative novel enediynes whose was demonstrated by the cloning of the enediyne PKS and structures are yet to be determined from other actinomycete its accessory enzymes for 3, 8, and 9 biosynthesis from the strains that previously were not known as enediyne corresponding producer, respectively [65]. The availability of producers. Since this method does not rely on specific the enediyne PKS loci has now set the stage to use them as biosynthetic machinery, it should be applicable to gene probes to clone the entire biosynthetic gene clusters. clusters of both the 9- and 10-membered enediynes as well 2322 Current Medicinal Chemistry, 2003, Vol. 10, No. 21 Shen et al.

Fig. (2). (A) Domain organization and sequence comparison between representative enediyne PKSs for a 9-membered enediyne core such as 7 (SgcE) and a 10-membered enediyne core such as 2 (CalE8). aa, amino acid (B) A unified paradigm for enediyne biosynthesis featuring the enediyne PKSs to account for the linear unsaturated polyketide intermediates, enediyne PKS accessory enzymes to branch into the 9- or 10-membered enediyne cores, and tailoring enzymes to imbue additional structural diversity. Atoms that were incorporated intact from the acyl CoA precursors to the enediyne cores are shown in bold.

A UNIFIED PARADIGM FOR ENEDIYNE suggest a common polyketide pathway for the biosynthesis BIOSYNTHESIS INVOLVING AN ITERATIVE of both 9- and 10-membered enediyne cores, despite the fact TYPE I PKS that their incorporation pattern by 13C-labeled acetate feeding experiments are distinct [47,64-66]. Common to all Although feeding experiments with 13C-labeled enediyne biosynthetic gene clusters known to date is the precursors clearly established that both the 9- and 10- enediyne PKS—an unprecedented type I PKS consisting of a membered enediyne cores were derived (minimally) from ketoacyl synthase (KS), an acyltransferase (AT), an acyl eight head-to-tail acetate units [47,64-66], controversy carrier protein (ACP), a ketoreductase (KR), a dehydratase remained until the recent cloning and characterization of the (DH), and a C-terminal domain (TD) (also proposed to biosynthetic gene clusters of 7 [53] and 2 [61] whether the encode a phosphopantetheinyl transferase) (Figure 2). enediyne cores are assembled by de novo polyketide Flanking the enediyne PKS gene are a group of five to ten biosynthesis or degradation from a fatty acid precursor. genes that are organizationally conserved among all Elucidation of the 7 and 2 biosynthetic gene clusters and characterized enediyne gene clusters. These genes encode the pathways, together with the cloning and sequence analysis of enediyne PKS accessory enzymes that are unusual the enediyne PKS loci for 1, 3, 8, and 9, now clearly oxidoreductases or proteins of unknown functions that are Biosynthesis and Prospect Towards Engineering Novel Antitumor Agents Current Medicinal Chemistry, 2003, Vol. 10, No. 21 2323 only associated with enediyne biosynthesis. Also identified the 9-membered enediyne biosynthetic pathways [48,52,53]. within the enediyne clusters are genes that encode a myriad Thorson and co-workers have successfully developed a of pathway-specific tailoring enzymes for the biosynthesis of genetic system for M. echinospora ssp. calichensis by PEG- the other structural moieties and their attachment to the mediated protopolast transformation and applied it to enediyne cores [48,53,61,64]. inactivate the calE8 enediyne PKS gene. The resultant calE8 Although these findings fell short of revealing the precise mutant lost its ability to produce 2, demonstrating the mechanism for enediyne core biosynthesis, they have feasibility of manipulating the biosynthesis of 10-membered provided sufficient data to formulate a unified paradigm for enediynes such as 2 in vivo [61]. enediyne biosynthesis. The enediyne PKSs could be Complementary to manipulating natural product envisaged to catalyze the assembly of a linear biosynthesis in their native producing organisms, progress polyunsaturated intermediate from the acyl CoA precursors has also been made to develop bacterial artificial in an iterative process. The nascent intermediate is chromosome (BAC) that can shuttle between E. coli and subsequently desaturated to furnish the two yne groups and Streptomyces species for expression of the target gene cluster cyclized to afford either a 9- or 10-membered enediyne core in a heterologous host. The entire biosynthetic gene cluster intermediate. The enediyne PKS accessory enzymes serve as could be cloned directly from chromosomal DNA [70-73] or the excellent candidates for these processing reactions. reconstructed from previously isolated overlapping cosmids Preliminary phylogenetic analysis of the enediyne PKSs and [74-76]. The resultant BAC could be mobilized into their accessory enzymes showed that they could be grouped genetically amenable host strains, such as Streptomyces into two distinct sub-families according to the 9- or 10- coelicolor or Streptomyces lividans, allowing genetic membered enediyne core structures. This finding suggested analysis and manipulation of biosynthetic gene clusters that that it might be even possible to predict the enediyne core are otherwise intractable genetically in their native producers. structure on the basis of sequence analysis of its biosynthetic While holding a great promise, this strategy has yet to gene cluster [65]. Further decoration of the enediyne cores demonstrate efficient expression of the entire gene cluster with the other structural moieties affords the structural and production of the encoded natural product in the diversity of the enediyne family of natural products, and the heterologous host. Attempts to clone the entire biosynthetic pathway specific tailoring enzymes are responsible for these clusters of 1 or 7 into BAC for heterologous expression were modifications. This emerging paradigm for enediyne not successful so far. In contrast, we have recently biosynthesis, consisting of (a) enediyne PKS-catalyzed discovered that the biosynthetic gene clusters for 1 and 7 synthesis of a linear polyketide intermediate, (b) enediyne reside on giant linear plasmids. By protoplast fusion, we PKS accessory enzymes-mediated processing of the enediyne successfully mobilized the giant linear plasmid for 1 core, and (c) pathway specific tailoring enzymes-driven biosynthesis from S. carzinosaticus into S. lividans and structural diversification of the enediyne natural products, confirmed 1 production in the heterologous host [Zhang, J.; has now laid the foundation to ask the fundamental Nonaka, K.; Shen, B. unpublished data]. These findings mechanistic questions for enediyne biosynthesis and to provided an exciting alternative to manipulate enediyne explore the potential of engineering novel enediyne biosynthesis in one of the best-characterized Strteptomyces compounds by applying combinatorial biosynthesis methods hosts. to the enediyne biosynthetic machinery. Although the engineering of novel enediynes with altered enediyne core has to wait for the fundamental understanding COMBINATORIAL BIOSYNTHESIS OF NOVEL of how the enediyne PKS and its accessory enzymes catalyze ENEDIYNES the enediyne core biosynthesis, the proof-of-principle for combinatorial biosynthesis of novel enediynes has already A prerequisite for applying combinatorial biosynthesis been demonstrated by the production of deshydroxy-C-1027 methods to engineer novel natural products is to have a (11) [53]. Characterization of the 7 biosynthetic gene cluster genetic system in place for the producing organism. Four and elucidation of the 7 biosynthetic pathway established general methods are available for introducing plasmid DNA that the C22-OH group of the β-amino acid moiety of 7 is into a Streptomyces species: (a) polyethyleneglycol (PEG)- introduced by the SgcC hydroxylase. Inactivation of the mediated protoplast transformation, (b) electroporation, (c) sgcC gene resulted in the production of a novel E. coli-Streptomyces conjugation, and (d) phage infection chromoprotein complex, the chromophore of which was [69]. However, the applicability of each method to a given confirmed to be 11. It was noteworthy that 11 is not only organism, cannot be predicted a priori, and the effectiveness biologically active as judged by bioassay with 7 as a of these methods to a particular species has to be determined positive control but also at least fivefold more stable than 7 empirically. In spite of these difficulties, significant progress at 25 °C in respect to undergoing the Bergman cyclization has been made in the past a few years for several enediyne [53]. The latter property could be potentially explored in producing organisms. We have developed efficient genetic developing 7 into clinically useful enediyne drugs. Taking systems for both the 7-producing S. globisporus and 1- together, these results not only demonstrated the feasibility producing S. carzinostaticus strains by either PEG-mediated of generating novel enediynes by manipulating the genes protoplast transformation or E. coli- Streptomyces governing their biosynthesis but also suggested that the conjugation. These methods were successfully applied to tailoring enzymes could be as equally rewarding as the carry out in vivo genetic characterization of 7 or 1 enediyne PKS and its accessory enzymes as the targets for biosynthesis by gene disruption, replacement, or engineering novel enediynes. Given the relatively conserved complementation experiments, setting the stage to engineer enediyne core structures yet distinct periphery moieties among the enediynes, it is tempting to speculate that some 2324 Current Medicinal Chemistry, 2003, Vol. 10, No. 21 Shen et al. of the tailoring enzymes could cross-talk among the various CA78747. B.S. is a recipient of National Science enediyne pathways, further expanding the enediyne structural Foundation CAREER Award MCB9733938 and National diversity accessible by combinatorial biosynthesis methods. Institutes of Health Independent Scientist Award AI51687.

CONCLUSION AND PROSPECTIVE REFERENCES

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