Drug Discovery Today: Technologies
Vol. 3, No. 3 2006
Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Medicinal chemistry
Progress in combinatorial biosynthesis for drug discovery Steven G. Van Lanen1, Ben Shen1,2,3,* 1
Division of Pharmaceutical Sciences, University of Wisconsin, Madison, WI 53705, USA University of Wisconsin National Cooperative Drug Discovery Group, Madison, WI 53705, USA 3 Department of Chemistry, University of Wisconsin, Madison, WI 53705, USA 2
Combinatorial biosynthesis, the process of genetic manipulations of natural product biosynthetic machinery for structural diversity, depends on several factors, and discussed here are two critical factors: access to genetic information and biochemical characterization
Section Editors: Li-he Zhang – School of Pharmaceutical Science, Peking University, Beijing, China Kaixian Chen – Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
of enzymes. Examples of the former include using predictions for the biosynthesis of unusual chemical entities such as aminohydroxybenzoic acid starter units, methoxymalonylate extender units, the enediyne core and bacterial aromatic polyketides. The latter aspect includes the continued elucidation of domain functionalities of modular polyketide synthases and nonribosomal peptide synthases and novel biochemical pathways such as the biosynthesis of a cyclopropyl unit and a b-hydroxyl acid. Finally, examples of successful combinatorial biosynthesis for daptomycin and indolocarbozole compounds are discussed. Introduction Natural products are a vital source of current clinical drugs. The Actinomycetales have clearly been the richest microbial source of bioactive compounds [1], and more often than not natural products are of polyketide or nonribosomal peptide origin. As a result, the biosynthetic enzymes responsible for the de novo construction of these structurally diverse and complex compounds have been intensely examined, and *Corresponding author: B. Shen ([email protected]) 1740-6749/$ Published by Elsevier Ltd.
DOI: 10.1016/j.ddtec.2006.09.014
research has established relatively few paradigms to account for the entirety of polyketides and nonribosomal peptides [2–5]. Consequently, the rational alteration, replacement, or genetic manipulation of the DNA encoding natural product biosynthetic machinery, which encompasses a strategy termed combinatorial biosynthesis, provides a conceptually feasible approach to produce an unlimited library of ‘unnatural’ natural products for drug discovery. The success of combinatorial biosynthesis depends on several factors, including the availability of the genetic information and the fundamental understanding of biosynthetic enzymes. Herein is discussed the progress related to these two concepts with the use of a select set of examples, primarily from natural products isolated from Actinomycetales. Several excellent reviews have been published elsewhere describing combinatorial biosynthesis related to specific families of natural products [6–8] and the progress of combinatorial biosynthesis for drug discovery [9–11].
Access to genetic information Combinatorial biosynthesis relies first and foremost on the availability of genetic information. Since the beginning of bioinformatics, databases have now expanded to include more than 100 gigabases of sequence data from 165,000 285
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organisms (http://www.nlm.nih.gov/news/press_releases/ dna_rna_100_gig.html), which includes nearly 400 whole genomes sequenced, and these numbers are increasing at steady rates. In addition, the observation that biosynthetic, resistance, and regulatory genes encoding natural product production in bacteria are clustered within one region of the microbial chromosome has simplified annotation of genes with respect to natural product biosynthesis. Therefore, it seems reasonable to assume that access to genetic information should not be limiting for combinatorial biosynthesis. Nevertheless, genome-sequencing projects to date have excluded a large majority of drug producers, with only a handful of Actinomycetales targeted. Therefore, a variety of strategies have been developed and refined to fish out characteristic DNA sequences that can ultimately be used to identify the biosynthetic machinery for families of compounds. The methodology that is typically the primary option for localizing gene clusters is using conserved regions within a polyketide synthase (PKS) and/or nonribosomal peptide synthase (NRPS) enzymes [12], or conserved regions of 4,6-dehydratase genes required for deoxysugar biosynthesis. By contrast, more specific approaches have recently appeared, which generally are targetted for a specific chemical feature of a family of compounds (Fig. 1). As discussed below, this has resulted in the cloning of the gene clusters for rifamycin B and ansamitocin P-3 [13], tautomycin and oxazolomycin [14,15], enediyne polyketides esperamicin and maduropeptin [16], and the spirotetronate chlorothricin [17].
3-Amino-5-hydroxybenzoic acid biosynthetic locus for cloning gene clusters Biosynthesis of the rifamycins and ansamitocins is initiated by a unique aromatic starter unit, 3-amino-5-hydroxybenzoic acid (AHBA), which in turn is biosynthesized by a branch of the shikimic acid pathway (Fig. 1a). By utilizing conserved regions of known AHBA synthase, the gene involved in 1 biosynthesis was cloned and subsequently used as a probe to identify the entire cluster [18]. In a similar manner, the gene cluster for the related compound 2 was also identified and cloned [19]. This strategy, therefore, can theoretically be applied to the entire ansamycin family of antibiotics, and likewise extended to other natural products containing similar shikimic acid-derived moieties.
Methoxymalonyl-acyl carrier protein biosynthetic locus for cloning gene clusters Numerous natural products contain a C2 unit within the polyketide chain that is derived from a glycolitic intermediate [20]. Cloning of the gene cluster for a few of these natural products has revealed a subcluster of four open reading frames (ORF) involved in the C2 precursor biosynthesis: a bifunctional glycerate phosphatase: transferase [21], an acyl carrier protein (ACP), and two dehydrogenases [22]. In some 286
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instances, such as the natural products 3 and 4 (Fig. 1b) a fifth ORF resides in this locus and functions as an O-methyltransferase. Labeling experiments and gene activations and complementation have verified the four ORFs involvement in the C2 units biosynthesis beginning from a reduced glycolytic intermediate [20]. By generating degenerate primers for the conserved acyl-CoA and b-hydroxyl-CoA dehydrogenases within this subcluster, both the 3 and 4 gene clusters were localized [14,15]. In these examples, using PCR amplification with degenerate primers based on modular PKS ketosynthase (KS) conserved regions would have not been expedient, if at all possible, because of the large number of PKS loci within the producer, a general phenomenon that has been observed upon whole genome sequencing and past cloning attempts in our laboratory. It is foreseen that this strategy can readily be used to localize other polyketide families with similar units, in other words polyketides containing a hydroxymalonate or methoxymalonate extender unit.
New bacterial polyketide synthases for cloning gene clusters Two final illustrations of alternative strategies in identifying gene clusters have coincided with the discovery of nontraditional PKS paradigms within bacteria. Typically, polyketide biosynthesis occurs by a noniterative, modular PKS (type I), a multienzyme complex of iteratively acting PKS activities (type II), or a homodimeric, iteratively acting PKS that is ACP independent (type III) [2,23]. However, a few type I PKS have now been identified harboring only one complete set of catalytic activities that are utilized in an iterative manner. As a first example, the enediyne polyketide family contains a single, unique 2000 amino acid PKS and a conserved set of flanking genes involved in the biosynthesis of the enediyne core. These conserved ORFs were subsequently used to prepare degenerate primers to PCR amplify the minimal enediyne PKS genes [16]. This approach was successfully applied for localizing and cloning the gene clusters of the enediynes 5 and 6 (Fig. 1c), providing rapid access to the minimal enediyne PKS gene cassettes without prior knowledge of the producing organism or pathway. By taking advantage of a separate bacterial iterative type I PKS that is different from the enediyne PKS family, a similar strategy was used in part to identify the chlorothricin gene cluster (Fig. 1d) [17]. Chlorothricin contains a 6-methylsalicylic acid (6-MSA) moiety, and probes were designed using PCR amplification of the KS and acyltransferase (AT) domains that are harbored within motifs identified by sequence alignments of three known bacterial iterative type I PKS (AviM, GenBank accession no. AAK83194 responsible for the orsellinic acid of avilamycin, CalO5 GenBank accession no. AAM70355 responsible for the orsellinic acid of calicheamicin, and NcsB, GenBank accession no. AAM77986 responsible for the naphthoic acid of neocarzinostatin), which are quite
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Figure 1. Natural products with recently cloned gene clusters. Structures for natural products whose gene clusters have recently been localized and cloned using knowledge of (a) AHBA biosynthesis for rifamycin B and ansamitocin P-3, (b) methoxymalonate biosynthesis for tautomycin(3) and oxazolomycin, (c) enediyne PKS for esperamicin and maduropeptin, and (d) bacterial iterative type I PKS for chlorothricin.
distinct from the archetype modular type I PKS. PCR using degenerate primers for these regions resulted in amplification of only a single iterative type I PKS (ChlB1, GenBank accession no. DQ116941) with the expected size, and the function of this PKS was subsequently confirmed by gene inactivation and complementation. ChlB1 now represents the fourth case
within bacteria of this type of unusual iterative type I PKSs responsible for aromatic polyketide biosynthesis. By targeting ORFs necessary for the biogenesis of specific chemical entities to localize and clone a gene cluster as illustrated by the examples above can be readily extrapolated to a variety of predicted enzyme chemistries and not just www.drugdiscoverytoday.com
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those mentioned. This approach nicely compliments classical strategies, which when used independently, have the disadvantage of resulting in several false positives because of the presence of unaccountable NRPS, PKS, and 4,6-dehydratase enzymes. Notable alternative strategies not discussed here are the generation of ‘perfect probes’ to identify all PKS and NRPS gene clusters [24] and the genome scanning approach to survey all natural product biosynthetic gene clusters without having the complete genome sequence as exemplified by identifying numerous cryptic enediyne biosynthetic loci from organisms that were previously not known as enedyine producers [25].
Functional predictions to biochemical characterization Combinatorial biosynthesis depends on the accurate predictions of the function of gene products, and ideally, in the absence of any prior knowledge of a structure of the natural product. For this goal to be fulfilled, the exact function of catalytic domains of NRPS and PKS and the biochemical characterization of other enzymes of a pathway are critical. Often new or unexpected chemistry is discovered during this process, which helps to rework current paradigms and define new strategies for natural product biosynthesis.
New activitites within NRPS and PKS NRPS and PKS domains catalyzing novel chemistry continue to be biochemically characterized. Often NRPS and PKS systems contain domains that are ‘non-essential’ to peptide or C–C bond formation but modify the nascent peptide or polyketide intermediate to imbue additional functionalities. As a result these domains can be considered to enrich the chemistry of the final product. For example, the mycosubtilin gene cluster consists of a hybrid PKS-NRPS system, and located at the interface of a large PKS-NRPS protein (MycA, GenBank accession no. AAF08795) is a predicted amino transferase (AMT) domain [26]. A recombinant protein construct containing four internal domains including the AMT domain was found to co-purify with the cofactor pyridoxal 50 -phosphate, and indeed was determined to be active as an AMT, transaminating the b-carbonyl of the ACP-bound growing polyketide intermediate (Fig. 2a). A second recent example is the biochemical elucidation of formylase (F) domain found at the N-terminus of the first NRPS module (LgrA, GenBank accession no. CAD92849) involved in linear gramicidin A biosynthesis (Fig. 2b) [27]. Although N-formylation is common in ribosomal translation initiation in bacteria, the F domain is unprecedented in NRPS. As was determined for the AMT domain, the formylase activity of the F domain was found to work in cis with the cognate peptidyl carrier protein (PCP) domain of the NRPS modular. By defining the AMT and F domain activities, this sets the stage to examine communication elements to downstream 288
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carrier proteins and overall substrate specificities, and therefore uncover the utility of the domains in NRPS and PKS engineering.
Cryptic enzyme activities in natural product biosynthetic pathways In many situations the prediction of a particular enzymatic conversion is not obvious from examination of the natural product. A case in point is the natural product coronotine, which contains a cyclopropyl ring that originates from the non-proteinogenic allo diastereomer of L-isoleucine, and only recently the strategy of cyclopropyl ring biosynthesis was unraveled [28]. Following loading of L-allo-isoleucine to a PCP, an a-ketoglutarate, iron-dependent halogenase (CmaB, GenBank accession no. AAC46036) incorporates chlorine into the inactivated, aliphatic g-carbon (Fig. 2c). A second, previously unknown enzyme (CmaC, GenBank accession no. AAQ93486), then uses the halogenated species to eliminate the chlorine concomitant with the formation of the cyclopropyl ring. A second example of hidden enzymatic conversions occurs during the biosynthesis of 6. Unexpectedly, the gene cluster for 6 contains homologs (minimum of 43% identity) for the six biosynthetic proteins involved in (S)-3-chloro-4,5-dihydroxy-b-phenylalanine biosynthesis of the enediyne C-1027 (9), and the pathway for 9 has been completely reconstituted in our laboratory using in vitro analysis of recombinant enzymes and in vivo gene inactivations. The initial step is conversion of L-a -tyrosine to (S)-b-tyrosine, catalyzed by an unusual aminomutase, followed sequentially by loading of (S)-b-tyrosine to a type II PCP, halogenation, hydroxylation, and incorporation into the enediyne core [29]. The first step for the biosynthesis of the analogous moiety found in 6 has now been confirmed, and, similar to C-1027 biogenesis, the first reaction is indeed formation of (S)-b-tyrosine catalyzed by MdpC4 (Fig. 2d). The next step is likely activation and incorporation of (S)-b-tyrosine to a type II PCP as catalyzed by MdpC1. However, in contrast to the C-1027 pathway, 6 not only contains different regiochemistry respective to halogenation and hydroxylation but also three further bioconversions, two of which afford the b-hydroxyl moiety via a transamination catalyzed by MdpC7 followed by reduction. Although the precise timing remains unclear, this pathway represents an alternative approach to introduce a hydroxyl functionality, which typically originates from a substrate precursor or from molecular oxygen by the activity of oxygenases during natural product biosynthesis. In addition to the discovery of cryptic halogenation in the biosynthesis of 8, the unprecedented strategy for b-hydroxyl acid biosynthesis by an aminomutase, a transaminase, and a reductase unveiled from 6 should ultimately aid designing de novo biosynthesis of novel natural products in combinatorial biosynthesis.
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Figure 2. Characterization of enzyme activities in natural product biosynthesis. Enzyme activities that have recently been characterized exemplified by (a) new domain activities from modular hybrid PKS-NRPS or (b) NRPS systems, (c) the newly discovered cryptic halogenase pathway for cyclopropyl unit biosynthesis, and (d) b-hydroxyl acid biosynthesis by an aminomutase-transaminase-reductase strategy in maduropeptin in comparison with b-amino acid biosynthesis in C-1027 (the exact timing of catalysis, excluding MdpC4, remains unknown). Shown in blocks are the recombinant protein constructs that were characterized. Abbreviations are ACP, acyl carrier protein; AMT, amino transferase; C, condensation; PCP, peptidyl carrier protein; PLP, pyridoxal-50 -phosphate; Gln, glutamine; F, formylase; and A, adenylation.
Novel types of bacterial polyketide synthases Finally, even the standard, accepted blueprints for nonribosomal peptide and polyketide biosynthesis have not been immune to discoveries that reside outside the current para-
digms. For example, classical modular type I PKS systems contain a minimum of 3 domains: a KS, an AT, and an ACP. However, many systems have been found where the AT domain is, first, not present within the modular PKS, and www.drugdiscoverytoday.com
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second, is located as a discrete protein that functions in trans [30,31]. These trans AT systems present a new way to load acyl-CoA units to an ACP, and also present a means to engineer new natural products by altering the loading and extender unit-specificity by genetically manipulating the trans AT. A second type of new PKS system has recently been discovered as previously mentioned with the uncovering of bacterial iterative type I PKS systems involved in aromatic polyketide biosynthesis. Prior efforts have lead to the general consensus that an iterative type II PKS is responsible for the production of aromatic polyketides in bacteria. However, heterologous expression of NcsB clearly revealed the PKS catalyzes multiple rounds of decarboxylative condensation, likely with regiospecific ketoduction, to afford the naphthoic acid moiety of NCS [32]. This new paradigm was also confirmed by the production of 6-MSA of 7 by the heterologous expression of ChlB1 [33].
Successful combinatorial biosynthesis It is evident from the examples above that there is much to be gained by the biochemical characterization of individual enzymes within natural product biosynthetic pathways. As a result, combinatorial biosynthesis has primarily been limited to metabolic engineering of single biosynthetic pathways. In other words, most examples in generating structural diversity of a natural product have been achieved by gene disruption or gene inactivation, therefore, changing the corresponding chemical property in the final product. However, there have been two notable examples where combinatorial biosynthesis in its truest form has been applied and successful: the engineering of new daptomycin-like lipopeptide antibiotics [34] and indolocarbazole compounds [35].
Combinatorial biosynthesis using daptomycin Daptomycin (9), produced by Streptomyces roseosporus, is a 13amino acid peptide with a linked saturated fatty acid (Fig. 3a), and recently 9, under the trade name of Cubicin1, has been approved to treat against gram positive pathogens. A genetic system was developed in the producer to generate NRPS mutant strains that could be complemented in trans. This allowed for the exchange of NRPS modules encoding the biosynthesis of congeners A54145 from Streptomyces fradiae and calcium dependent antibiotic from Streptomyces coelicolor, which contain a similar structure as 9 but have substitutions in several the 13 amino acid positions. Hence, by exchanging a module, the adenylation-amino acid specificity was changed to introduce different amino acids along the peptide chain, and barring that downstream events were forgiving, new 9-derivatives were isolated. Alternatively, the fatty acid side chain was substituted to introduce alternative chemical functionality. In the end, 72 new lipopeptides were produced and the specific bioactivity of these new compounds should be available soon. 290
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Figure 3. Natural products targeted for combinatorial biosynthesis. Structures of natural products whose biosynthetic gene clusters have been targeted for combinatorial biosynthesis resulting in (a) a library of daptomycin analogs, and (b) a library of indolocarbazole analogs.
Combinatorial biosynthesis using indolocarbazole compounds A second, successful example of combinatorial biosynthesis was recently described within the indolocarbazole family of antibiotics [35]. The indolocarbozoles are antitumor compounds that include rebeccamycin (10) and staurospaurine (11, Fig. 3b). The entire ten gene cluster was introduced into the heterologous host Streptomyces albus by both an integrative vector and a replicative vector, and production of 10 was confirmed. This allowed the capability to delete specific genes contained within the replicative plasmid and monitor potential intermediates, or cross-complement with similar genes from other biosynthetic pathways such as the congener 11. Alternatively, tryptophan halogenase genes that incorporate chlorine with different regiospecificity were also used. In total, >30 indolocarbazole derivatives were prepared, and although similar to the engineering of new 9-like antibiotics, the genetic engineering of new indolocarbazoles extends the utility of combinatorial biosynthesis by demonstrating the feasibility to incorporate genetic elements from unrelated families of natural products to produce new compounds.
Conclusions Combinatorial biosynthesis, which has been primarily limited to gene manipulation of a single natural product biosyn-
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thetic pathway, has now come to encompass its truest definition by the successful gene swapping from different pathways, even from non-related families of natural products as shown for the indolocarbazoles. As highlighted above, the methodology relies in part on access to genetic information and the accurate predictions of gene functions, which is often attained during the process of combinatorial biosynthesis. Factors not mentioned here but have been discussed elsewhere [36] include an expedient genetic system for the native producer, function expression of the entire cluster in a heterologous host [37], optimizing culture conditions [38], and reliable isolation and detection procedures for the compound of interest and its derivatives. Of the factors discussed for successful combinatorial biosynthesis, the accessibility to genetic information is clearly playing a less significant role compared to the past. Whole genome sequences continue to be fully annotated at a steady rate, and using predicted chemistries to ‘fish’ out gene clusters has become commonplace in the current literature, as exemplified by the cloning of 1–7 gene clusters. The strategy of using a specific probe to look for unusual chemistry is especially relevant considering the finding of numerous unknown PKS and NRPS genes upon whole genome sequencing of the model Streptomyces organisms, S. coelicolor and S. avertimilis [39]. This phenomenon clearly has given the use of PKS and NRPS regions as probes less utility because often unwanted clusters result in false positives. Biochemical characterization of natural product biosynthetic pathways continues to unveil novel biochemistry, enzymology, and strategies utilized by the organism to produce complex, bioactive molecules. Knowledge of new enzyme activities enhances the combinatorial biosynthesis toolbox, and it is foreseen that new chemistries, such as the AMT domain of MycA and the F domain of LgrA, can be used as a means to introduce a chemical handle for further chemical modification. Although the engineered handles may lack the in vitro potency displayed by the parental natural product, as is typically the case, what is often overlooked is the impact on other properties that may lower cytotoxicity, for example solubility, stability, specificity, and drug uptake, which can be significantly altered and improved via engineering. Finally, the characterization of biochemical pathways should ultimately lead to predictions of structure of compounds based solely on the genetic information, a glimpse of which has been seen with the structural deciphering of coelochelin [40]. In conclusion, combinatorial biosynthesis continues to incrementally progress toward an ideal scenario: genetic engineering as a universal application that is both programmable and predictable. Considering that the first description of combinatorial biosynthesis was only about two decades ago [13], considerable progress has been made. Undoubtedly, the next two decades will bring numerous examples of
Drug Discovery Today: Technologies | Medicinal chemistry
successful combinatorial biosynthesis, and among the libraries of compounds generated, a better drug will be realized.
Acknowledgements Current studies on natural product biosynthesis and engineering described from the Shen laboratory are supported in part by National Institutes of Health (NIH) grants CA94426, CA78747, CA106150, and CA113296. S.V.L is a recipient of an NIH postdoctoral fellowship CA1059845, and B.S is the recipient of an NIH Independent Scientist Award AI51689.
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Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Medicinal chemistry
Progress in combinatorial biosynthesis for drug discovery Steven G. Van Lanen1, Ben Shen1,2,3,* 1
Division of Pharmaceutical Sciences, University of Wisconsin, Madison, WI 53705, USA University of Wisconsin National Cooperative Drug Discovery Group, Madison, WI 53705, USA 3 Department of Chemistry, University of Wisconsin, Madison, WI 53705, USA 2
Combinatorial biosynthesis, the process of genetic manipulations of natural product biosynthetic machinery for structural diversity, depends on several factors, and discussed here are two critical factors: access to genetic information and biochemical characterization
Section Editors: Li-he Zhang – School of Pharmaceutical Science, Peking University, Beijing, China Kaixian Chen – Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
of enzymes. Examples of the former include using predictions for the biosynthesis of unusual chemical entities such as aminohydroxybenzoic acid starter units, methoxymalonylate extender units, the enediyne core and bacterial aromatic polyketides. The latter aspect includes the continued elucidation of domain functionalities of modular polyketide synthases and nonribosomal peptide synthases and novel biochemical pathways such as the biosynthesis of a cyclopropyl unit and a b-hydroxyl acid. Finally, examples of successful combinatorial biosynthesis for daptomycin and indolocarbozole compounds are discussed. Introduction Natural products are a vital source of current clinical drugs. The Actinomycetales have clearly been the richest microbial source of bioactive compounds [1], and more often than not natural products are of polyketide or nonribosomal peptide origin. As a result, the biosynthetic enzymes responsible for the de novo construction of these structurally diverse and complex compounds have been intensely examined, and *Corresponding author: B. Shen ([email protected]) 1740-6749/$ Published by Elsevier Ltd.
DOI: 10.1016/j.ddtec.2006.09.014
research has established relatively few paradigms to account for the entirety of polyketides and nonribosomal peptides [2–5]. Consequently, the rational alteration, replacement, or genetic manipulation of the DNA encoding natural product biosynthetic machinery, which encompasses a strategy termed combinatorial biosynthesis, provides a conceptually feasible approach to produce an unlimited library of ‘unnatural’ natural products for drug discovery. The success of combinatorial biosynthesis depends on several factors, including the availability of the genetic information and the fundamental understanding of biosynthetic enzymes. Herein is discussed the progress related to these two concepts with the use of a select set of examples, primarily from natural products isolated from Actinomycetales. Several excellent reviews have been published elsewhere describing combinatorial biosynthesis related to specific families of natural products [6–8] and the progress of combinatorial biosynthesis for drug discovery [9–11].
Access to genetic information Combinatorial biosynthesis relies first and foremost on the availability of genetic information. Since the beginning of bioinformatics, databases have now expanded to include more than 100 gigabases of sequence data from 165,000 285
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organisms (http://www.nlm.nih.gov/news/press_releases/ dna_rna_100_gig.html), which includes nearly 400 whole genomes sequenced, and these numbers are increasing at steady rates. In addition, the observation that biosynthetic, resistance, and regulatory genes encoding natural product production in bacteria are clustered within one region of the microbial chromosome has simplified annotation of genes with respect to natural product biosynthesis. Therefore, it seems reasonable to assume that access to genetic information should not be limiting for combinatorial biosynthesis. Nevertheless, genome-sequencing projects to date have excluded a large majority of drug producers, with only a handful of Actinomycetales targeted. Therefore, a variety of strategies have been developed and refined to fish out characteristic DNA sequences that can ultimately be used to identify the biosynthetic machinery for families of compounds. The methodology that is typically the primary option for localizing gene clusters is using conserved regions within a polyketide synthase (PKS) and/or nonribosomal peptide synthase (NRPS) enzymes [12], or conserved regions of 4,6-dehydratase genes required for deoxysugar biosynthesis. By contrast, more specific approaches have recently appeared, which generally are targetted for a specific chemical feature of a family of compounds (Fig. 1). As discussed below, this has resulted in the cloning of the gene clusters for rifamycin B and ansamitocin P-3 [13], tautomycin and oxazolomycin [14,15], enediyne polyketides esperamicin and maduropeptin [16], and the spirotetronate chlorothricin [17].
3-Amino-5-hydroxybenzoic acid biosynthetic locus for cloning gene clusters Biosynthesis of the rifamycins and ansamitocins is initiated by a unique aromatic starter unit, 3-amino-5-hydroxybenzoic acid (AHBA), which in turn is biosynthesized by a branch of the shikimic acid pathway (Fig. 1a). By utilizing conserved regions of known AHBA synthase, the gene involved in 1 biosynthesis was cloned and subsequently used as a probe to identify the entire cluster [18]. In a similar manner, the gene cluster for the related compound 2 was also identified and cloned [19]. This strategy, therefore, can theoretically be applied to the entire ansamycin family of antibiotics, and likewise extended to other natural products containing similar shikimic acid-derived moieties.
Methoxymalonyl-acyl carrier protein biosynthetic locus for cloning gene clusters Numerous natural products contain a C2 unit within the polyketide chain that is derived from a glycolitic intermediate [20]. Cloning of the gene cluster for a few of these natural products has revealed a subcluster of four open reading frames (ORF) involved in the C2 precursor biosynthesis: a bifunctional glycerate phosphatase: transferase [21], an acyl carrier protein (ACP), and two dehydrogenases [22]. In some 286
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instances, such as the natural products 3 and 4 (Fig. 1b) a fifth ORF resides in this locus and functions as an O-methyltransferase. Labeling experiments and gene activations and complementation have verified the four ORFs involvement in the C2 units biosynthesis beginning from a reduced glycolytic intermediate [20]. By generating degenerate primers for the conserved acyl-CoA and b-hydroxyl-CoA dehydrogenases within this subcluster, both the 3 and 4 gene clusters were localized [14,15]. In these examples, using PCR amplification with degenerate primers based on modular PKS ketosynthase (KS) conserved regions would have not been expedient, if at all possible, because of the large number of PKS loci within the producer, a general phenomenon that has been observed upon whole genome sequencing and past cloning attempts in our laboratory. It is foreseen that this strategy can readily be used to localize other polyketide families with similar units, in other words polyketides containing a hydroxymalonate or methoxymalonate extender unit.
New bacterial polyketide synthases for cloning gene clusters Two final illustrations of alternative strategies in identifying gene clusters have coincided with the discovery of nontraditional PKS paradigms within bacteria. Typically, polyketide biosynthesis occurs by a noniterative, modular PKS (type I), a multienzyme complex of iteratively acting PKS activities (type II), or a homodimeric, iteratively acting PKS that is ACP independent (type III) [2,23]. However, a few type I PKS have now been identified harboring only one complete set of catalytic activities that are utilized in an iterative manner. As a first example, the enediyne polyketide family contains a single, unique 2000 amino acid PKS and a conserved set of flanking genes involved in the biosynthesis of the enediyne core. These conserved ORFs were subsequently used to prepare degenerate primers to PCR amplify the minimal enediyne PKS genes [16]. This approach was successfully applied for localizing and cloning the gene clusters of the enediynes 5 and 6 (Fig. 1c), providing rapid access to the minimal enediyne PKS gene cassettes without prior knowledge of the producing organism or pathway. By taking advantage of a separate bacterial iterative type I PKS that is different from the enediyne PKS family, a similar strategy was used in part to identify the chlorothricin gene cluster (Fig. 1d) [17]. Chlorothricin contains a 6-methylsalicylic acid (6-MSA) moiety, and probes were designed using PCR amplification of the KS and acyltransferase (AT) domains that are harbored within motifs identified by sequence alignments of three known bacterial iterative type I PKS (AviM, GenBank accession no. AAK83194 responsible for the orsellinic acid of avilamycin, CalO5 GenBank accession no. AAM70355 responsible for the orsellinic acid of calicheamicin, and NcsB, GenBank accession no. AAM77986 responsible for the naphthoic acid of neocarzinostatin), which are quite
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Figure 1. Natural products with recently cloned gene clusters. Structures for natural products whose gene clusters have recently been localized and cloned using knowledge of (a) AHBA biosynthesis for rifamycin B and ansamitocin P-3, (b) methoxymalonate biosynthesis for tautomycin(3) and oxazolomycin, (c) enediyne PKS for esperamicin and maduropeptin, and (d) bacterial iterative type I PKS for chlorothricin.
distinct from the archetype modular type I PKS. PCR using degenerate primers for these regions resulted in amplification of only a single iterative type I PKS (ChlB1, GenBank accession no. DQ116941) with the expected size, and the function of this PKS was subsequently confirmed by gene inactivation and complementation. ChlB1 now represents the fourth case
within bacteria of this type of unusual iterative type I PKSs responsible for aromatic polyketide biosynthesis. By targeting ORFs necessary for the biogenesis of specific chemical entities to localize and clone a gene cluster as illustrated by the examples above can be readily extrapolated to a variety of predicted enzyme chemistries and not just www.drugdiscoverytoday.com
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those mentioned. This approach nicely compliments classical strategies, which when used independently, have the disadvantage of resulting in several false positives because of the presence of unaccountable NRPS, PKS, and 4,6-dehydratase enzymes. Notable alternative strategies not discussed here are the generation of ‘perfect probes’ to identify all PKS and NRPS gene clusters [24] and the genome scanning approach to survey all natural product biosynthetic gene clusters without having the complete genome sequence as exemplified by identifying numerous cryptic enediyne biosynthetic loci from organisms that were previously not known as enedyine producers [25].
Functional predictions to biochemical characterization Combinatorial biosynthesis depends on the accurate predictions of the function of gene products, and ideally, in the absence of any prior knowledge of a structure of the natural product. For this goal to be fulfilled, the exact function of catalytic domains of NRPS and PKS and the biochemical characterization of other enzymes of a pathway are critical. Often new or unexpected chemistry is discovered during this process, which helps to rework current paradigms and define new strategies for natural product biosynthesis.
New activitites within NRPS and PKS NRPS and PKS domains catalyzing novel chemistry continue to be biochemically characterized. Often NRPS and PKS systems contain domains that are ‘non-essential’ to peptide or C–C bond formation but modify the nascent peptide or polyketide intermediate to imbue additional functionalities. As a result these domains can be considered to enrich the chemistry of the final product. For example, the mycosubtilin gene cluster consists of a hybrid PKS-NRPS system, and located at the interface of a large PKS-NRPS protein (MycA, GenBank accession no. AAF08795) is a predicted amino transferase (AMT) domain [26]. A recombinant protein construct containing four internal domains including the AMT domain was found to co-purify with the cofactor pyridoxal 50 -phosphate, and indeed was determined to be active as an AMT, transaminating the b-carbonyl of the ACP-bound growing polyketide intermediate (Fig. 2a). A second recent example is the biochemical elucidation of formylase (F) domain found at the N-terminus of the first NRPS module (LgrA, GenBank accession no. CAD92849) involved in linear gramicidin A biosynthesis (Fig. 2b) [27]. Although N-formylation is common in ribosomal translation initiation in bacteria, the F domain is unprecedented in NRPS. As was determined for the AMT domain, the formylase activity of the F domain was found to work in cis with the cognate peptidyl carrier protein (PCP) domain of the NRPS modular. By defining the AMT and F domain activities, this sets the stage to examine communication elements to downstream 288
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carrier proteins and overall substrate specificities, and therefore uncover the utility of the domains in NRPS and PKS engineering.
Cryptic enzyme activities in natural product biosynthetic pathways In many situations the prediction of a particular enzymatic conversion is not obvious from examination of the natural product. A case in point is the natural product coronotine, which contains a cyclopropyl ring that originates from the non-proteinogenic allo diastereomer of L-isoleucine, and only recently the strategy of cyclopropyl ring biosynthesis was unraveled [28]. Following loading of L-allo-isoleucine to a PCP, an a-ketoglutarate, iron-dependent halogenase (CmaB, GenBank accession no. AAC46036) incorporates chlorine into the inactivated, aliphatic g-carbon (Fig. 2c). A second, previously unknown enzyme (CmaC, GenBank accession no. AAQ93486), then uses the halogenated species to eliminate the chlorine concomitant with the formation of the cyclopropyl ring. A second example of hidden enzymatic conversions occurs during the biosynthesis of 6. Unexpectedly, the gene cluster for 6 contains homologs (minimum of 43% identity) for the six biosynthetic proteins involved in (S)-3-chloro-4,5-dihydroxy-b-phenylalanine biosynthesis of the enediyne C-1027 (9), and the pathway for 9 has been completely reconstituted in our laboratory using in vitro analysis of recombinant enzymes and in vivo gene inactivations. The initial step is conversion of L-a -tyrosine to (S)-b-tyrosine, catalyzed by an unusual aminomutase, followed sequentially by loading of (S)-b-tyrosine to a type II PCP, halogenation, hydroxylation, and incorporation into the enediyne core [29]. The first step for the biosynthesis of the analogous moiety found in 6 has now been confirmed, and, similar to C-1027 biogenesis, the first reaction is indeed formation of (S)-b-tyrosine catalyzed by MdpC4 (Fig. 2d). The next step is likely activation and incorporation of (S)-b-tyrosine to a type II PCP as catalyzed by MdpC1. However, in contrast to the C-1027 pathway, 6 not only contains different regiochemistry respective to halogenation and hydroxylation but also three further bioconversions, two of which afford the b-hydroxyl moiety via a transamination catalyzed by MdpC7 followed by reduction. Although the precise timing remains unclear, this pathway represents an alternative approach to introduce a hydroxyl functionality, which typically originates from a substrate precursor or from molecular oxygen by the activity of oxygenases during natural product biosynthesis. In addition to the discovery of cryptic halogenation in the biosynthesis of 8, the unprecedented strategy for b-hydroxyl acid biosynthesis by an aminomutase, a transaminase, and a reductase unveiled from 6 should ultimately aid designing de novo biosynthesis of novel natural products in combinatorial biosynthesis.
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Figure 2. Characterization of enzyme activities in natural product biosynthesis. Enzyme activities that have recently been characterized exemplified by (a) new domain activities from modular hybrid PKS-NRPS or (b) NRPS systems, (c) the newly discovered cryptic halogenase pathway for cyclopropyl unit biosynthesis, and (d) b-hydroxyl acid biosynthesis by an aminomutase-transaminase-reductase strategy in maduropeptin in comparison with b-amino acid biosynthesis in C-1027 (the exact timing of catalysis, excluding MdpC4, remains unknown). Shown in blocks are the recombinant protein constructs that were characterized. Abbreviations are ACP, acyl carrier protein; AMT, amino transferase; C, condensation; PCP, peptidyl carrier protein; PLP, pyridoxal-50 -phosphate; Gln, glutamine; F, formylase; and A, adenylation.
Novel types of bacterial polyketide synthases Finally, even the standard, accepted blueprints for nonribosomal peptide and polyketide biosynthesis have not been immune to discoveries that reside outside the current para-
digms. For example, classical modular type I PKS systems contain a minimum of 3 domains: a KS, an AT, and an ACP. However, many systems have been found where the AT domain is, first, not present within the modular PKS, and www.drugdiscoverytoday.com
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second, is located as a discrete protein that functions in trans [30,31]. These trans AT systems present a new way to load acyl-CoA units to an ACP, and also present a means to engineer new natural products by altering the loading and extender unit-specificity by genetically manipulating the trans AT. A second type of new PKS system has recently been discovered as previously mentioned with the uncovering of bacterial iterative type I PKS systems involved in aromatic polyketide biosynthesis. Prior efforts have lead to the general consensus that an iterative type II PKS is responsible for the production of aromatic polyketides in bacteria. However, heterologous expression of NcsB clearly revealed the PKS catalyzes multiple rounds of decarboxylative condensation, likely with regiospecific ketoduction, to afford the naphthoic acid moiety of NCS [32]. This new paradigm was also confirmed by the production of 6-MSA of 7 by the heterologous expression of ChlB1 [33].
Successful combinatorial biosynthesis It is evident from the examples above that there is much to be gained by the biochemical characterization of individual enzymes within natural product biosynthetic pathways. As a result, combinatorial biosynthesis has primarily been limited to metabolic engineering of single biosynthetic pathways. In other words, most examples in generating structural diversity of a natural product have been achieved by gene disruption or gene inactivation, therefore, changing the corresponding chemical property in the final product. However, there have been two notable examples where combinatorial biosynthesis in its truest form has been applied and successful: the engineering of new daptomycin-like lipopeptide antibiotics [34] and indolocarbazole compounds [35].
Combinatorial biosynthesis using daptomycin Daptomycin (9), produced by Streptomyces roseosporus, is a 13amino acid peptide with a linked saturated fatty acid (Fig. 3a), and recently 9, under the trade name of Cubicin1, has been approved to treat against gram positive pathogens. A genetic system was developed in the producer to generate NRPS mutant strains that could be complemented in trans. This allowed for the exchange of NRPS modules encoding the biosynthesis of congeners A54145 from Streptomyces fradiae and calcium dependent antibiotic from Streptomyces coelicolor, which contain a similar structure as 9 but have substitutions in several the 13 amino acid positions. Hence, by exchanging a module, the adenylation-amino acid specificity was changed to introduce different amino acids along the peptide chain, and barring that downstream events were forgiving, new 9-derivatives were isolated. Alternatively, the fatty acid side chain was substituted to introduce alternative chemical functionality. In the end, 72 new lipopeptides were produced and the specific bioactivity of these new compounds should be available soon. 290
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Figure 3. Natural products targeted for combinatorial biosynthesis. Structures of natural products whose biosynthetic gene clusters have been targeted for combinatorial biosynthesis resulting in (a) a library of daptomycin analogs, and (b) a library of indolocarbazole analogs.
Combinatorial biosynthesis using indolocarbazole compounds A second, successful example of combinatorial biosynthesis was recently described within the indolocarbazole family of antibiotics [35]. The indolocarbozoles are antitumor compounds that include rebeccamycin (10) and staurospaurine (11, Fig. 3b). The entire ten gene cluster was introduced into the heterologous host Streptomyces albus by both an integrative vector and a replicative vector, and production of 10 was confirmed. This allowed the capability to delete specific genes contained within the replicative plasmid and monitor potential intermediates, or cross-complement with similar genes from other biosynthetic pathways such as the congener 11. Alternatively, tryptophan halogenase genes that incorporate chlorine with different regiospecificity were also used. In total, >30 indolocarbazole derivatives were prepared, and although similar to the engineering of new 9-like antibiotics, the genetic engineering of new indolocarbazoles extends the utility of combinatorial biosynthesis by demonstrating the feasibility to incorporate genetic elements from unrelated families of natural products to produce new compounds.
Conclusions Combinatorial biosynthesis, which has been primarily limited to gene manipulation of a single natural product biosyn-
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thetic pathway, has now come to encompass its truest definition by the successful gene swapping from different pathways, even from non-related families of natural products as shown for the indolocarbazoles. As highlighted above, the methodology relies in part on access to genetic information and the accurate predictions of gene functions, which is often attained during the process of combinatorial biosynthesis. Factors not mentioned here but have been discussed elsewhere [36] include an expedient genetic system for the native producer, function expression of the entire cluster in a heterologous host [37], optimizing culture conditions [38], and reliable isolation and detection procedures for the compound of interest and its derivatives. Of the factors discussed for successful combinatorial biosynthesis, the accessibility to genetic information is clearly playing a less significant role compared to the past. Whole genome sequences continue to be fully annotated at a steady rate, and using predicted chemistries to ‘fish’ out gene clusters has become commonplace in the current literature, as exemplified by the cloning of 1–7 gene clusters. The strategy of using a specific probe to look for unusual chemistry is especially relevant considering the finding of numerous unknown PKS and NRPS genes upon whole genome sequencing of the model Streptomyces organisms, S. coelicolor and S. avertimilis [39]. This phenomenon clearly has given the use of PKS and NRPS regions as probes less utility because often unwanted clusters result in false positives. Biochemical characterization of natural product biosynthetic pathways continues to unveil novel biochemistry, enzymology, and strategies utilized by the organism to produce complex, bioactive molecules. Knowledge of new enzyme activities enhances the combinatorial biosynthesis toolbox, and it is foreseen that new chemistries, such as the AMT domain of MycA and the F domain of LgrA, can be used as a means to introduce a chemical handle for further chemical modification. Although the engineered handles may lack the in vitro potency displayed by the parental natural product, as is typically the case, what is often overlooked is the impact on other properties that may lower cytotoxicity, for example solubility, stability, specificity, and drug uptake, which can be significantly altered and improved via engineering. Finally, the characterization of biochemical pathways should ultimately lead to predictions of structure of compounds based solely on the genetic information, a glimpse of which has been seen with the structural deciphering of coelochelin [40]. In conclusion, combinatorial biosynthesis continues to incrementally progress toward an ideal scenario: genetic engineering as a universal application that is both programmable and predictable. Considering that the first description of combinatorial biosynthesis was only about two decades ago [13], considerable progress has been made. Undoubtedly, the next two decades will bring numerous examples of
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successful combinatorial biosynthesis, and among the libraries of compounds generated, a better drug will be realized.
Acknowledgements Current studies on natural product biosynthesis and engineering described from the Shen laboratory are supported in part by National Institutes of Health (NIH) grants CA94426, CA78747, CA106150, and CA113296. S.V.L is a recipient of an NIH postdoctoral fellowship CA1059845, and B.S is the recipient of an NIH Independent Scientist Award AI51689.
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