enzyme technologies (metagenomics, evolution, biocatalysis, and biosynthesis) || drug discovery and...

PART C

BIOSYNTHETIC APPLICATIONS

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8DRUG DISCOVERYAND DEVELOPMENT BYCOMBINATORIAL BIOSYNTHESIS

Matthew A. DeSieno and Carl A. DenardDepartment of Chemical and Biomolecular Engineering and Institute for GenomicBiology, University of Illinois at Urbana-Champaign, Urbana, Illinois

Huimin ZhaoDepartment of Chemical and Biomolecular Engineering and Institute for GenomicBiology, and Departments of Chemistry, Biochemistry, and Bioengineering, Universityof Illinois at Urbana-Champaign, Urbana, Illinois

I. INTRODUCTION

Over the past 50 years, natural products have been one of the key sources of ther-apeutic agents in the pharmaceutical industry. Between 1981 and 2002, 49% ofnew chemical entities introduced into clinical use were of either natural productorigin or inspiration. That number rises up to approximately 75% when con-sidering the drugs used in the treatment of severe or life-threatening conditions[1]. Some of the clinically important natural products include the antimicrobialserythromycin, oleandomycin, tylosin, and vancomycin, the immunosuppressantdrugs such as cyclosporine, FK506, and rapamycin; and the antitumor drugsdoxorubicin, bleomycin, and the epothilones [2]. Natural products contain a verycomplex architecture and rich functionality, which undoubtedly results in theirhigh potency and selectivity, making them attractive drug candidates [3].

Originally, natural products used in therapeutic formulations typically camefrom relatively crude plant extracts [4]. These extracts were first screened for any

Enzyme Technologies: Metagenomics, Evolution, Biocatalysis, and Biosynthesis,Edited by Wu-Kuang Yeh, Hsiu-Chiung Yang, and James R. McCarthyCopyright © 2010 John Wiley & Sons, Inc.

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254 DRUG DISCOVERY AND DEVELOPMENT BY COMBINATORIAL BIOSYNTHESIS

antibacterial inhibition before being further pursued as potential therapeutics.At that time, the producing organisms were seen only as black boxes, sincescientists were unaware of the biosynthetic clusters within the genome whichwere responsible for the production of these novel compounds [5]. The “GoldenEra” of natural product discovery began shortly after the large-scale productionof penicillin and search for new antibiotics during World War II. During thistime period, many new natural products were discovered, including streptomycin,gentamicin, and tetracycline, as pharmaceutical companies targeted antibacterials,antifungals, and many other infectious diseases [4]. Industry slowly began todeemphasize natural product research during the 1980s and 1990s, as it wasperceived as an obsolete technology [3]. Although still able to give high-qualitydrug target leads, natural product research began to lose out to the highly efficientlarge chemical libraries created by combinatorial chemistries, since traditionalnatural product discovery was considerably more sluggish and time consumingand required extensive resources [6].

The major problem that both the pharmaceutical industry and academic labora-tories currently face is that the number of antibiotic-resistant bacteria is growingat an alarming rate. The onset of resistance diminishes the effectiveness of currentdrugs, which has now created a large demand for the discovery and productionof new antibiotics for the treatment of infectious diseases [7]. Combinatorialbiosynthesis is one such method that is being used to find these highly valuablecompounds. This method involves the use of recombinant DNA technology tomanipulate the genetic machinery encoding the biosynthesis of natural productsto create diverse libraries of new compounds. Such genetic machinery is typicallyclustered within the genome of the native producer. Combinatorial biosynthesishas the potential to create variant compounds that may be more effective againstproblematic or resistant strains of bacteria [8].

Natural products are an excellent starting point for combinatorial biosynthe-sis since they have already been selected through evolution as a way for theirnative producers to gain an advantage in the environment. Evolution has selectedthe secondary metabolites that best balance the energy cost of production andthe physiological or ecological benefit [9]. These benefits can be in the formof defense compounds, which help ward off predators; signaling compounds,which attract other organisms; or inhibitors of the growth or proliferation ofrival organisms [10]. Also, natural products are likely to have already evolvedto be capable of cell membrane penetration, allowing interaction with specifictargets within the cell [11]. Combinatorial biosynthesis enables the enormousbiodiversity present in nature to be fully accessed by combining genes thatwould never get the chance to meet under natural conditions. Manipulation ofthe genetic machinery may deliver compounds that would not be under evo-lutionary stress by the influence of selection pressures, habitat, or biochemicallimitations [9].

The sheer complexity and large molecular masses of many natural compoundsmakes chemical modification extremely difficult to optimize for therapeutic usein humans [12]. They contain a great number of reactive groups which require

INTRODUCTION 255

difficult and selective protection reactions to ensure specificity for both synthesisand modifications of existing molecules. As a result, this necessitates a bet-ter understanding of the biosynthetic pathways for these natural products [13].Although synthesis from combinatorial libraries is faster than natural produc-tion isolation, combinatorial biosynthesis is able to extract the maximum valuefrom hard-won natural product leads. The directed libraries of modified naturalproducts are far more expansive than can be obtained from organic syntheses[14]. Overall, natural products still remain the superior choice to obtain bioac-tive lead compounds for further combinatorial biosynthesis, due to their variousinteractions with a wide range of organisms.

As discussed previously, successful combinatorial biosynthesis requires a largenumber of lead compounds. Actinomycetes, in particular Streptomyces , and fungiare the leading producers of natural products and continue to be good sourcesof combinatorial biosynthesis. The soil-dwelling bacteria Streptomyces is thelargest antibiotic-producing genus in the world, generating a very diverse groupof secondary metabolites. It has been estimated that approximately 99% of thebacterial species has yet to be fully explored, meaning the potential for discover-ing more bioactive compounds is very high [15]. Many strains of Streptomyceswere originally believed to be unculturable, but recent attempts using culture-independent molecular methods have since shown that a majority are in factculturable [16–18]. Additionally, the genome of Streptomyces coelicolor wasrecently sequenced and published [19]. After decades of study, four novel sec-ondary metabolites produced by S. coelicolor were identified under laboratoryconditions, but the genome suggests that potentially up to 20 different metabolitescould be produced [5]. These metabolites, along with numerous other naturalproducts produced from strains of Streptomyces , are excellent candidates forcombinatorial biosynthesis.

Fungi are also abundant producers of natural products, a majority of which arepolyketides. One survey estimated that of the 1500 fungal secondary metabolitesisolated and characterized between 1993 and 2001, approximately half displayedantibacterial, antifungal, or antitumor activity [20]. The best characterized ofthis group of compounds are napthopyrone, aflatoxin, and lovastatin, althoughthere are many other diverse metabolites [21,22]. For example, Brady and co-workers studied the edophytic fungus CR115 from Costa Rica, which is knownto produce a family of related but structurally diverse bioactive compounds. Thisfungus produces guanacastepene A, a diterpene, that has demonstrated antibioticactivity against drug-resistant strains of Staphylococcus aureus and Enterococcusfaecalis [23].

There are many excellent review articles on combinatorial biosynthesis[2,24–26]. In this chapter we highlight some recent advances in combinatorialbiosynthesis, including new tools for manipulating biosynthetic pathways ofinterest and selected examples of combinatorial biosynthesis for four majorclasses of natural products: macrolides, cyclic lipopeptides, carotenoids, andalkaloids. In addition, we address some of the challenges and future directionsfor this expanding field.


II. TOOLS FOR COMBINATORIAL BIOSYNTHESIS

A. Heterologous Hosts

The most critical tool in combinatorial biosynthesis is the utilization of heterolo-gous hosts for the production of both original natural products as well as those thathave been modified [27]. The native producers of these compounds are generallyslow-growing or genetically intractable, making them unsuitable for large-scaleindustrial production. Thus, choosing a heterologous host becomes absolutelycritical to the combinatorial biosynthesis of natural products. Two of the mostcommon organisms chosen for heterologous production of polyketides or non-ribosomal peptides are Escherichia coli and Streptomyces coelicolor , althoughmany other candidates are available, including other bacteria, fungi, and plants.Several factors must be considered when selecting a heterologous host, and thereis by no means a universal choice, so decisions should be made on a case-by-casebasis [28].

One consideration with the use of heterologous hosts is potential expressionproblems of these large enzyme complexes. A range of issues will arise with poorexpression, such as inclusion body formation, increased amino acid misincorpo-ration, and incomplete posttranslational modification, all of which are probablya direct result of poor codon usage [28]. However, with advances in genomics,codon usage for both the native and heterologous hosts can be identified, resultingin codon-optimized genes for combinatorial biosynthesis [3].

Once all the biosynthetic genes have been functionally expressed within aheterologous host, the substrate pool in the organism may require further adjust-ments. In many cases, the available substrate pool within the heterologous host isnot sufficient for polyketide or nonribosomal peptide formation. Polyketide syn-thases (PKSs) utilize a broad range of substrates, such as acetyl-CoA, propionyl-CoA, malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA, isobutyryl-CoA,and isovaleryl-CoA. Nonribosomal peptide synthases (NRPSs) use the natu-rally occurring amino acids as well as p-aminobenzoic acid, cyclohexenoylcarboxylic acid, and dozens of other α- and β-amino acids [29]. For a majorityof PKSs, the only substrates that are utilized are acetyl-CoA, propionyl-CoA,malonyl-CoA, and (2S)-methylmalonyl-CoA, so any heterologous host possess-ing a readily available pool of these substrates would be an excellent choice. Twooptions remain available for hosts that do not contain these acyl-CoA substrates;either respective 1,3-dicarboxylic acids could be directly added to the media, ormetabolic engineering could be used to synthesize these rare substrates. In thefirst case, malonyl-CoA synthetase has a broad enough specificity that it canaccept other 1,3-dicarboxylic acids and convert them into their α-carboxylatedCoA thioesters, which can then be utilized by PKSs. For the second option,there is now a wealth of knowledge for metabolic pathways capable of creatingthese CoA thioesters; for example, there are four known pathways to create (2S)-methylmalonyl-CoA [30]. Both the direct addition of substrate and the metabolicroute would be suitable for the introduction of unnatural amino acids in NRPSs[28]. It is worth noting that the use of heterologous hosts that do not originally

TOOLS FOR COMBINATORIAL BIOSYNTHESIS 257

have these substrates would ensure a reduction in the amount of side reactions,compared to native producers, where these precursors are more prevalent, leadingto higher yields of the desired compounds [31].

Finally, self-resistance to production of bioactive natural products must alsobe addressed when using heterologous hosts, as there may be growth inhibitionupon formation of the desired compound. Fortunately, the self-resistance genesare typically present within the same natural gene cluster as the biosyntheticgenes, making coexpression an easy solution [32–34]. However, when both themode of action and the target for the compound are unknown, the resistancegenes within the original gene cluster may no longer be of any use in protectingthe heterologous host [28].

B. Genome Mining Tools

Another important tool leading to the growth of combinatorial biosynthesis isthe increasing availability and amount of genetic information. Sequence datafrom over 165,000 organisms, including nearly 400 whole genomes, are readilyaccessible online at the NCBI Genbank (www.ncbi.nlm.nih.gov). One advantageof these biosynthetic clusters is that in most cases, all the genes required forbiosynthesis, resistance, and regulation are all together on the microbial chro-mosome, making searching and annotation much easier. Unfortunately, genomesequencing projects of organisms known to produce natural compounds have beenrather limited, with only a few strains of Streptomyces being completed [35].

As a result, new techniques have been developed that effectively locate polyke-tide synthase or nonribosomal peptide synthase gene clusters without an entiresequenced genome. The first option would be to search for conserved regionswithin the PKS or NRPS domain using degenerate primers, but more specificmethods have been used that can effectively target specific chemical features ofthe final compounds [35]. One example is the biosynthesis of rifamycins andansamitocins, where 3-amino-5-hydroxybenzoic acid (AHBA) is used as the rarestarter unit. Conserved regions of a known AHBA synthase were used, and onceidentified it was then used as a probe to find the entire gene cluster for the naturalproduct [36].

Another distinct approach to finding gene clusters utilizes high-throughputphage display of a shotgun library of the bacterial genome. This method relieson the ability of Sfp phosphopantetheinyl transferase from Bacillus subtilis tocovalently modify carrier-protein domains from either NRPS or PKS enzymeswith a biotin-coenzyme A substrate. The shotgun library is displayed on thesurface of phage and then iteratively selected for streptavidin-bound Sfp-modifiedfragments. Once identified, the clones selected can be sequenced, and further useof polymerase chain reaction (PCR) can reveal the full-length PKS or NRPSgene cluster. This method was successful in finding several clusters within thegenomes of B. subtilis and Myxococcus xanthus [37]. These two methods, alongwith many others, are extremely powerful in locating PKS or NRPS gene clusterspresent in the unsequenced genomes.


Genome mining has not only allowed for the discovery of gene clusters fornovel natural products but also has the potential to identify novel therapeutictargets. The recent completion of the human genome project has allowed formore specific screening of drug candidates created by combinatorial biosynthesis[38]. This approach is increasingly important when dealing with disease modelswithin the human body, although this methodology is not limited to humans andcan be expanded to other targets of interest. One example of potential therapeutictargets that has gained a great deal of interest lately are the enzymes within theG-protein coupled receptor (GPCR) superfamily [39]. Increased knowledge andaccess to genome mining tools play an important dual role in finding new naturalproducts and targets for combinatorial biosynthesis.

C. Synthetic Biology Tools

Advances in synthetic biology tools have also been valuable in the progressof combinatorial biosynthesis. The large size of PKSs and NRPSs has madegenetic manipulation particularly difficult when transferring into heterologoushosts. As a result, a new method for gene synthesis was required that couldproduce an entire PKS or NRPS cluster rapidly with high accuracy and effi-ciency. Kodumal and co-workers developed a new method able to synthesizea contiguous 32-kb polyketide synthase gene cluster. The authors began withshort DNA sequences approximately 500 bp in length called synthons , obtainedthrough common polymerase chain reaction (PCR) methods. The next step wasthe efficient combination of synthons into segments approximately 5 kb long bya method called ligation by survival (LBS). This method relied on the growingsynthons being cloned on either a donor or an acceptor plasmid. Through diges-tion of these plasmids and subsequent ligation together, growth on a specificdouble selection marker would ensure that two synthons were spliced together[40]. After three cycles of parallel processing and LBS, a DNA fragment of 5 kbwas obtained. These fragments were then combined using conventional cloningmethods to form the entire 32-kb gene cluster encoding 6-deoxyerythronolideB synthase (DEBS), whose functionality was shown by gene expression andpolyketide production in E. coli [41]. This approach is just one example of genesynthesis, and many different methods exist.

Shao and co-workers presented an alternative approach for the synthesis oflarge pieces of recombinant DNA through the use of DNA assembler, a single-step assembly method relying on in vivo homologous recombination in Sac-chromyces cerevisiae. The authors demonstrated the rapid construction of twopathways, the three-gene D-xylose utilization pathway (ca. 9 kb) and the 11-genepathway combining D-xylose utilization and zeaxanthin biosynthesis (ca. 19 kb).An expression cassette was constructed for each gene in the pathway by overlap-extension PCR, consisting of a promoter, structural gene, and terminator. The5′-end of each cassette was designed either to overlap with a vector or part ofa helper fragment carrying a selection marker and overlap with a targeted locusfor chromosome integration. The 3′-end of the cassette was designed to overlap

TOOLS FOR COMBINATORIAL BIOSYNTHESIS 259

1

2

n−1

n−3

n−2

n−1

n

vector

chromosomeδ δ site

δ1

1

2

δ2helper fragment n

(A)

(B)

FIGURE 1 Scheme for one-step assembly and integration of a biosynthetic pathwayusing in vivo homologous recombination into (A) vector and (B) δ site on a S. cere-visiae chromosome. n represents the number of DNA fragments. (See insert for colorrepresentation of the figure.)

with the second cassette, and each successive cassette should then overlap withthe two flanking ones. All the linearized gene cassettes were transformed intoS. cerevisiae, yielding functional pathways at high efficiencies (Fig. 1) [42]. Thismethod could easily be extended to combinatorial biosynthesis, which requiresthe gene synthesis and assembly of the large megasynthases responsible for theproduction of polyketides and nonribosomal peptides.

After these large DNA sequences are efficiently cloned, combinatorial biosyn-thesis of large gene clusters can now begin. One of the preferred methodscurrently in use is the multiplasmid approach due to the ease of mixing andmatching different modules on compatible plasmids. For example, Xue and co-workers developed a three-plasmid system for Streptomyces used to create alibrary of 6-dEB variants. The authors engineered mutant modules on one of the


individual plasmids and then combined them in different combinations with theremaining two plasmids through transformation, facilitating the process of combi-natorial biosynthesis [43]. Hypothetically, a single PKS module could have eightpossible mutations, two from the acyltransferase (AT) specificity (either malonyl-or methylmalonyl-CoA) and four possible β-carbon modifications (either noreduction, ketoreduction, ketoreduction + dehydration, or ketoreduction + dehy-dration + enoylreduction). A two-plasmid system would yield 82 = 64 possiblemutations and subsequently 64 potentially novel polyketides. For the same exper-imental effort, a single-expression plasmid system would provide a maximumof only 8 × 2 = 16 potentially novel polyketides. The multiplasmid approachrepresents a powerful tool for combinatorial biosynthesis [44].

D. Protein Engineering Tools

Two protein engineering tools, rational design and directed evolution, have alsobeen applied as tools in the development of combinatorial biosynthesis. In thepast, these methods have allowed for the creation of enzymes with altered or novelproperties [45,46]. Rational design approaches rely on a detailed understandingof the enzyme structure and catalytic mechanism followed by site-directed muta-genesis; directed evolution mimics the process of natural evolution in the testtube and does not rely on structural and mechanistic understanding of the tar-get enzyme. The directed evolution method has not been utilized frequently incombinatorial biosynthesis because the assays required to screen the large num-ber of mutant pathway enzymes have proven to be difficult [3]. However, bothrational design and directed evolution have been used to alter the substrate speci-ficity of single enzymes and also help gain insights into the mechanisms of thosemultidomain enzymes.

Reeves and co-workers utilized a rational design approach to alter the substratespecificity of one acyltransferase (AT) domain in DEBS. The authors showedpreviously that the AT domain from modules 1, 2, 3, 5, and 6 could all besubstituted with the AT domain from module 2 of the rapamycin cluster, andpolyketide production was still monitored, but substitution in module 4 of DEBSresulted in no polyketide production [47]. Three more malonyl-CoA-specific ATdomains were also substituted in place of DEBS AT4, each with higher-sequencehomology to DEBS AT4 than to rapAT2, but again, no detectable polyketide wasmeasured, suggesting that this domain is highly sensitive to any perturbations.Alignments with modular PKS AT domains and the E. coli fatty acid synthaseFabD revealed three primary regions believed to confer substrate specificity tomethylmalonyl-CoA. Site-specific mutations were made to switch these regionsto sequences seen in malonyl-CoA-specific domains. Changing each region or allthree regions together yielded the natural product 6-deoxyerythronolide B (frommethylmalonyl-CoA) and the new analog, 6-desmethyl-6-deoxyerythronolide B(from malonyl-CoA). This result was the first reported case of extender unitspecificity of a PKS module being altered by site-specific mutagenesis [48].

Within each NRPS module, the adenylation (A) domain is responsible for thespecific binding, activation, and covalent tethering of the amino acid monomer

EXAMPLES OF COMBINATORIAL BIOSYNTHESIS 261

[49]. Thus far there has been limited success in the swapping of heterologous Adomains to create unnatural products, a result of a severe reduction in the activityof these chimeric NRPSs [50]. Fischbach and co-workers were able to restore theactivity of impaired NRPSs using directed evolution. The authors replaced thevaline-specific A domain of AdmK, a protein required for andrimid biosynthe-sis, with CytC1, a 2-aminobutyrate-incorporating A domain with reported broadspecificity [51]. The AdmK-CytC1 chimera produced andrimid at a 32-fold lowerlevel than the wild-type AdmK. Mutagenic PCR was used to introduce mutationsand after several rounds, a clone was isolated that could produce andrimid at a10.7-fold higher level than the original AdmK-CytC1. The introduction of theCytC1 domain into a functionally restored chimera allowed for the productionof andrimid derivatives by introducing nonproteogenic amino acids. Addition ofL-2-aminobutyrate or D-2-aminobutyrate to the culture medium allowed for theproduction of novel derivatives in high ratios to andrimid. Restoration of chimeraactivity required only modest library sizes (103 to 104 clones) and three rounds ofscreening, suggesting that this method would probably be broadly applicable [52].

In another demonstration of protein engineering in combinatorial biosynthe-sis, Schmidt-Dannert and co-workers created new metabolic pathways for theproduction of novel carotenoids in E. coli [53]. The phytoene desaturases fromErwinia uredovora and Erwinia herbicola , normally capable of introducing onlysmall amounts of six double bonds into phytoene, were used in DNA shuffling.This library of desaturases was screened and one chimera was found that wouldefficiently produce the fully conjugated carotenoid, 3,4,3′, 4′-tetrahydrolycopene.A second shuffled library, this time of lycopene cyclases, was used to extend thepathway to produce a variety of colored products. One of these new pathwayswas able to produce torulene, a cyclic carotenoid never previously synthesized inE. coli . An approach similar to this manipulation could be used in combinatorialbiosynthesis to generate novel products in heterologous hosts that are originallyinaccessible from natural sources.

III. EXAMPLES OF COMBINATORIAL BIOSYNTHESIS

A. Combinatorial Biosynthesis of Macrolides

The macrolide antibiotics erythromycin and tylosin, among several other polyke-tide antibiotics, are important in medicine and animal health (Fig. 2) [54]. Ery-thromycin is comprised of variants A through D (not shown in Fig. 2), all of whichare similar in structure and antibacterial properties. Erythromycin’s antibacte-rial activity comes from the ability to inhibit translation during bacterial proteinbiosynthesis by binding the 50S ribosomal units. In recent years, derivativesof macrolides such as clarythromycin, roxythromycin (14-member macrolideswith nitrogen in the cycle), and azithromycin (15-member macrolide) have beenused most frequently as chemotherapeutic agents in the treatment of infections,including those of the respiratory tract [55]. In light of the high practical valueof macrolide polyketides, particularly erythromycin, several attempts have beenmade to further modify erythromycin.


O

O

O

OO

HO

N

O

OO

HO

O

OO

OH

OH

O

tylosin

O

O

O

OH

OO

HO NOH

OH

O

OOH

erythromycin

OO

O O

O

O

OHNH3

+

OH

HO

NH

ONHCH3

NH

OHHO

HN

HN

OH

NH

O

O

HN

O

OH

HO

−O

O

O

NH2

OO

Cl

Cl

HO

vancomycin

HN

O

O

H

N N

O

NH

HO ON

NH

N

O OH

HOO

HO

Cl Cl

rebeccamycinstaurosporine

O

FIGURE 2 Chemical structures of selected natural products.

The development of genetic engineering and combinatorial biosynthesis toolshave allowed researchers to manipulate the structures of polyketides by modifyingthe domains or modules of the PKS. The erythromycin PKS DEBS has beenthe most extensively studied for the development of combinatorial biosynthesismethods and the generation of polyketide derivatives of erythromycin by geneinactivation; “domain swaps,” substitution or addition of domains or modules tomodify the aglycon; and glycosylation or methylation patterns [56–59]. Severalexemplary reviews that cover the extensive work done on polyketide biosynthesismay be found in the literature but are not covered here [2,24,54,60]. The impactof the 6-deoxyerythronolide B (6-dEB) and erythromycin analogs obtained hasbeen widespread and is evident in the expansion of the technology to other PKSsystems. In the following section we cover some recent achievements [2].


Most recently, combinatorial biosynthesis has been geared toward the engi-neering of hybrid modules from different PKSs to create hybrid lactones. Tangand McDaniel at Kosan Biosciences combined subunits from the pikromycin,erythromycin, and oleandomycin polyketide synthases to create heterologouscomplexes functionally assembled to create hybrid polyketide pathways [61].First, the authors co-transformed the pikAI-II genes encoding subunits 1 and2 (modules PikA1-4) of the pikromycin PKS and the eryAIII encoding sub-unit 3 of the DEBS (cloned in two Streptomyces compatible expression vec-tors) into S. lividans and produced about 10 mg/L of the hybrid macrolactone3-hydroxynarbonolide. Substitution of the eryAIII gene with oleA3 from the ole-andomycin cluster encoding the same module (46% identity between the twogenes) also produced 3-hydroxynarbonolide, showing that both the DEBS3 andOleA3 fully complemented the PikAIII and PikAIV subunits. Combining theabove PikPKS subunits with modified heterologous DEBS3 subunits (products ofdomain deletion and substitutions) also afforded new “unnatural” hybrid macro-lactones. This work was impressive and promising because it showed that naturalas well as modified subunits from heterologous PKSs can be assembled function-ally to create novel compounds, providing the possibility of engineering novelbiosynthetic routes to create high titers of structurally related compounds.

Another example of combinatorial biosynthesis of macrolides involved theengineering of Streptomyces fradiae strains derived from a tylosin producer toproduce 16-membered hybrid lactones [62]. A hybrid PKS operon was con-structed that contained the first two subunits of the chalcomycin cluster, chmGI-II , and the last three subunits from the spiramycin cluster, srmGIII-V . In addition,the ChmGII C-terminal docking site was replaced with that of SrmGIII to ensurefunctional interaction between the two modules. Methoxymalonyl-acyl-carrierprotein precursor genes were also introduced, as they were needed to producethe hybrid lactones. Under the action of the strong tylosin PKS promoter tylGIp,the recombinant strain produced 2 g/L of the polyketide. Further engineeringof the glycosylation pathway (glycosylation by mycarose) generated a triglyclo-sylated hybrid macrolide related to tylosin.

The knowledge gathered from the molecular engineering of polyketide andnonribosomal peptide synthetases has been extended to the biosynthesis ofnovel glycopeptide antibiotics. Vancomycin, like daptomycin, is important in thetreatment of gram-positive bacterial infections, particularly methicillin-resistantStaphylococcus aureus (MRSA) (Fig. 2) [63]. Oritavancin, a semisyntheticderivative of chloroeremomycin active against vancomycin-resistant enterococci(VRE), along with several other glycopeptides antibiotics are currently inclinical development [64].

Researchers have studied variations in the glycosylation patterns and in theheptapeptide core structures of glycopeptides with the help of recently cloned,sequenced, and analyzed biosynthetic clusters [65]. A number of biosyntheticpathways for deoxysugars have been described in recent years, including thosefor erythromycin A, oleandomycin, pikromycin, mithramycin, megalomycin, lan-domycin, urdamycin, and chromomycin [66]. In many cases, biosynthetic gene


clusters for common 6-deoxyhexoses have been observed. The in vivo efficacy ofglycopeptide antibiotics is greatly determined by the sugar residues that deriva-tize the peptide core, since they participate in molecular recognition of the drugtarget site.

Glycosylation steps usually occur late in biosynthesis by transferring thedeoxysugar to the aglycon from an NDP-sugar activated form. Some of theseglucosyltransferases (Gtfs) have relaxed substrate specificity, such as the Gtfsin urdamycin biosynthesis (D- and L-rhodinose in place of D-olivose), where theOleG2 Gtf can transfer a rhamnosyl group instead of mycarosyl to yield 3-O-L-rhamnosylnarbonolide and the misglycosylation of the tylosin scaffold by the TylM2 Gtf using TDP-D-desosamine to give 5-O-desosaminyltylactone [67]. Thisdiscovery has opened the door to the generation of many hybrid glycopeptidesin vivo and in vitro by combinatorial biosynthesis [67–69].

Walsh and co-workers demonstrated in vitro that both GtfE and GtfD canattach various NDP-sugar donors and accept different substrates to produce newglycopeptides. GtfE can use several deoxy- and amino-substituted analogs ofglucose and attach them to vancomycin and teicoplanin aglycons. GtfD, in turn,can accept some monoglycosylated compounds as substrates for the addition ofepivancomycin. Disruption of genes involved in the biosynthesis of sugars usinggene inactivation in the native producer allowed for the isolation of differentderivatives containing deoxysugar intermediates of erythromycin, mithramycin,and methymycin/pikromycin [70]. Urdamycin derivatives have also been isolatedin S. fradiae containing an inactivation of the urdR gene, which catalyzes the4-ketoreductase addition in the final step of dNDP-D-olivose biosynthesis. A newsugar moiety (D-rhodinose), processed by the deoxygenation at C3 and reductionat C4 of an accumulated 4-keto intermediate by UrdQ and UrdZ3, respectively,was also introduced [71–73].

Production of glycosylated derivatives has also been accomplished by geneinactivation and gene expression in a heterologous host. Several methymycin andpikromycin derivatives were produced by expressing genes from a calicheamicingene cluster in a desI mutant [74]. By overexpressing the TylM2 glycosyltrans-ferase gene from the tylosin producer S. fradiae in the erythromycin-deficientSaccharopolyspora erythrea triple mutant (in which the EryBV, EryCIIIglycosyltransferases, and erythromycin polyketide synthase genes were deleted),Leadlay and co-workers created the new compound 5-O-desosaminyl-tylactoneby feeding tylonolide [67]. McDaniel and co-workers took advantage of theability of the DesVII desosaminyltransferase to recognize different acceptorsubstrates to create over 20 different 14-membered desosaminyltransferasemacrolides. A Streptomyces lividans host that synthesized dNDP-D-desosaminewas created by integration of a set of nine sugar biosynthesis genes from thepikromycin/methymycin cluster into the chromosome, along with the pikromycindesosaminyltransferase. This recombinant strain was transformed with a libraryof expression plasmids encoding genetically modified polyketides [75]. Byusing a reverse of this approach, that is, providing the S. lividans host with theability to produce different sugars, elloramycin derivatives were produced from


the same aglycon [76]. Recently, nine derivatives of the antitumor antibioticmithramycin have also been produced by altering the glycosylation pattern,from which seven new compounds were obtained, all showing antitumoractivity against tumor cell lines [77,78]. Two new compounds, identified asdemycarosyl-3D-β-d-digitoxosyl-MTM and deoliosyl-3C-β-d-mycarosyl-MTM,may be potential clinical trial candidates. Both showed improved activityagainst the estrogen receptor–positive human breast cancer cell line MCF-7compared with the parent drug MTM. In addition, higher apoptosis of theestrogen receptor-negative human breast cancer cell line MDA-231, for whichchemotherapeutic agents are urgently needed, was observed for two additionalcompounds [77].

B. Combinatorial Biosynthesis of Cyclic Lipopeptides

Interest in cyclic lipopeptide antibiotics began in the 1950s with the isolationof amphomycin and various closely related lipopeptides. In 1987, Debono andco-workers elucidated the cyclic nature of members of the A21978C complex,composed of a 10-membered cyclic peptide coupled by an ester bond betweenthe C-terminus of L-kynurenine13 (Kyn) and the hydroxyl group of L-Thr4 toform a 10-amino acid ring with a three-amino acid tail coupled by an amidelinkage of the N-terminus of Trp1 to different fatty acids (Fig. 3) [79]. The threemajor components of A21978C1−3 have 11-, 12-, and 13-carbon branched fattyacid chains attached to Trp1 [79]. Daptomycin (Fig. 3), produced by Strepto-myces roseosporus , contains the common 13-amino acid core cyclized by anintramolecular ester bond to make a 10-membered ring with a three-residue sidechain. A n-decanoyl fatty acid side chain is attached to Trp1, synthesized eitherchemically or by feeding decanoic acid to the fermentation [63]. One of the mainfeatures of daptomycin and the other A21978 members is the presence of bothD- and L-amino acids. Over the years, a number of 10-membered cyclic lipopep-tide antibiotics related to daptomycin, all of which are secondary metabolitesproduced by actinomycetes, have been identified. These antibiotics comprise thetwo other cyclic depsipeptides, the calcium-dependent antibiotic (CDA) producedby Streptomyces coelicolor , A54145 produced by Streptomyces fradiae, and thecyclic peptides amphomycin, laspartomycin, and friulimicin [80].

Daptomycin (Cubicin) was approved in the United States in 2003 for thetreatment of gram-positive bacterial skin infections. It was reported to be activeagainst 15 gram-positive genera, including 35 species, but most important,against methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistantStaphylococcus epidermidis (MRSE), vancomycin-resistant enterococci (VRE),and penicillin-resistant Streptococcus pneumoniae (PRSP) [81]. Even thoughdaptomycin is efficacious, generation of analogs can further expand its clinicaluses, such as the treatment of community-acquired pneumonia. A number ofderivatives of daptomycin modified at D-ornithine6 or D-serine11, or substitutedwith different fatty acid tails, have been synthesized and evaluated; however,none has proven superior to daptomycin [82]. A54145, amphomycin, and laspar-tomycin have also been subjected to semisynthetic derivatization using similar


Asp9

Gly10

D-Ser11

3mGlu12

Kyn13

Thr4

Gly5

Orn6

Asp7

D-Ala8

Asp3

D-Asn2

R -Trp1

R-Ser3

R = 2,3 -epoxyhexanoyl

A54145 (S. fradiae)CDA (S. coelicolor )A21978 (S. roseosporus)

R = n-decanoyl (daptomycin) anteiso-undecanoyl (A21978C1) iso-dodecanoyl (A21978C2) anteiso -tridecanoyl (A21978C3)

R = n-decanoyl iso-dodecanoyl anteiso -un decanoyl

dptA dptBC dptD dptG H I J dptMN-EF

(A)

(B)

Asp9

Gly10

D-hAsn11

3mGlu12

Trp13D-Trp5

Asp6

Asp7

D-hPG8

Thr4

mOAsp9

Gly10

D-Asn11

Glu/3mGlu12

Ile/Val13Sar5

Ala6

D-Lys8

hAsn3

D-Glu2

R -Trp1

Thr4

Asp7

FIGURE 3 (A) Chemical structures of lipopeptide A21978, CDA, and A54145. hAsn,hydroxyasparagine; hPG, hydroxyphenylglycine; Sar, sarcosine; OAsp, methoxy-Asp.(B) Subunit and module organizations of the daptomycin gene cluster.

peripheral modification strategies [83]. Other chemical modifications, such asthe substitution of different amino acids in the core cyclic peptide, have beenproven difficult to explore using chemical methods, due to the complex nature ofdaptomycin, although a chemoenzymatic approach to generating small amountsof derivatives containing readily available amino acids remains promising [84].Total synthesis and semisynthetic production of daptomycin derivatives, in addi-tion, are hampered by the lack of commercially available 3-methylglutamic acid[63]. In light of these difficulties, combinatorial biosynthesis to generate analogsof daptomycin and other cyclic lipopeptides represent an important alternative.

Sequence analysis of the daptomycin biosynthetic gene cluster (dpt) revealedthree genes, dptA, dptBC , and dptD , which encode the NRPS subunits DptA,DptB, and DptC, respectively, for the assembly of the peptide core [85] (Fig. 3).The catalytic domains in the subunits couple five, six, and two amino acids,respectively. Additional genes are likely to be involved in the coupling ofbranched fatty acids to the N-terminal of Trp1 (dptE, dptF ), error correction(dptH ), or incorporation of 3mGlu12 and Kyn13 (dptI, dptJ ). It was shownrecently that an epimerase domain (E-domain) was present in the secondmodule, suggesting that daptomycin contained a D-Asn instead of an L-Asn as


reported previously [81]. More recently, the dpt cluster was cloned on a BACvector and expressed in S. lividans [81,86].

By combinatorial biosynthesis, derivatives of daptomycin were first generatedby exchanging subunit DptD with subunits LptD and CdaPS3, correspondingto the third subunit of the A54145 and CDA biosynthetic clusters, respectively.Whereas DptD incorporates Kyn13, A54145 and CDA are responsible for incor-porating Ile (Val)13 and Trp13, respectively. Expressing the subunits LptD orCdaPS3 in the �dptD deletion mutant showed that the heterologous subunits,expressed under the ermEp promoter, completely trans-complemented the mutantand gave yields of hybrid lipopeptide of about 50% for CdaPS3 and 25% forLptD of the control strains. This method was expanded to a double knockoutmutant (�dptA �dptD), which showed that the dptA and dptD genes can beconveniently expressed from the ermEp promoter from ectopic positions [54].

A complete module exchange between D-Ala8 and D-Ser11 has also beenaccomplished successfully, and yields of 60% and 20% of the respective controlswere obtained for D-Ser8 and D-Ala11. Both novel antibiotics had antibacterialactivity [80]. Furthermore, Nguyen and co-workers observed that a �dptI mutant(dptI encodes Glu-3-methyltransferase) produced daptomycin analogs lacking themethyl group of 3mGlu12 [63]. It was found that the absence of the methyl groupof 3m-Glu12 (Glu12) in A54145 mutants resulted in mutants with less antibacterialactivity; however, they were considerably less toxic in mouse LD50 tests. There-fore, the 3mGlu12-to-Glu12 variant may be beneficial to some analogs, but futureinvestigations remain to be completed. One advantage exploited by Miao andco-workers was that S. roseosporus produces a mixture of compounds contain-ing three different fatty acid side chains—anteiso-undecanoyl, iso-dodecanoyl,and anteiso-tridecanoyl—which accumulate in fair amounts during fermentation[79]. Therefore, a library of 72 daptomycin analogs was generated and tested, allof which showed antibacterial activity [80]. In some cases, the yields were low,so additional strain and fermentation development will undoubtedly be needed.

Many other possible modifications of the daptomycin peptide core via combi-natorial biosynthesis can be envisioned. The combinatorial biosynthesis method-ology can also be coupled with further chemical modifications of the lipid sidechain and the L-Orn residue of the daptomycin core peptide to further optimizethe novel lipopeptides to generate candidates for clinical development. This cou-pling of combinatorial biosynthesis with chemical modifications can, in theory, beextended to related lipopeptides as well as to other peptides produced by NRPSprocesses, to generate libraries of compounds that would be difficult to produce byde novo chemical syntheses in quantities sufficient for clinical development [80].

C. Combinatorial Biosynthesis of Carotenoids

Carotenoids are naturally occurring pigments that are important nutraceuticalcompounds, and these natural lipophilic antioxidants are synthesized as hydro-carbons (carotenes) or their oxygenate derivatives (xanthophylls) by plants andmicroorganisms. In the cell, their natural function is to protect against oxidative


damage by quenching photosensitizers, interacting with singlet oxygen moleculesand scavenging of peroxy radicals [87,88]. Over 700 carotenoids with diversechemical structures have been identified in bacteria, algae, and plants. The chem-ical structure of carotenoids dictates their biological properties because it deter-mines how they interact with other molecules and integrate into membranes [89].Spheroidene, plectaniaxanthin, and lutein are examples of carotenoids producedin bacteria, fungi, and plants, respectively.

Carotenoids are produced in thousands of plants and microbial species, differ-ing in the number of conjugated double bonds, the structure of the end groups,and the oxygen-containing substituents [90]. Most are based on a C40 phytoenecarbon produced by condensation of two molecules of geranylgeranyldiphos-phate (GGDP; C20PP) catalyzed by the CrtB gene, carotenoid synthase [91]. Asmall number of bacteria (e.g., Staphylococcus and Heliobacterium) contain a C30

pathway, starting from the condensation of two molecules of farnesyldiphosphate(FDP; C15PP) by CrtM synthase to produce C30 carotenoids, or apocartenoids.C50 carotenoids, or homocarotenoids, also exist in bacteria; however, their biosyn-thesis starts with a C40 backbone, but with the addition of two C5 isopreneunits [91].

Although highly controversial, carotenoids may play an important role inthe prevention of cardiovascular diseases and cancer [92–94]. Delays in tumorgrowth in mice and rat models have been observed with canthaxanthin andβ-carotene. In addition, carotenoids have industrial and pharmaceutical appli-cations as nutrient supplements, food colorants, and animal feeds. The actualsale of carotenoids is estimated at about $500 million per year [95]. Althoughsome carotenoids can be extracted from their natural producers, the major-ity of carotenoids accumulate as trace amounts as biosynthetic intermediates,making them cumbersome for extraction, purification and application. There-fore, most carotenoids used industrially are chemically synthesized, as in thecases of β,β-carotene, astaxanthin, and lycopenes. One possibility of overcomingthe lack of availability of carotenoids has been the heterologous expression ofcarotenoid genes from various organisms in a suitable noncarotenogenic microor-ganism such as E. coli and the yeast species Candida utilis and Saccharomycescerevisiae [90]. These hosts, particularly E. coli , have been used to producerare and novel carotenoid derivatives, including the unusual acyclic carotenoids,hydroxycarotenoids, with improved antioxidant properties and carotenoids withnovel carbon backbone chain length (C35 or greater than C40) using combina-torial biosynthesis tools. Due to the limited availability of precursors, metabolicengineering to increase their formation though the early terpenoid pathway isnecessary. Two of the most recent successes in the combinatorial biosynthesisof carotenoids will be highlighted: the biosynthesis of novel hydroxycarotenoidsby gene recombination in E. coli [95] and the evolution of a pathway to novellong-chain carotenoids [91].

Considerable progress has been made in successfully cloning and expressingthe necessary carotenogenic genes in E. coli [96,97]. To achieve the production ofa carotenoid in E. coli , several steps are crucial: selection of the necessary genes


that cover the entire pathway desired, construction of the expression plasmids,transformation of a suitable E. coli strain with a combination of plasmids, andgrowth under optimized carotenoid production conditions.

Combinatorial biosynthesis was used to synthesize novel lipophilic carotenoidsthat have powerful antioxidant properties by coexpression in E. coli of threedifferent carotenoid desaturases in combination with a carotenoid hydratase, acyclase, and a hydroxylase on plasmids. This effort resulted in 12 differentcarotenoid derivatives, four of which have never been detected previously bio-logically or synthesized chemically [95].

Specifically, the authors created an E. coli strain harboring plasmids pACCRT-EBal-1, pACCRT-EBIEu, and pACCAR25�crtX, which mediated the synthesis of3,4-didehydrolycopene, lycopene, and products of lycopene cyclization, respec-tively. Starting with either construct, the genes crtC and crtD , which encodea C-1,2 hydratase and a C-3,4-desaturase, respectively, were introduced. Theresulting carotenoids were separated via high-pressure liquid chromatography andidentified by their retention times, spectroscopic properties (NMR), and molec-ular weights. Absorption maxima in methanol and trimethylsilylation (TMS)derivatization further characterized the four novel hydroxycarotenoids. Theywere named 3,1-(HO)2-γ-carotene, 1-HO-3′,4′-didehydrolycopene, 1,1′-(HO)2-3,4-didehydrolycopene, and 1,1′-(HO)2-tetradehydrolycopene, and the reactionsequence for the formation of each of the new carotenoids was hypothesized.One drawback of the biosynthesis, which resulted in low levels of carotenoids,was the emergence of competing pathways, since the novel carotenoids werenot the only products in E. coli transformants. By-product zeanxanthin wasformed along with 3,1-(HO)2-γ-carotene; undesired by-products 1-(HO)- and1,1′-(HO)2-lycopene also accumulated in the E. coli transformants carrying onlythe structural genes. Plasmids carrying genes encoding required precursors, thatis, the isopentenyl pyrophosphate (IPP) isomerase (idi ) and the dxs gene for1-deoxy-D-xylulose-5-phosphate synthase, were transformed into E. coli , and atwo-fold increase in novel carotenoid levels was obtained.

Using combinatorial biosynthesis, Umeno and Arnold created new pathwaysfor the biosynthesis of carotenoids with backbones longer than C40 by focusingon engineering the C30 carotenoid synthase crtM to accept longer diphosphatesubstrates by site-directed mutagenesis [91]. Previously, using random mutage-nesis, single-amino-acid substitutions in the C30 synthase CrtM (F26L, F26S,W38C, and E180G) conferred C40 synthase activity to the enzyme [98,99]. Fur-thermore, by analyzing the effects of each mutation at positions 26, 38, and 180on the C30 and C40 synthase activities, coupled with a crystal structure compari-son with squalene synthase, it was found that only E180G positively affects bothC30 and C40. The mutations at positions 26 and 38, which caused a gain of C40

activity, came at the cost of C30 synthase activity.When supplied with the precursor farnesylgeranyl diphosphate (FGDP;

C25) produced by a Y81A BsFDS enzyme variant, several CrtM variantswith additional mutations at positions 26 and 38 generated C35, C40, and two


novel compounds: 16-isolpentenylphytoene 1 (C45 backbone: C20 plus C25)

and a 16,16′-diisopentenylphytoene 2 (C50 backbone; C25 plus C25). When themutation E180G was introduced, the highest production of both novel productswas observed, 215 μg/g (DCW) and 147 μg/g (DCW), for products 1 and 2,respectively. In conclusion, it is likely that once a novel carotenoid backboneis created, subsequent enzymes in the carotenoid biosynthetic pathway, eithernatural or engineered, can accept the new substrate and process the remainingsteps. Protein engineering may be needed to increase or broaden the substratespecificity of carotenoid-modifying enzymes, including desaturases, cyclases,and hydroxylases.

D. Combinatorial Biosynthesis of Alkaloids

Alkaloids are an important natural product family with interesting structural fea-tures and pharmaceutical properties. Ajmaline, an important plant-derived phar-maceutical which is commercially isolated from Rauvolfia roots, has been used inthe therapy of heart disorders, while the benzylisoquinoline alkaloids morphineand rebeccamycin are potent analgesics and show antitumor and antibacterialactivities [9,100]. Also, vincaleucoblastine and vincristine are used in medicineas cytostatics, and reserpine is a neuroleptic and antihypertensive [100,101].Alkaloids are produced primarily by plants, and for reasons mentioned above,characterization and engineering of their biosynthetic pathways are complicated[102].

Combinatorial biosynthesis of alkaloids has only been successful in a fewcases [100,103,104]. This is due in part to the fact that their biosynthetic path-ways involve many steps (30 enzymes for the indole alkaloid vincristine andmore than 17 for morphine). Considerable progress, however, has been madein the attempt to elucidate and characterize the various steps in the biosynthe-sis of the benzylisoquinoline alkaloids morphine and berberine. To date, almostall 17 steps of morphine biosynthesis have been either elucidated or expressedin E. coli or insect cells and characterized, while the biosynthesis of berberinehas been almost successfully elucidated [105,106]. In the near future, success-ful combinatorial biosynthesis of berberine in a heterologous host will surelybe tested. The complex biosynthesis of vincristine and vinblastine, which aremonoterpenoid indole alkaloids from Catharanthus roseus , has also been underinvestigation in recent years. The entire biosynthesis requires at least 30 biosyn-thetic and two known regulatory genes, which encode around 35 intermediates.Furthermore, intracellular trafficking of intermediates poses a major challenge incombinatorial biosynthesis [107]. Nevertheless, researchers have identified twoimportant genes in the early phase of their biosynthetic pathway, tryptophandecarboxylase and strictosidine synthase genes. Recently, feeding studies of theprecursors tryptamine and secologanin to a S. cerevisiae host carrying the trypto-phan decarboxylase and strictosidine synthase genes produced a sufficient amountof strictosidine, which afforded the function of these genes [108]. In the following


sections, the combinatorial biosynthesis of indolocarbadozole derivatives ofrebeccamycin and staurosporine (Fig. 2) is highlighted.

Numerous efforts are being directed toward the generation of indolocarbazolederivatives with improved properties for the treatment of cancer, neurodegen-erative disorders and diabetes-associated pathologies. Several indolocarbazoleanalogs have already entered clinical trials [109,110]. Previously, the biosyn-thetic gene cluster of rebeccamycin from Lechevaliera aerocolonigenes and stau-rosporine from Streptomyces sp. TP-A0274 have been cloned, but only the formerhaving been characterized [111,112]. With these tools available, the authors usedcombinatorial biosynthesis to dissect and reconstitute the rebeccamycin pathway.By combining genes from different microorganisms with rebeccamycin genes,they developed an experimental strategy and produced over 30 indolocarbazolederivatives in the heterologous host Streptomyces albus [103].

Briefly, genes rebODCPGMHFT pertaining to the rebeccamycin biosyntheticpathway were previously identified and protein functions were assigned [112].The authors reconstituted the pathway first by expressing rebO and rebD inS. albus , which yielded the compound 3,4-bis(indol-3-yl)pyrrole-2,5-dicarboxylicacid. Coexpression of the additional genes rebC and rebP were required toproduce the indolopyrrolocarbazole core, commonly known as arcyriaflavin A.Coexpression of rebODCPG yielded a glycosylated version of arcyriaflavin,which was also achieved by feeding arcyriaflavin to S. albus expressing rebG .Dideschlororebeccamycin was obtained upon the introduction of rebM to therebODCPG combination. It was further shown that the chlorination catalyzed byrebH can occur in the early steps of the pathway (rebODH and rebODCPH ) andthat rebF encoding a flavin reductase could be replaced by ubiquitous FADH2-dependent halogenases in the cell. Finally, rebT encodes an integral membranetransporter which transports rebeccamycin outside the cell; in the absence of sucha gene, rebeccamycin inhibits the growth of S. albus .

The structural differences between rebeccamycin and staurosporine, along withbioinformatic analysis of their biosynthetic clusters, suggested that the newlydiscovered gene from staurosporine pathway staC , which shows high homologyto rebC , was responsible for the structural difference between the two com-pounds. Expression of either rebODP + staC or rebOD + staCP yielded astaurosporine aglycone derivative, identical to the natural product K252c. Addi-tion of the remaining rebeccamycin genes, rebG and rebM , afforded additionalK252c derivatives. Finally, the size of the combinatorial library was furtherincreased by introducing the tryptophan 5-halogenase pyrH from Streptomycesrugosporus or the tryptophan 6-halogenase, thaI , from Streptomyces abogriseo-lus . This introduction should produce 5- and 6-chlorotryptophan intermediates,respectively, resulting from combination products from rebOD + pyrH (thaI),rebODCP + pyrH (thaI), and rebODP + staC + pyrH (thaI). However, only5-chlorotryptophan intermediates were obtained, possibly due to lack of substraterecognition of the rebeccamycin enzymes to use the thaI halogenase efficiently[103].


IV. CONCLUSIONS AND FUTURE PROSPECTS

A. Current Challenges

Despite the great successes of combinatorial biosynthesis in the discovery anddevelopment of new drugs, many challenges remain. One potential limitation ofcombinatorial biosynthesis is low yields of the desired combinatorial productscompared to those of the original compound produced by the native producer.Although this limitation is addressed as a single problem here, it is probablythe result of several combined problems. First, alteration of domains or moduleswithin a biosynthetic pathway may negatively affect the expression and proteinfolding of downstream enzymes, thus lowering the overall effectiveness of theentire pathway. More important, however, the unchanged enzymes within thepathway may have strict specificity and may not be able to accept altered sub-strates. The specificity problem is discussed in further detail shortly [10]. It isworth noting that the problem of low yields becomes magnified if the novel com-pound shows promise as a therapeutic drug and production must be increasedto meet industrial standards. The yields of natural products required for initialisolation and characterization are much lower than those for scaled-up fermen-tations in drug formulations. As a result, many of these biosynthetic pathwaysrequire additional metabolic engineering to increase the yields of the compoundsof interest [3]. This can prove to be challenging at times, but there have beensuccessful reports of metabolic engineering, as in the case of artemisinin [113].

Combinatorial biosynthesis still remains a relatively time-consuming andlabor-intensive process in many cases [10]. However, the methods and toolsutilized in combinatorial biosynthesis today, in particular many microbiologytechniques, are far superior to those in use only a short while ago. Forexample, development was originally limited due to the inefficient methods forcreating recombinant microorganisms which relied on gene replacement andcomplementation of pathway mutants [26]. Some of the recent advances inmore effective methods that facilitate the process of creating natural productcombinatorial libraries will be discussed later.

Another major problem facing combinatorial biosynthesis is the relatively poorunderstanding of the structure and function relationship within the megasynthasesused to produce these natural products. Several questions remain unanswered forPKS and NRPSs, including the three-dimensional structure of these enzymes,how modules and domains recognize and dock to each other, and how selectiveindividual domains are for their substrates [27]. The three-dimensional struc-ture of an entire PKS complex has yet to be determined, although a model wassuggested where the ketosynthase (KS), acyltransferase (AT), acylcarrier protein(ACP), and thioesterase (TE) domains all form dimers along a central core of thesynthase (Fig. 4). The accessory reduction domains ketoreductase (KR), dehy-dratase (DH), and enoylreductase (ER) meanwhile were proposed to remain asmonomers along loop positions at the periphery of the synthase [114]. The crystalstructures of two individual thioesterase domains were solved and both were infact dimers with substrate channels probably providing their substrate specificity

CONCLUSIONS AND FUTURE PROSPECTS 273

AT ACP KS AT KR KS AT KRACP ACP

AT ACP KS AT KR KS AT KRACP ACP

AT ACP KS AT KR KS AT KR TEACP ACP

TE

TE

AT ACP AT KR KS AT KR TEACP ACPKS

eryLM ery module 1 ery module 2 eryTE

eryLM rap module 11 rap module 12 eryTE

eryLM ery module 1

rap module 12

eryTEKS2

eryLM

rap module 11

eryTEKS1 rap module 12

O

OH

O

O

OH

OH

OH

O

O O

O O

FIGURE 4 Domain organization of hybrid bimodular PKS enzymes from erythromycin(ery) and rapamycin (rap) clusters. (See insert for color representation of the figure.)

[115,116]. Although the original model cannot account for the unusual domainorganization seen in some PKSs, the piece-by-piece solving approach of solv-ing structures along with sophisticated homology modeling may yield by far themost knowledge on the three-dimensional structure of these exceedingly com-plex megasynthases [27]. Between each of the modules and domains are specificlinkers, which based simply on their sequence alone, are not “molecular strings”but actually contain a certain degree of stiffness, which undoubtedly confersspecificity between each module or domain [117]. The challenges that arise frommodule and domain recognition through these critical linkers will be discussedin detail later.

As mentioned above, one of the most obvious and daunting challenges thatcombinatorial biosynthesis must address is the substrate specificity of down-stream enzymes. This method relies on relaxed substrate specificity of enzymesas unnatural intermediates are fed into portions of the natural biosynthetic path-ways, but many enzymes have fairly strict specificity, making it increasinglydifficult [10]. As mentioned above, there is a clear, yet not totally understoodrelationship between the structure of the synthase modules and their substratespecificity. One of the more studied domains in regard to substrate specificityis the acyltransferase domain of PKSs, since AT domains choose a starter unitto initiate the assembly process and the units used during chain extensions. The


extender ATs tend to exhibit an overall stricter specificity, although from a chem-ical standpoint, the extended units must contain an α-carboxylate group for thecondensation reaction, so they are typically malonyl-CoA or methylmalonyl-CoA [27]. For example, the DEBS synthase will prime only with propionyl-CoAfollowed by six elongations with (2S)-methylmalonyl-CoA [118].

In summary, adjusting the specificity of AT domains and switching entiredomains or modules are two attractive targets for combinatorial biosynthesis.However, researchers must be careful not to disrupt the overall architecture of themegasynthases, which could potentially lower the overall yield of the compoundor render the entire pathway inactive. A deeper understanding of the structureand function of these enzymes will unquestionably make efforts in combinatorialbiosynthesis more efficient and productive.

B. Future Directions

One possible future direction for combinatorial biosynthesis is the pursuit of nat-ural product discovery outside the two main species of Streptomyces and fungi,which would allow for the introduction of genetic machinery of novel naturalproducts toward combinatorial biosynthesis. The most likely candidate for thispursuit would be marine organisms, which until now have been almost totallyuncharacterized. There has been approximately 600 peptide or peptidic metabo-lites described from various marine taxa, many of which have potential as clinicaltherapeutics, such as the bengamides, milnamides, hemiasterlines, dolastatins,brystatins, and discodermolide [119,120].

Until now, a majority of the compounds have been isolated from Oscillato-riales and Nostocales, with very few from Chroococcales, Stigonematales, andPleurocapsales. Unfortunately, this distribution does not reflect the species’ abil-ity to produce natural products, but rather, the availability of strains and theability to obtain exploitable biomass from the natural habitat. Herein lies theproblem with natural product isolation from marine organisms or cyanobacteria:although filled with a rich landscape of secondary metabolites, the collecting andculturing for many of these fascinating organisms is extremely laborious and timeconsuming [119].

Despite this challenge, there have recently been some interesting findings oncyanobacterial secondary metabolites. At this point, 14 cyanobacterial gene clus-ters have been sequenced and reported. One common attribute among all ofthese is that they contain both NRPS and PKS machinery within a single readingframe, almost as if cyanobacteria have already undergone combinatorial biosyn-thesis naturally. However, little is currently known about how cyanobacteria wereable to evolve these mixed gene clusters and even less is known about the eco-logical or physiological functions of the peptides they produce. As more geneticand biochemical data is obtained, the function of many of these metabolites maybe figured out [119,120].

One example of a cyanobacterial metabolite is barbamide, which can beextracted from Lyngbya majuscule strain 19L, an Oscillatoriale. The biosynthe-sis of this compound has several unique features, including a tri-chloroleucine

CONCLUSIONS AND FUTURE PROSPECTS 275

starter unit which undergoes deamination, extension by a diketide with E-doublebond formation and oxidative carboxylation of the terminal cysteine to form athiazole ring [120]. The important insights gained from barbamide as well asother cyanobacterial secondary metabolites will aid in the development of newdrug targets by the incorporation of new types of machinery into combinatorialbiosynthesis.

Combinatorial biosynthesis may ultimately progress into the rational de novodesign and construction of specific natural products. This advance would involvethe creation of an assembly line of synthetic polyketide synthase or nonriboso-mal peptide synthase modules, which would create a designed metabolite. All theproblems mentioned above currently limit the widespread use of de novo con-struction. Most predominant is the structure and function relationship betweendifferent modules, but progress has been made toward this ultimate goal. Thefollowing discussion focuses on PKSs, but similar problems also face NRPSbiosyntheses. As mentioned earlier, the growth of the polyketide chain requiresthe transfer from the ACP of one module to the ketosynthase of the next. Sincethese modules are typically on separate proteins, both interpolypeptidyl (betweenthe C-terminus of one module and the N-terminus of the next) and intrapolypep-tidyl (between the ACP of one module and the KS of the next) acyl chain transfersmust occur, each of which demands very specific linkers [121,122].

Recent studies have confirmed the importance of these problematiclinker regions. In one case, bimodular PKSs were constructed based on theerythromycin derivative DEBS1-TE, where only the first two modules andthioesterase domain were present. Hybrid polyketide synthases were constructedby replacing one of the modules with its counterpart from the rapamycinPKS cluster. As expected, the authors determined that preservation of theintact acyl-carrier protein-ketosynthase (ACP-KS) didomain between moduleswas the best way to retain activity and produce the desired triketide lactone.For example, if the desired triketide was formed by the first module of theerythromycin cluster and the second from rapamycin, the compound would onlybe produced when the KS domain from the second erythromycin module wasattached to the remaining reduction domains from the rapamycin, ensuring asuccessful ACP-KS transfer between the two modules (Fig. 4) [123].

Another impressive example of the de novo construction of a bimodular PKSwas a more generic approach in which a facile cassette assembly method for theinterchange of modules and domains was utilized [124]. A sequence alignment of140 modules from 14 PKS gene clusters revealed a six-base-pair recognition sitewithin the conserved linker regions. This universal design turned each moduleand linker into synthetic building blocks which are flanked by unique restrictionsites. Two modules, one “donor” and one “acceptor,” were cloned with linkerregions into two separate plasmids and then cotransformed into a strain of E. coli ,yielding a total of 154 possible bimodular combinations. Approximately half ofthese combinations were able to produce the triketide lactone, ultimately revealingthe specificity of each linker. This study demonstrated that all the modules,


domains, linkers, and thioesterase required for natural product biosynthesis canall be moved easily by this truly combinatorial approach.

Acknowledgments

We thank the National Institutes of Health (GM077596) for financial support inour combinatorial biosynthesis studies. M.A.D. acknowledges support from theNational Institutes of Health under Ruth L. Kirschstein National Research Award5 T32 (GM070421) from the National Institute of General Medical Sciences.

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