hybrid peptide polyketide natural products: biosynthesis ... · polyketides with diverse structures...

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Metabolic Engineering 3, 78�95 (2001)

REV

Hybrid Peptide�Polyketide NatuProspects toward Engin

Liangcheng Du, Ce� sar S

Department of Chemistry, University of Californ

Received February 7, 2000; accepted October

The structural and catalytic similarities between modular non-ribosomal peptide synthetase (NRPS) and polyketide synthase(PKS) inspired us to search for hybrid NRPS�PKS systems. Byexamining the biochemical and genetic data known to date for thebiosynthesis of hybrid peptide�polyketide natural products, we show(1) that the same catalytic sites are conserved between the hybridNRPS�PKS and normal NRPS or PKS systems, although theketoacyl synthase domain in NRPS�PKS hybrids is unique, and (2)that specific interpolypeptide linkers exist at both the C- and N-terminiof the NRPS and PKS proteins, which presumably play a criticalrole in facilitating the transfer of the growing peptide or polyketideintermediate between NRPS and PKS modules in hybrid NRPS�PKS systems. These findings provide new insights for intermodularcommunications in hybrid NRPS�PKS systems and should now betaken into consideration in engineering hybrid peptide�polyketidebiosynthetic pathways for making novel ``unnatural'' natural products.� 2001 Academic Press

INTRODUCTION

Nonribosomal peptides refer to linear, cyclic, or branchedpeptides, mostly consisting of less than 20 amino acidresidues, which are often modified by acylation, glycosyla-tion, epimerization, heterocyclization, or N-methylation ofthe amide nitrogen. Many of the nonribosomal peptides areclinically important drugs, such as cyclosporin A (1),penicillin, and vancomycin. Nonribosomal peptides are syn-thesized by nonribosomal peptide synthetases (NRPSs)that can incorporate into the peptide products bothproteinogenic and nonproteinogenic amino acids��over 300different amino acids are known to date (Kleinkauf and vonDo� hren, 1990). NRPS possesses a modular structure, andeach module is a functional building block responsible forthe incorporation and modification of one amino acid unit.

doi:10.1006�mben.2000.0171, available online at http:��www.idealibrary.com

1 To whom correspondence and reprint requests should be addressed.Fax: (530) 752-8995. E-mail: shen�chem.ucdavis.edu.

781096-7176�01 �35.00Copyright � 2001 by Academic PressAll rights of reproduction in any form reserved.

EW

ral Products: Biosynthesis andering Novel Molecules

a� nchez, and Ben Shen1

a, One Shields Avenue, Davis, California 95616

3, 2000; published online December 29, 2000

The order and number of the modules on an NRPS2 proteindictate the sequence and number of amino acids in the resul-tant peptide product. A typical NRPS module consists mini-mally of an adenylation (A) domain responsible for aminoacid activation, a thiolation (T) domain, also known as pep-tidyl carrier protein (PCP), for thioesterification of theactivated amino acid, and a condensation (C) domain fortranspeptidation between the aligned peptidyl and aminoacyl thioesters to elongate the growing peptide chain(Fig. 1A)(Cane, 1997; Cane et al., 1998; Cane and Walsh,1999; Konz and Marahiel, 1999; von Do� hren et al., 1999).Additional domains have also been identified for themodification of the amino acyl and�or peptidyl substratesduring this process, such as an epimerization (E) domainfor the conversion of an l- to d- configuration of an aminoacid (Marahiel et al., 1997), a methylation (MT) domain forN-methylation of the amide nitrogen (Marahiel et al.,1997), a cyclization (Cy) domain for the formation ofheterocyclic rings (Konz et al., 1997), a reduction (R)domain for reductive release of an aldehyde product(Ehmann et al., 1999), and oxidation domains for the con-version of a thiazoline to a thiazole (Ox) (Du et al., 2000a,b;Julien et al., 2000; Molnar et al., 2000; Shen et al., 1999) orfor :-hydroxylation of the incorporated amino acid (Ox$)(Silakowski et al., 1999).

Polyketides are one of the largest groups of naturalproducts and are derived from sequential condensations of

on

2 Abbreviations used: A, adenylation; ACP, acyl carrier protein;AdoMet, S-adenosylmethionine; AL, acyl CoA ligase; AMT, amino trans-ferase; AT, acyltransferase; Bmt, (4R)-4-[(E)-2-butenyl]-4-methyl-l-threonine; C, condensation; CoA, coenzyme A; Cy, cyclization; DEBS,6-deoxyerythronolide B synthase; DH, dehydratase; E, epimerization; ER,enoyl reductase; FAS, fatty acid synthase, KR, ketoreductase; KS, ketoacylsynthase; MT, methyltransferase; NRPS, nonribosomal peptide syn-thetase; O-MT, O-methyltransferase; Ox and Ox$, oxidation; PCP, pep-tidyl carrier protein; PKS, polyketide synthase; PPTase, 4$-phospho-pantetheinyl transferases; R, reduction; T, thiolation; TE, thioesterase.

h
FIG. 1. Modular organization of NRPS and PKS and comparison tobiosynthesis catalyzed by two hypothetical NRPS modules. (B) C�C bondmodules. (C) Posttranslational modification of apo-ACP or apo-PCP into
Review

hpeptide�polyketide biosynthesis catalyzed by a hypothetical NRPS�PKS hycatalyzed by a hypothetical PKS�NRPS hybrid. Abbreviations are defined in

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ybrid NRPS�PKS and PKS�NRPS. (A) C�N bond formation for peptideformation for polyketide biosynthesis catalyzed by two hypothetical PKSolo-ACP or holo-PCP by a PPTase. (D) C�C bond formation for hybrid

Metabolic Engineering 3, 78�95 (2001)doi:10.1006�mben.2000.0171

brid. (E) C�N bond formation for hybrid polyketide�peptide biosynthesisa footnote.

short carboxylic acids. Many of the polyketides are clini-cally valuable drugs, such as daunorubicin, erythromycin,lovastatin, and rapamycin (2). Polyketide biosynthesis iscatalyzed by polyketide synthases (PKSs). Two types ofmicrobial PKSs are known. Type II PKSs are multienzymecomplexes that carry a single set of iteratively used activitiesand consist of several monofunctional proteins for the syn-thesis of aromatic polyketides, such as daunorubicin andtetracycline. Type I PKSs are multifunctional proteins thatharbor sets of noniteratively used distinct active sites, ter-med modules, for the catalysis of each cycle of polyketidechain elongation in biosynthesis of reduced polyketides,such as the macrolide and polyene antibiotics. A typicalPKS module consists minimally of an acyltransferase (AT)domain for extender unit selection and transfer, an acylcarrier protein (ACP) for extender unit loading, and aketoacyl synthase (KS) domain for decarboxylative con-densation between the aligned acyl thioesters to elongatethe growing polyketide chain (Fig. 1B) (Cane, 1997; Cane etal., 1998; Cane and Walsh, 1999; Staunton and Wilkinson,1998; Shen, 2000). Additional domains have also been iden-tified for the modification of the initial ;-carbonyl group,such as a ketoreductase (KR) domain for a ;-hydroxylgroup (Cane, 1997), a methyl transferase domain (O-MT)for O-methylation of the ;-hydroxyl or enol group(Silakowski, 1999), a dehydratase (DH) domain for analkene moiety (Cane, 1997), an enoyl reductase (ER)domain for an alkane moiety (Cane, 1997), an MT domainfor the introduction of a methyl branch into : position (Duet al., 2000b; Kennedy et al., 1999; Molnar et al., 2000;Pelludat et al., 1998), and an acyl CoA ligase (AL) domainfor the priming of the loading module with an unusual star-ter unit (Duitman et al., 1999). An amino-transferase(AMT) domain for the conversion of an activated fatty acidinto an amino acid has also been noted, which is uniquelylocated between a PKS and a NRPS module (Duitman etal., 1999; Kaebernick et al., 2000; Tillett and Neilan, 1999;Tillett et al., 2000).

NRPSs and PKSs apparently use a very similar strategyfor the biosynthesis of two distinct classes of naturalproducts. In addition to sharing a modular organization,both systems use carrier proteins��PCP for NRPS andACP for PKS��to tether the growing chain. BothPCP and ACP are posttranslationally modified by a4$-phosphopantetheine prosthetic group, and this modifica-tion is catalyzed by a family of 4$-phosphopantetheinyltransferases (PPTases) (Fig. 1C) (Lambalot et al., 1996;Walsh et al., 1997). During the entire elongation process,the growing intermediates remain covalently attached to thecarrier proteins, in a thioester linkage via the sulfhydryl


group of the 4$-phosphopantetheine group. Once reachingits full length, the peptide or polyketide product is released

80

from the proteins, as catalyzed by a thioesterase (TE)domain that is usually found at the distal C-terminus of theNRPS or PKS enzymes (Cane, 1997; Cane et al., 1998). Anadditional discrete TE has also been found to associate withseveral NRPS and PKS gene clusters, which has beenimplicated in liberating the mischarged NRPS or PKS whenthe latter is blocked by an unspecific thioesterification at thePCP (de Ferra et al., 1997; Marahiel et al., 1997) or ACPdomain (August et al., 1998; Butler et al., 1999), respec-tively.

The modular structure of NRPS and PKS has greatlyfacilitated rational engineering of metabolic pathways forboth nonribosomal peptide and polyketide biosynthesis.Numerous novel peptide and polyketide metabolites withpredicted structural alterations have been produced bytargeted domain substitution, deletion, addition, reposition,or by introduction of other mutations into NRPSs andPKSs, as well as by exploiting intermodular communica-tions to facilitate the transfer of biosynthetic intermediatesbetween unnaturally linked PKS modules (Cane, 1997;Cane et al., 1998; Cane and Walsh, 1999; Gokhale et al.,1999). One particularly successful system is the 6-deoxyery-thronolide B synthase (DEBS), from which most of ourcurrent understandings of structure and mechanism of PKSwere derived. Thus, on one hand, polyketides with definedsize and functional groups can be designed by specificengineering of the DEBS proteins (McDaniel et al., 1999).On the other hand, genetic engineering of the DEBSproteins in a combinatorial manner by a multiplasmidapproach can result in the production of large libraries ofpolyketides with diverse structures (Xue et al., 1999).

While genetic engineering of PKS currently holds themost promise in generating novel structures, NRPS isemerging to offer equally promising potential for makingnovel metabolites (Cane, 1997; Cane et al., 1998; Cane andWalsh, 1999; Konz and Marahiel, 1999; von Do� hren et al.,1999). In their 1995 seminal work, Marahiel and co-workersfirst demonstrated that novel peptides with altered aminoacid sequence can be made by targeted substitution of an Adomain in the surfactin (3) synthetase SrfA of Bacillus sub-tilis with A domains of other NRPSs of either bacterial orfungal origin (Stachelhaus et al., 1995). More recently, theseresearchers defined general rules, also known as non-ribosomal codes, for the structural basis of substraterecognition in the A domain and showed that the aminoacid specificity of A domain could be altered rationally bysite-directed mutation of the nonribosomal code of the Adomain, rather than A domain substitution, providing afundamentally new strategy for engineered biosynthesis ofnovel peptides (Stachelhaus et al., 1999; Challis et al., 2000).

Review

Alternatively, Walsh and co-workers demonstrated that theediting function of an A domain can be bypassed by using

a PPTase to directly transfer the aminoacyl phosphopan-tetheine group from an amino acyl coenzyme A (CoA) tothe apo-PCP. Subsequent C domain-catalyzed peptidebond formation between the aligned aminoacyl-S-PCPsyielded several ``unnatural'' dipeptides (Belshaw et al.,1999). We recently described a type II PCP that can beaminoacylated by a totally unrelated A domain, providingyet another approach to bypass the editing function of Adomains for engineered biosynthesis of novel peptides (Duand Shen, 1999).

These striking structural and catalytic similaritiesbetween NRPS and PKS (Figs. 1A�1C) have inspired us(Du et al., 2000b; Shen et al., 1999) and others (Cane andWalsh, 1999; Gehring et al., 1998a,b; Quadri, 2000) tosearch for hybrid NRPS�PKS systems integrating bothNRPS and PKS modules (Figs. 1D and 1E). The fact thatindividual domains and modules of both NRPS and PKSare considerably tolerant toward genetic engineering sup-ports the wisdom of combining individual NRPS and PKSmodules for combinatorial biosynthesis. It is imagined thatthese hybrid NRPS�PKS systems will result in the produc-tion of novel metabolites by incorporating biosyntheticbuilding blocks of both amino acids and short carboxylicacids and that the genetic tools developed for engineeringNRPS and PKS should be directly applicable for engineer-ing hybrid NRPS�PKS systems. A great challenge will thenbe to understand the mechanism by which an NRPS-boundgrowing peptidyl intermediate is further elongated by aPKS module (Fig. 1D) or vice versa (Fig. 1E). Since naturalproducts of hybrid peptide�polyketide origin are known,the goal of this article is to examine the biosynthesis of thesecompounds to shed light on intermodular communicationsbetween NRPS and PKS modules and to illustrate thefeasibility of constructing hybrid NRPS�PKS systems forexpanding the size and diversity of ``unnatural'' naturalproduct libraries.

1. BIOSYNTHESIS OF HYBRID PEPTIDE�POLYKETIDE NATURAL PRODUCTS

Hybrid peptide�polyketide metabolites refer to naturalproducts that are biosynthetically derived from amino acidsand short carboxylic acids, and a few examples are depictedin Fig. 2. Based on the biosynthetic mechanisms by whichthe amino acid, or peptide, and carboxylic acid, orpolyketide, moieties are incorporated into these products,these hybrid peptide�polyketide natural products could bedivided into two classes��those whose hybrid pep-tide�polyketide backbone is assembled by a hybrid NRPS�PKS system that mediates the direct elongation of a NRPS-

Review

bound peptidyl intermediate by a PKS module or vice versa(Fig. 2B) and those whose hybrid peptide�polyketide back-

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bone is assembled via other mechanisms that do not requiredirect functional hybridization between NRPS and PKSproteins (Fig. 2A). While the hybrid NRPS�PKS system isthe focus of this paper, we would like to discuss the systemsthat do not involve direct functional hybridization betweenNRPS and PKS proteins briefly first, since they serve asexcellent examples to demonstrate nature's versatility inbiosynthesizing hybrid peptide�polyketide products.

1.1. Systems Not Involving DirectFunctional Hybridization between NRPS and PKS

Coronatine biosynthesis is an example in which theamino acid and polyketide moieties are synthesized byNRPS and PKS enzymes individually and coupled into ahybrid polyketide�amino acid metabolite by a discreteligase (Fig. 3). The phytotoxin coronatine (4), produced bymany pathovars of Pseudomonas syringae, contains two dis-tinct components��the polyketide moiety of coronafacicacid (14), and the amino acid moiety of coronamic acid(15). Variation at the amino acid moiety constitutes theother naturally occurring analogs of 4 (Bender et al., 1999).While acetate, butyrate and pyruvate as precursors for 14,and l-isoleucine as a precursor for 15 were established earlyby feeding experiments (Parry et al., 1994, 1996), it is therecent cloning and molecular characterization of thebiosynthesis gene cluster for 4 that confirm that 14 and 15are biosynthesized by PKS and NRPS, respectively(Rangaswamy et al., 1998). The gene cluster for 4 is com-posed of two loci, encoding PKS and NRPS enzymes,respectively, separated by a regulatory region. Both theNRPS and PKS loci have their own thioesterase, support-ing the hypothesis that both 14 and 15 are released from theNRPS and PKS enzymes before being coupled together toform 4. The latter reaction is most likely catalyzed by aligase, and two candidates for the latter function, Cfa5 andCfl, were indeed identified in the cloned gene cluster. Thefact that various amino acids have been identified in analogsof 4 is consistent with this model, indicative that the ligaseappears to have relaxed amino acid specificity.

Cyclosporin biosynthesis is an example in which apolyketide intermediate is first converted into an amino acidthat is subsequently incorporated into the natural productby an NRPS enzyme (Fig. 4). The cyclic peptide 1 containsan unusual amino acid of (4R)-4-[(E)-2-butenyl]-4-methyl-l-threonine (Bmt, 16) that is of polyketide origin.Although its gene is yet to be characterized, the BmtPKS has been partially purified and extensively studied(Offenzeller et al., 1996). The Bmt PKS appears to containall the enzymatic activities in a single protein. In vitro


incubation of acetyl CoA, malonyl CoA, NADPH, andS-adenosylmethionine (AdoMet) in the presence of Bmt

s
FIG. 2. Examples of hybrid peptide�polyketide natural products cyclo

microcystin (6), bleomycins (7), epothilones (8), myxothiazol (9), pristinamjunctions between the peptide and polyketide moieties are shaded. (A) The bition between NRPS and PKS proteins. (B) The biosynthesis of these compo

82

porin A (1), rapamycin (2), surfactin (3), coronatine (4), mycosubtilin (5),(10), TA (11), yersiniabactin (12), and nostopeptolides (13). The

Review

ycin IIB

osynthesis of these compounds does not require direct functional hybridiza-unds involves hybrid NRPS�PKS systems.

y
FIG. 3. Biosynthetic pathway for coronatine (4) in P. syringae. The pol(15) are coupled into 4 by a ligase.
PKS resulted in the synthesis of (3R)-hydroxy-(4R)-methyl-(6E)-octenoic acid as a CoA thioester (17), clearly estab-lishing the polyketide origin of 16. Further transformationsby additional enzymes convert 17 into 16, which is subse-quently incorporated into 1 by the CssA NRPS enzyme(Fig. 4). Cloning and molecular characterization of the cssAgene from Tolypocladium niveum has revealed that CssAcontains 11 NRPS modules, the fifth of which is predicted tobe responsible for the incorporation of 16 into 1 (Weber etal., 1994).

The fatty acid chains of lipopeptides, such as syringo-mycins, 3, and fengycins, have long been believed to beincorporated into the peptide products by direct transfer ofthe acyl group to the amino group of an NRPS-acti-vated amino acid. Subsequent condensations between theacylated aminoacyl-PCP with the rest of NRPS-activatedamino acids result in the synthesis of lipopeptides. Severalgene clusters for lipopeptide biosynthesis have been clonedand characterized recently (Cosmina et al., 1993; Guenzi etal., 1998; Konz and Marahiel, 1999; Tosato et al., 1997).Biochemical investigations of both SrfA-A (Vollenbroich etal., 1994) and SyrE1 (Guenzi et al., 1998) showed that thefirst module of the Srf and Syr NRPS complexes activatesGlu and Ser, respectively, a fact that strongly supports the

Review

FIG. 4. Biosynthetic pathway for cyclosporin (1) in T. niveum. Theconverted into the corresponding ;-amino acid 16 before its incorporation in

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ketide moiety coronafacic acid (14) and amino acid moiety coronamic acid

hypothesis that acylations in lipopeptide biosynthesis occurafter the activation of the amino acid. Although enzymescatalyzing the coupling reaction between the fatty acid andactivated amino acid are yet to be identified, a C domain,preceding the first NRPS module, has been observed in alllipopeptide NRPSs identified so far, serving as a goodcandidate for this activity.

In contrast to the aforementioned lipopeptides that con-tain a ;-hydroxy fatty acid chain, mycosubtilin (5) andmicrocystin (6) are members of a lipopeptide family thatcarry a ;-amino fatty acid modification, and theirbiosyntheses represent another variation in which a fattyacid or polyketide moiety��after its conversion into anamino acid��is incorporated into the hybrid peptide�polyketide product by an NRPS enzyme. It is important tonotice that, although an enzyme containing both FAS orPKS and NRPS modules is present in these systems, thereis not a direct transfer of a PKS-bound polyketide inter-mediate to a NRPS module; instead the transfer requires anintermediate step for the conversion of the polyketide inter-mediate into an amino acid. The biosynthesis gene clusterfor 5 has been recently cloned and characterized from B.subtilis ATCC6633, revealing features unique for FAS andNRPS (Duitman et al., 1999). The mycosubtilin synthase


polyketide moiety (3R)-hydroxy-(4R)-methyl-(6E)-octenoyl CoA (17) isto 1 by an NRPS enzyme.

subunit A (MycA) combines functional domains of bothFAS and NRPS, as well as an unprecedented AMT domain(Fig. 5A). It is proposed that myristate, activated as acylCoA by the AL domain, and malonyl CoA are first loadedto the two ACP domains of MycA. Condensation betweenmyristyl-ACP1 and malonyl-ACP2 , catalyzed by the KSdomain, results in a ;-ketoacyl-ACP2 intermediate, which issubsequently converted into a ;-aminoacyl-ACP2 by theAMT domain. In contrast to normal NRPS, the extra Cdomain between ACP2 and PCP1 seems to act as an amino-acyl transferase, catalyzing the transfer of the ;-amino-acyl group from ACP2 to PCP1 to yield the ;-amino-acyl-PCP1 intermediate, setting the stage for peptideelongation. Subsequent condensations between ;-amino-acyl-S-PCP1 and the remaining PCP-activated amino acidsare catalyzed by the MycA, MycB, and MycC NRPSenzymes, to finally furnish 5 (Fig. 5B). For the biosynthesisof 6, although only the NRPS region has been published(Meissner et al., 1996; Nishizawa et al., 1999), the DNAsequence of the entire gene cluster has been determined fromthe marine cyanobacterium Microcystis aeruginosaPCC7806 and deposited directly in GenBank (Kaebernicket al., 2000; Tillett and Neilan, 1999; Tillett et al., 2000).On the basis of the functions of similar domains in NRPSand PKS, as well as the unique AMT domain in the myco-subtilin NRPS (Duitman et al., 1999), a similar strategy forincorporating the polyketide-derived ;-amino acid (seeFig. 2A) by an NRPS enzyme could be envisaged for thebiosynthesis of 6.

1.2. Systems Involving Direct Functional Hybridizationbetween NRPS and PKS

Most of the hybrid peptide�polyketide metabolites whosebiosynthesis has been examined so far (Fig. 2B) are assem-bled by hybrid NRPS�PKS systems that mediate directtransfer of a NRPS-bound peptidyl intermediate to a PKSmodule or vice versa (Figs. 1D and 1E). On the basis offeeding experiments with isotope-labeled precursors andisolation of biosynthetic intermediates and shunt meta-bolites, such a model in fact was implicated long before thecharacterization of the modular structure of either NRPSor PKS. For example, by feeding 14C- and 13C-labeledbiosynthetic precursors, Fujii, Umezawa, Takita, and co-workers (Fujii, 1979; Nakatani et al., 1980; Takita, 1984;Takita and Muroka, 1990) showed that the aglycone ofbleomycin (7) was derived from a Ser, two Asn, a His, anAla, an acetate, a Thr, a ;-Ala, and two Cys in Streptomycesverticillus ATCC15003, a fact that supports a hybrid pep-


tide�polyketide biogenesis. Subsequent isolation and struc-tural determination of a series of biosynthetic intermediates,

84

such as P-3A (18), P-4 (19), and P-6m (20), from fermenta-tion cultures led them to propose the biosynthetic pathwayfor 7 as shown in Fig. 6. According to a hybrid NRPS�PKS�NRPS model, we could easily envisage the biosynthesisof the bleomycin aglycone in three stages��(I) NRPS-mediated formation of 18 from Ser, Asn, Asn, and His, (II)PKS-mediated elongation of 18 with malonyl CoA andAdoMet to 19, and (III) NRPS-mediated elongation of 19with ;-Ala, Cys, and Cys to yield 20��involving functionalhybridizations between NRPS and PKS for the transitionI�II and between PKS and NRPS for the transition II�III(Du et al., 2000b; Shen et al., 1999). While the processiveassembly of the bleomycin aglycone by a hybrid NRPS�PKS�NRPS system is evident from the isolation of thelinear peptide (18), peptide�polyketide (19), and pep-tide�polyketide�peptide (20) intermediates, such inter-mediates are rarely accumulated among most other NRPSand PKS systems studied so far (Yu et al., 1999).

Pristinamycins are members of the streptogramin Afamily of metabolites, whose biosynthesis from amino acidsand short carboxylic acids was established by extensivefeeding experiments (Kingston et al., 1983). According to ahybrid NRPS�PKS model, the pristinamycin IIB backbone(10) could be assembled by a hybrid NRPS�PKS systemthat mediates the transfer of the growing peptide orpolyketide intermediate between NRPS and PKS modulesthree times (Fig. 7A). Bamas-Jacques and co-workers(1997) have cloned and partially characterized the genecluster for the biosynthesis of 10 in Streptomycespristinaespiralis and revealed that snaD encodes the NRPSenzyme catalyzing the activation and incorporation of theSer residue into 10. Inactivation of snaD yielded apristinamycin-nonproducing S. pristinaespiralis mutant thataccumulated a series of linear polyketide and polyketide�peptide�polyketide intermediates, such as 21, 22, and 23.The structures of the latter metabolites were determined andare shown in Fig. 7B, providing direct evidence to supporta hybrid PKS�NRPS�PKS�NRPS system for the biosynthesisof 10 (Bamas-Jacques et al., 1997).

While the feeding experiments and isolation of biosyn-thetic intermediates and shunt metabolites certainly sup-port the hybrid NRPS�PKS hypothesis, it is the recentcloning and characterization of multiple gene clustersencoding hybrid peptide�polyketide metabolite biosynthesisthat provide the genetic and biochemical basis forinvestigating functional hybridization between NRPS andPKS proteins. To date, biosynthetic gene clusters that havebeen shown to involve direct functional hybridizationbetween NRPS and PKS include the Blm synthetase for 7from S. verticillus (Fig. 8A) (Du and Shen, 1999; Du et al.,

Review

2000b; Shen et al., 1999), the EPOS synthetase forepothilones (8) from Sorangium cellulosum (Fig. 8B) (Julien

o

m

FIG. 5. Biosynthetic pathway for mycosubtilin (5) in B. subtilis. (A) Dof the ;-amino acid moiety and its incorporation into 5 by NRPS enzymes.

FIG. 6. Biosynthetic pathway for bleomycin (7) in S. verticillus involvedbiosynthetic intermediates P-3A (18), P-4 (19), and P-6m (20) were isolated fro

Review

FIG. 7. Biosynthetic pathway for pristinamycin IIB (10) in S. pristinaespiraintermediate 21 and hybrid peptide�polyketide intermediates 22 and 23 were is

85

main organization of the MycA protein. (B) MycA-mediated biosynthesisAbbreviations are defined in the footnote.

a hybrid NRPS�PKS�NRPS system. The growing hybrid peptide�polyketidethe wild-type S. verticillus fermentation and their structures were determined.


lis involved a hybrid PKS�NRPS�PKS�NRPS system. The growing polyketideolated from a snaD-inactivated mutant and their structures were determined.

w
FIG. 8. Domain and modular organization and classification of knoS. verticillus, (B) epothilone (8) biosynthesis from So. cellulosum, (C) andbiosynthesis from S. hygroscopicus, (F) TA (11) biosynthesis from M. xa

nnostopeptolide (13) biosynthesis from Nostoc sp. GSV224. Abbreviations aris defined in comparison with similar domains identified in the belomycin an

86

n hybrid NRPS�PKS systems for (A) bleomycin (7) biosynthesis from(D) myxothiazol (9) biosynthesis from St. aurantiaca, (E) rapamycin (2)thus, (G) yersiniabactin (12) biosynthesis from Y. enterocolitica, and (H)

Review

e defined in a footnote. References are given in text. The Ox domain in Cd epothilone gene clusters.

et al., 2000; Molnar et al., 2000; Tang et al., 2000), the Mtasynthetase for myxothiazol (9) from Stigmatella aurantiacaDW4�3-1 (Figs. 8C and 8D) (Silakowski et al., 1999), theRap synthetase for 2 from Streptomyces hygroscopicus(Fig. 8E) (Konig et al., 1997), the Ta1 synthetase forantibiotic TA (11) from Myxococcus xanthus (Fig. 8F)(Paitan et al., 1999), the HMWPs for yersiniabactin (12)from Yersinia enterocolitica and Y. pestis (Fig. 8G)(Pelludat et al., 1998; Gehring et al., 1998a,b), and the Nossynthetase for nostopeptolides (13) from Nostoc sp.GSV224 (Hoffmann et al., 1999). Analogous to theclassification system used in FAS, NRPS, and PKS, thefunctional hybridization between NRPS and PKS could begrouped into two classes��type I and type II. As depicted inFig. 8, the interacting NRPS and PKS modules in type Ihybrids are covalently linked with all domains arranged ina linear order on the same protein, while the interactingNRPS and PKS modules in type II hybrids are physicallylocated on separate proteins. For a given hybrid pep-tide�polyketide biosynthetic pathway, the functionalhybridization between the NRPS and PKS modules couldbe either type I, such as the one for 11 (Fig. 8F), type II,such as the ones for 7 (Fig. 8A), 8 (Fig. 8B), 9 (Figs. 8C and8D), 2 (Fig. 8E), and 13 (Fig. 8H), or a combination of bothtypes, such as the ones for 9 (Fig. 8C) and 12 (Fig. 8G).

2. GENETIC EVIDENCE FOR FUNCTIONALHYBRIDIZATION BETWEEN NRPS AND PKS

In a PKS system, the elongation step is the C�C bond for-mation mediated by the KS domain that catalyzes (1) thetransfer of the growing polyketide intermediate of acyl-S-ACP from the upstream PKS module to the active site Cysof KS, and (2) the decarboxylative condensation betweenthe resulting acyl-S-KS and its cognate malonyl-S-ACP(Fig. 1B). In a hybrid NRPS�PKS system, however, the KSdomain should mediate the similar C�C bond formation bycatalyzing the transfer of the growing peptide intermediateof peptidyl-S-PCP from the upstream NRPS module to theactive site Cys of KS to form an peptidyl-S-KS species,followed by similar decarboxylative condensation with thecognate malonyl-S-ACP, resulting in the elongation of thepeptide chain with a short carboxylic acid (Fig. 1D).

In a NRPS system, the elongation step is the C�N bondformation mediated by the C domain that catalyzes thenucleophilic substitution between the acyl group of thegrowing peptide intermediate of peptidyl-S-PCP from theupstream NRPS module and the amino group of its cognateaminoacyl-S-PCP (Fig. 1A). Although the active site of the

Review

C domain has been mapped to a His residue (Stachelhaus etal., 1998), unlike KS in polyketide biosynthesis, it is not

87

known if the acyl group from the upstream peptidyl-S-PCPforms a transient intermediate with the C domain before itreacts with its cognate aminoacyl-S-PCP to form the pep-tide linkage. Similarly, in a hybrid PKS�NRPS system, theC domain should mediate the same C�N bond formationbut by catalyzing the nucleophilic substitution between theacyl group of the growing polyketide intermediate of acyl-S-ACP from the upstream PKS module and the amino groupof its cognate aminoacyl-S-PCP, resulting in the elongationof the polyketide chain with an amino acid (Fig. 1E).

While the above discussion provides the biochemicalbasis for hybrid NRPS�PKS and PKS�NRPS systems, italso suggests that the critical domains for functionalhybridizations should be the PCP domain of NRPS and theKS domain of PKS in a NRPS�PKS hybrid, and the ACPdomain of PKS and the C domain of NRPS in aPKS�NRPS hybrid, respectively. Therefore, it is imaginedthat detailed comparison of these domains between hybridand non-hybrid systems could shed light on the mechanismfor the altered catalytic activities and intermodular com-munications between the interacting NRPS and PKSmodules to constitute hybrid enzymes.

2.1. Hybrid PKS�NRPS Systems

Among all known PKS�NRPS hybrids, the ACP domainof PKS and the C domain of NRPS show no unique featurescompared to usual NRPS and PKS domains, with theexception of the ACPs from BlmVIII and NosB PKSs(Figs. 8A and 8H), which are more similar to PCPs than toACPs. This is despite the fact that C domains in PKS�NRPShybrids must elongate an acyl-S-ACP, instead of anaminoacyl-S-PCP, from the upstream module with theircognate aminoacyl-S-PCP. Actually this is in accord withthe recent findings that the C domain of tyrocidin syn-thetase shows low selectivity toward upstream aminoacyl-S-PCP��known as donor site, and high selectivity towardits cognate aminoacyl-S-PCP��known as acceptor site(Belshaw et al., 1999). If this finding could be confirmed asa general characteristic of the C domain, it will explain whythere is no evolutionary pressure to dramatically rearrangeC domains in the PKS�NRPS hybrids. Physical proximityof the active sites in combination with subtle changes in theC domain primary structure may be enough for it to acceptthe growing polyketide intermediate of acyl-S-ACP, insteadof aminoacyl-S-PCP, as a donor substrate.

Intermodular communications in PKS has been attri-buted to either the intermodular linkers, which exist betweenmodules within a protein, or the interpolypeptide linkers,


which exist between modules residing on two separateproteins (Gokhale et al., 1999; Gokhale and Khosla, 2000).

The regions of the interpolypeptide linkers were in factnoticed in early sequence analysis of both the rapamycin(Aparicio et al., 1996) and rifamycin PKSs (Tang et al.,1998). These regions were recognized to have the potentialto form amphipathic helices, which could mediate coiled-coil interactions between the interacting PKS enzymes(Aparicio et al., 1996). However, it is Khosla and co-workers who very recently put forth the linker hypothesisand suggested that the chain transfer between modules ispermissive as long as these evolutionarily optimized linkerscan provide the connectivity between the adjacent modules(Gokhale et al., 1999; Gokhale and Khosla, 2000). Byappropriate engineering of either the intermodular linkerbetween module 1 and 2 of DEBS and the interpolypeptidelinker between module 2 and 3 of DEBS, these researchersdemonstrated that it is possible to facilitate the transfer ofpolyketide intermediates of acyl-S-ACP between hetero-logous PKS modules derived from both the DEBS and therifamycin PKSs (Gokhale et al., 1999), providing afundamentally new strategy of using modules as buildingblocks for combinatorial biosynthesis.

Inspired by the linker hypothesis for PKS, we set out todo parallel sequence analyses for both NRPS and hybridNRPS�PKS systems to search for the molecular basisof intermodular communications in these systems. Wereasoned that identification of similar linkers in the lattersystems should be viewed as an evidence supporting thelinker hypothesis and that linkers may be a general solutionto provide suitable module connectivity for other multi-modular enzyme systems. We further imagined that any dif-ference of the linkers between the NRPS or PKS and thehybrid NRPS�PKS systems may provide insight into theevolution of modular NRPS and PKS into NRPS�PKSsystems.

To search for the linkers, we use the C-terminal bound-aries of the ACP and PCP domains, defined according tothe conserved active site Ser (Figs. 9A and 9C), and theN-terminal boundaries of the KS and C domains, definedaccording to the conserved active site Cys and core motifC-1 (Konz et al., 1997), respectively (Figs. 9D and 9B). Incontrast to PKS, no apparent intermodular linker could beidentified between both NRPS�NRPS modules and PKS�NRPS modules (type I hybrids). Since the intermodularlinkers for PKS (shaded in Fig. 9E) are very short (between17 and 21 amino acid residues) and poorly conserved (withPro as the only conserved residue) (Gokhale et al., 1999),our inability to identify any intermodular linker for bothNRPS and hybrid PKS�NRPS systems does not necessarilyexclude its existence. On the other hand, lack of inter-modular linker in the latter systems may reflect that the


mechanism for intermodular communications between thetwo aligned NRPS modules or PKS and NRPS modules is

88

embedded in the primary structure of the NRPS or PKS�NRPS proteins, respectively, when the interacting modulesare physically arranged in the same protein. The localvicinity of two active sites of ACP and C at the junction ofa type I PKS�NRPS hybrid may be sufficient to shift andintegrate the chemistry of hybrid polyketide�peptidebiosynthesis. For such systems, there is probably littlepressure for the fusion protein to evolve a specific molecularrecognition or intermodular communication mechanismbetween the active sites of the PKS and NRPS modules.

Interpolypeptide linkers, on the other hand, are identifiedfor both NRPS (Figs. 9B and 9C) and hybrid PKS�NRPSsystems (type II) (Figs. 9A and 9B), suggesting that thecorrect pairing of two interacting NRPS proteins or PKSand NRPS proteins in type II PKS�NRPS hybrids appearsto require specific protein-protein recognition. The N-ter-minal linkers for NRPS proteins in PKS�NRPS hybrids(boxed in Fig. 9B) are 23�76 amino acids long, rich in basicand acidic residues, such as Arg, Glu, and Asp, and veryhydrophilic. Intriguingly, the C-terminal linkers for thePKS proteins in the PKS�NRPS hybrids (boxed in Fig. 9A)are 15�54 amino acids long, rich in acidic residues, such asAsp and Glu, and also generally very hydrophilic. It is,therefore, tempting to propose that these interpolypeptidelinkers may play a critical role in protein-protein recogni-tion, possibly by electrostatic interactions, to constitute afunctional PKS�NRPS hybrid.

2.2. Hybrid NRPS�PKS Systems

In NRPS�PKS hybrids, the PCP of NRPS seems to haveno relevant difference compared to other PCPs, while theKS domain of PKS is unique in comparison with other KSs.The latter finding contrasts to the apparent lack of specialfeatures shown by the C domain of PKS�NRPS hybrids.During our analysis of the biosynthesis gene cluster for 7 inS. verticillus (Du and Shen; 1999, Du et al., 2000b; Shen etal., 1999), we noticed that the KS domain of the BlmVIIIPKS protein (see Fig. 8A) displayed lower similarity to allknown streptomycete KSs than to a particular set of KSdomains from other bacteria. Intrigued by this observation,we decided to set a phylogenetic analysis of KS domainsfrom bacterial multidomain PKSs, with special interest inthose sequences with higher similarity to KS of BlmVIII. Allthe analyzed KSs for gene clusters encoding macrolidebiosynthesis in Streptomyces and Saccharopolyspora fallinto two distinct clusters��KS domains from loadingmodules, also known as KSQ (Bisang et al., 1999), and KSdomains from the extending modules, abbreviated as

Review

``MACRO'' in Fig. 10. The only known Streptomyces KSdomain from a NRPS�PKS hybrid system, the KS of

I
FIG. 9. Amino acid sequences of the interpolypeptide linkers for type I
Review

and amino acid sequences of the intermodular linkers for type I NRPS�PKSPKS are shaded. Newly identified intermodular linkers and interpolypeptidesuch as S for ACP or PCP, Cys for KS, and SxxQ for C, used to establish do

89

PKS�NRPS hybrids (A and B) and type II NRPS�PKS hybrids (C and D)


hybrids (E). Known intermodular linkers and interpolypeptide linkers forlinkers for hybrid NRPS�PKS systems are boxed. The conserved residues,main boundaries, are shaded. References for all sequences are given in text.

c

uo

i,

o

FIG. 10. Phylogenetic relationships among KSs from bacterial multifunactions. Total number of analyzed domains was 107, boundaries of whichincluded as an outgroup sequence of known crystal structure. Multiple seq(1000 resampled data sets) were performed by using the CLUSTAL X prTREEVIEW (Page, 1996). For simplicity, those nodes with a bootstrap supstitutions is proportional to the length of the horizontal lines. The word ``Mmacrolide PKSs (except KSQ domains Nid-Q, Pik-Q, and Tyl-Q): avermectet al., 1997), niddamycin (Kakavas et al., 1997), pikromycin (Xue et al.No. U78289) gene clusters. AviM, avilamycin (Streptomyces viridochromogepothilone (Sorangium cellulosum); HMWP1, yersiniabactin (Yersinia enterKolattukudy, 1992); McyD�E�G, mycrocystins (Mycrocystis aeruginosa);


(Bacillus subtilis); NosB, nostopeptolides (Nostoc sp. GSV224); PksC�E�F aculosis, respectively); PksK�P, pksX locus (unknown product, B. subtilis); Tahere.

90

tional PKSs. Domains in the shaded box are involved in NRPS�PKS inter-were defined as in Aparicio et al. (1996). The E. coli KASII sequence wasence alignment and phylogenetic analysis using the bootstrapping methodgram (Thompson et al., 1997), and the resulting tree was displayed usingport <500 were collapsed in the final tree. The number of amino acid sub-ACRO'' represents KS sequences from Streptomyces or Saccharopolysporan (Ikeda et al., 1999), erythromycin (Donadio and Katz, 1992), ``hyg'' (Ruan1998), rapamycin, rifamycin (Tang et al., 1998), and tylosin (Accession

enes; Gaisser et al., 1997); BlmVIII, bleomycin (S. verticillus); EPOSA-D,colitica); MAS, mycocerosic acid (Mycobacterium tuberculosis; Mathur andMtaB�D�E�F, myxothiazol (Stigmatella aurantiaca); MycA, mycosubtilin

Review

nd PpsA�B�C�D, phthiocerol�phenolphthiocerol (M. leprae and M. tuber-1, antibiotic TA (Myxococcus xanthus). See text for references not included

BlmVIII, does not fall into either of the aforementionedgroups, but belongs to a family that includes sequences fromdifferent gene clusters of diverse bacterial species. Strikingly,close examination of the latter family of sequences revealedthat it contains all the KS domains known to interactwith NRPS modules in both type I and type II NRPS�PKS hybrids, including the KSs of BlmVIII (Fig. 8A),EPOS B (Fig. 8B), MtaD (Fig. 8C), Ta1 (Fig. 8F),HMWP1 (Fig. 8G), and NosB (Fig. 8H), as well as the firstKS domains of PksK and PksP. Although the latter twoPKS genes belong to two gene clusters of unknown functionfrom B. subtilis (Albertini et al., 1995; Kunst et al., 1997),they are respectively located on multifunctional proteinsdownstream of an NRPS module that most likely activatesGly (sequence analysis not shown), a genetic organizationstrongly suggesting that they could be part of a type INRPS�PKS hybrid. However, it should be pointed that thisfamily also includes a few KSs that unlikely belong toNRPS�PKS hybrids, such as the KS of McyG (Kaebernicket al., 2000; Tillett and Neilan, 1999; Tillett et al., 2000),PksF (GenBank Accession No. U00023), and PpsE (alsoknown as Pps5; Azad et al., 1997).

On the basis that the KS domains in all known NRPS�PKS hybrids contain the highly conserved catalytic residuesCys-163, His-303, and His-340 [numbering follows theEscherichia coli KASII sequence (Huang et al., 1998)], wepropose that the transfer of the peptidyl intermediate fromthe aminoacyl-S-PCP of the upstream NRPS module to theCys residue of the KS domain of the PKS module and thesubsequent decarboxylative condensation with its cognatemalonyl-S-ACP are catalyzed by the KS domain in a similarmechanism as in normal PKS. On the other hand, we havenoticed that sequence dissimilarities among KS are espe-cially concentrated in regions which, in the dimeric KASIIenzyme, are involved in monomer-monomer interactionand substrate binding. X-ray crystal structural analysis ofthe KS enzymes, KASI, KASII, and KASIII, of the FAScomplex from E. coli has unveiled recently that the use ofdimer interface to modulate substrate specificity indeedseems to be a conserved feature in condensing enzymes(Huang et al., 1998; Moche et al., 1999; Olsen et al., 1999;Qiu et al., 1999). It is, therefore, tempting to propose thatthe KS in NRPS�PKS hybrids, while using the samecatalytic sites conserved in all condensing enzymes, mayalter its substrate binding site to adapt the peptidyl inter-mediate in hybrid peptide�polyketide biosynthesis. However,a few considerations must be taken into account as deducedfrom our phylogenetic analysis, although we are certainlyaware that the conclusions are based on the analysis ofsequences from a very limited number of hybrid systems,

Review

and they must be taken cautiously. It seems that the evolu-tion of a KS domain to function in a hybrid NRPS�PKS is

91

an unusual event��as far as we know, it only happened once(as represented by the starred node in the phylogenetic treeof Fig. 10), and all KSs interacting with a NRPS module arederived from that single event. On the other hand, evolutionin the opposite direction (a KS belonging to this familyadapting back to PKS�PKS interactions) is possible, asexemplified by PksF and PksE. This is interesting for futuregenetic engineering of hybrid peptide�polyketide biosyn-thetic pathways, raising the question of whether any KSdomain from type I PKS will be able to function in achimeric NRPS�PKS hybrid, provided the proper inter-modular or interpolypeptide linkers (see discussion below),or if only those KSs from the aforementioned family can beused to contrast new hybrids.

Finally, similar to the PKS�NRPS hybrids, the inter-modular linkers in type I NRPS�PKS hybrids are notapparent. In reference to the intermodular linkers of PKS(shaded in Fig. 9E) (Gokhale et al., 1999; Gokhale andKhosla, 2000), a putative region of variable sequencebetween 22 and 62 amino acid residues is identified for typeI NRPS�PKS hybrids (boxed in Fig. 9E). Although it is notpossible to draw any conclusion based on the limited datacurrently available, the sequences in this region are clearly dif-ferent from those of the intermodular linkers in PKS, imply-ing that they may play a role in facilitating the transfer of agrowing peptidyl intermediate, instead of a polyketide inter-mediate, to the PKS module in type I NRPS�PKS hybrids.

On the other hand, putative interpolypeptide linkers fortype II NRPS�PKS hybrids are readily identified at theC-termini of the NRPS proteins (Fig. 9C) and the N-terminiof the PKS proteins (Fig. 9D). Intriguingly, the lattersequences (boxed in Fig. 9D) are much shorter (6�26 aminoacids long) in comparison with the corresponding inter-polypeptide linkers in PKS (shaded in Fig. 9D), and are richin acidic residues such as Asp and Glu. In contrast, theC-termini of NRPS in the type II NRPS�PKS hybrids aregenerally longer than those of normal NRPS proteins(Fig. 9C). As boxed in Fig. 9C, the sequences in this regionare 25�50 amino acids long and very rich in basic residuessuch as Arg. We would like to propose that both theC-terminal region of the NRPS proteins (boxed in Fig. 9C)and the N-terminal region of the PKS proteins (boxed inFig. 9D) act together as interpolypeptide linkers in type IINRPS�PKS hybrids. The fact that the linkers at the C-ter-mini of NRPS are rich in basic residues and those at theN-termini of PKS are rich in acidic residues and that bothtypes of linkers are hydrophilic suggests once again thatthey may play a critical role in protein-protein recognitionby electrostatic interactions to correctly pair the NRPS andPKS proteins in type II NRPS�PKS hybrids.


It should be emphasized that these sequence-basedspeculations will have to be experimentally assessed in the

future. However, they are very compelling based on theavailable genetic and biochemical data and should be takeninto consideration in experimental designs to investigate themolecular basis for intermodular communications in hybridNRPS�PKS systems and in attempts to engineer hybridpeptide�polyketide biosynthetic pathways for making novelunnatural natural products.

3. PROSPECTS TOWARD ENGINEERING HYBRIDPEPTIDE�POLYKETIDE METABOLITES

The field of hybrid peptide�polyketide biosynthesis haswitnessed an exponential growth, with multiple biosynthesisgene clusters cloned and characterized in the past 2 years,and we anticipate many more clusters to be cloned andcharacterized in the next few years. Although biochemicaland mechanistic characterizations of hybrid NRPS�PKSsystems are clearly lacking, sequence analysis of the clonedhybrid peptide�polyketide biosynthesis genes and func-tional comparison of the deduced domains and moduleswith the better-characterized NRPS and PKS systems arestarting to shed light on hybrid peptide�polyketidebiosynthesis. While nature certainly exhibits its versatility inmaking hybrid peptide�polyketide metabolites, it is thehybrid NRPS�PKS systems that are most likely amenablefor combinatorial biosynthesis. A great challenge in study-ing the biosynthesis of hybrid peptide�polyketide naturalproducts, therefore, lies at revealing the basic catalytic andmolecular recognition features and structure�function rela-tionships of these remarkable systems, without which thepotential of combinatorial biosynthesis for the productionof novel peptide�polyketide metabolites cannot be fullyrealized. However, based (1) on the similar domain andmodule functions and similar modular organization ofindividual NRPS and PKS modules between known hybridNRPS�PKS systems and NRPS and PKS, (2) on thedemonstrated role of the intermodular linkers and inter-polypeptide linkers played in PKS, and (3) on the proposedfunctions of the putative intermodular linkers and inter-polypeptide linkers identified for hybrid NRPS�PKSsystems, we could envisage future endeavor in engineeringhybrid NRPS�PKS systems for the production of novelstructures along the following directions. First, the methodo-logies developed for NRPS and PKS engineering should bedirectly applicable to hybrid NRPS�PKS engineering, aslong as the natural intermodular or interpolypeptide linkersare maintained in the resultant NRPS�PKS hybrids. Thisstrategy should allow the introductions of perturbationsinto both the peptide and polyketide moieties of a hybrid


peptide�polyketide metabolite. The ever-growing inventoryof hybrid peptide�polyketide biosynthesis gene clusters

92

should provide a selection of platforms to engineer novelmetabolites with tailored structural features. Second, itshould be possible to construct chimeric PKS�NRPShybrids by the use of appropriately designed linkers.Although we are unable to identify the intermodular linkersfor type I PKS�NRPS hybrids, several interpolypeptidelinkers are defined and available for such investigations.Without knowing the specificity of individual linkers, thenatural pair of interpolypeptide linkers, such as those iden-tified at the C-terminus of BlmVIII and N-terminus ofBlmVII, should be used in the initial experiments. On theother hand, in strategic analogy to the PKS interpolypep-tide linker engineering, the N-terminal interpolypeptidelinkers alone could also be tested and may be sufficient topromote protein�protein recognition and to facilitate inter-mediate transfer in the resultant chimeric PKS�NRPShybrid. Third, chimeric NRPS�PKS hybrids could also beconstructed by the use of appropriately engineered linkerswith or without the inclusion of the unique KS domainsidentified in the natural NRPS�PKS hybrids. Since bothintermodular and interpolypeptide linkers for NRPS�PKShybrids have been identified, it is conceivable that thechimeric NRPS�PKS hybrids could be constructed in eithera type I or a type II structure, depending on the choice of thelinkers used. For the chimeric type II NRPS�PKS hybrid, itwould be wise to use the natural pair of interpolypeptidelinkers in the initial experiments, such as those identified atthe C-terminus of BlmIX and N-terminus of BlmVIII.Should the linkers along prove to be insufficient, the uniqueKS domain from natural NRPS�PKS hybrids should thenbe included, which may provide the needed selectivity forthe elongation of a peptidyl intermediate by a PKS modulein NRPS�PKS hybrids.

ACKNOWLEDGMENTS

Studies on peptide and polyketide biosynthesis in our laboratories havebeen supported in part by an IRG grant from the American Cancer Societyand the School of Medicine, University of California, Davis; NationalScience Foundation Grant MCB9733938; National Institutes of HealthGrants AI40475 and CA78747; a University of California BioSTAR grant;and the Searle Scholars Program�Chicago Community Trust.

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