MINI-REVIEW

Tentative biosynthetic pathways of some microbialdiketopiperazines

Binbin Gu & Shan He & Xiaojun Yan & Lixin Zhang

Received: 13 June 2013 /Revised: 1 August 2013 /Accepted: 2 August 2013 /Published online: 18 August 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Cyclodipeptides and their derivatives, thediketopiperazines (DKPs), constitute a large class of naturalproducts that exhibit various biological properties. Until recent-ly, there are a few characterized DKP biosynthetic pathways. Inall these cases, the formation of the cyclodipeptides that harborthe DKP scaffold is catalyzed either by nonribosomal peptidesynthetases or by cyclodipeptide synthases. This reviewfocuses on the DKP biosynthetic pathways and their associ-ated molecular mechanisms.

Keywords Diketopiperazine . NRPS . CDPS . Biosyntheticgene clusters

Introduction

The diketopiperazines (DKPs) are comprised of a broad arrayof secondary metabolites that are mainly produced by micro-organisms (Huang et al. 2010). They were first discovered in1880 and later studied by E. Fischer (1906). DKPs were longdisregarded because many cyclodipeptides formed from pro-tein hydrolysates were considered to be the by-products ofprotein degradation. During the last 40 years, however, morecomplex molecules bearing the DKP scaffold have receivedan increasing amount of attention, as many of these com-pounds display diverse and noteworthy biological activities.For example, thaxtomin A (1 ) acts as a phytotoxin (Healy

et al. 2000; King and Calhoun 2009). Cyclo(L-Phe-L-Pro) (2 )and cyclo(L-Phe-trans-4-OH-L-Pro) (3 ) are antifungal com-pounds (Ström et al. 2002). Erythrochelin (4 ), coprogen (5 ),and dimerumic acid (6 ) are siderophores (Lazos et al. 2010;Oide et al. 2006; Wilhite et al. 2001). Roquefortine C (7 ) andacetylaszonalenin (8 ) are mycotoxins (García-Estrada et al.2011; Yin et al. 2009). Bicyclomycin (9 ) and albonoursin (10)are antibacterial agents (Fukushima et al. 1973; Kohn andWidger 2005). Brevianamide S (11 ) exhibits selectiveantibacterial activity against Bacillus Calmette–Guérin, sugges-tive of antitubercular potential (Song et al. 2012; Zhang et al.2007). Ambewelamides A (12) and B (13), phenylahistin (14),and verticillin A (15) exhibit antitumor properties (Chu et al.1995; Kanoh et al. 1999a, b; Williams et al. 1998). Gliotoxin(GT) (16) and sirodesmin PL (17) have antibacterial, antiviral,and immunosuppressive properties (Sutton et al. 1994, 1996;Waring and Beaver 1996). Additionally, GT is also a potentinducer of apoptotic and necrotic cell death (Hurne et al. 2002;Pardo et al. 2006; Waring et al. 1988).

Although DKPs have diverse biological activities, thephysiological role of the DKPs in the organisms that producethem remains unclear. It has been suggested that DKPs act asdiffusible molecules involved in cell-to-cell communication.They may constitute a new class of quorum-sensing signals(Degrassi et al. 2002; Holden et al. 1999; Park et al. 2006) orinterspecies signals (Li et al. 2011) in bacteria. Besides, asDKPs have bioactive effects on the plant or animal hosts, arole in transkingdom signaling has also been suggested (Ortiz-Castro et al. 2011; Prasad 1995).

Until now, there are a few characterized DKP biosyntheticpathways. In almost all known cases, nonribosomal peptidesynthetases (NRPSs) are involved in the synthesis ofcyclodipeptides that constitute the DKP scaffold, except inthe albonoursin, pulcherriminic acid (18) (further convertedinto pulcherrimin (19)), mycocyclosin (20), and cyclo(L-Trp-L-Trp)-Me/Me2 (cWW-Me/Me2, 21 /22) pathways, in whichcyclodipeptide synthases (CDPSs), a family of class-Ic aa-

B. Gu : S. He (*) :X. YanKey Laboratory of Applied Marine Biotechnology of the Ministryof Education, Ningbo University, Ningbo 315211, Chinae-mail: heshan@nbu.edu.cn

L. Zhang (*)Key Laboratory of Pathogenic Microbiology and Immunology,Institute of Microbiology, Chinese Academy of Sciences,Beijing 100190, Chinae-mail: zhanglixin@im.ac.cn

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tRNA synthetase (aaRS)-like enzymes, use aa-tRNAs as sub-strates to catalyze the formation of the DKP peptide bonds(Belin et al. 2012; Gondry et al. 2009; Giessen et al. 2013).NRPS are modularly organized multienzyme complexes ofremarkable size, and each module is responsible for the incor-poration of one amino acid into the final peptide. Each modulecan further be subdivided into domains, which representthe enzymatic units that catalyze the individual steps ofnonribosomal peptide synthesis. These domains are theadenylation domain (A), the peptidyl carrier protein domain(T), and the condensation domain (C) (Schwarzer et al. 2003).Cyclodipeptides can be formed by dedicated NRPSs, as inbrevianamide F (23 ) (Maiya et al. 2006), erythrochelin(Lazos et al. 2010), ergotamine (24) (Correia et al. 2003),roquefortine C (García-Estrada et al. 2011), acetylaszonalenin(Yin et al. 2009), thaxtomin A (Healy et al. 2000), GT (Balibarand Walsh 2006), or sirodesmin PL (Gardiner et al. 2004)biosynthesis. Alternatively, cyclodipeptides can be formed byNRPSs during the synthesis of longer peptides, as truncatedside products, as in cyclo(D-Phe-L-Pro) (25) or cyclomarazineA (26) biosynthesis (Gruenewald et al. 2004; Schultz et al.2008; Schwarzer et al. 2001). In most of these DKP sideproducts, the formation of the DKP ring in these molecules isprimarily activated via a covalent thioester linkage to the en-zyme and for which the cis conformation of the peptide bond,necessary for the cyclization reaction, is favored by the presenceof a prolyl (Schwarzer et al. 2001) or N-methyl-amino acylresidue (Schultz et al. 2008). CDPSs constitute a new familyof peptide bond-forming enzymes that use aa-tRNAs as sub-strates to form various cyclodipeptides. They are associated withcyclodipeptide-tailoring enzymes (Belin et al. 2009; Cryle et al.2010; Giessen et al. 2013; Gondry et al. 2001) in the biosyn-thetic pathways and dedicated to the biosyntheses of DKPs.

Dedicated NRPS-dependent pathways for DKPsbiosyntheses

GT biosynthetic pathway

The fungal metabolite GT is the prototype of a class ofepipolythiodioxopiperazines (ETPs), which is characterizedby the presence of an internal disulfide bridge (Gardineret al. 2005), that are produced by several fungal species,e.g., Aspergillus fumigatus , Penicillium , and Trichodermaspecies (Macdonald and Slater 1975; Shah et al. 1995;Wilhite et al. 2001). Because of the extraordinary structureand activity of GT, this fungal metabolite has been the subjectof many investigations. There are two reported modes ofaction for their toxicity. Firstly, they act as redox-active toxinsgenerating reactive oxygen species by cycling between theiroxidized disulfide and reduced dithiol forms. Secondly, theyform mixed disulfides with proteins that have accessible thiol

groups (Chai and Waring 2000; Munday 1987). The genesresponsible for GT biosynthesis are organized in a clusterwithin the A . fumigatus genome (Patron et al. 2007). Thiscluster comprises 13 genes (Gardiner and Howlett 2005;Schrettl et al. 2010) (Fig. 1a), and the borders of the clusterwere determined by functionally assigning individual genes toGT biosynthesis. In recent years, the key steps of the GTbiosynthesis pathway have been elucidated (Fig. 1b). GliP isa three-module (A1–T1–C1–A2–T2–C2–T3) DKP synthe-tase, which is responsible for the biosynthesis of the DKPscaffold (Fig. 1c). It has been shown that the C2–T3 domainsare neither necessary for dipeptide formation nor for release(Balibar andWalsh 2006). The glutathione S -transferase GliGand the cytochrome P450 (P450) monooxygenase GliC ac-count for the C–S bond formation, and the GToxidase GliT isinvolved in the formation of the disulfide bridge (Scharf et al.2012). However, several open questions in GT biosynthesisremain to be solved. The GT cluster displays similarities toother ETP biosynthesis gene clusters such as the one codingfor sirodesmin PL biosynthesis (Gardiner et al. 2004; Patronet al. 2007). It is, thus, likely that the core enzymes, which areresponsible for the biosynthesis of the ETP backbone, aresimilar. Recently, nine novel GT-related intermediates wereidentified by comparing the metabolite extracts of the wildtype and a gliZ (transcriptional regulator) knockout strain ofA . fumigatus using 2D nuclear magnetic resonance spectros-copy (Forseth et al. 2011). Althoughmost of these compoundsare shunt products, their structures support the model of GTbiosynthesis (Gardiner and Howlett 2005).

Prenylated indole DKP alkaloids biosynthetic pathways

Prenylated indole alkaloids, which are found mostly in fungi ofgeneraAspergillus andPenicillium , are hybrid natural productscontaining both aromatic and isoprenoid moieties (Stockinget al. 2000; Williams et al. 2000). Besides L-tryptophan, theprecursor of indole rings, most of these substances contain asecond amino acid to form DKPs (Williams et al. 2000). Inrecent years, there are only three dedicated NRPS-dependentpathways (roquefortine, acetylaszonalenin, and fumitremorginbiosynthetic pathways), which are implicated in the prenylatedindole DKP alkaloids biosyntheses. An early phylogeneticanalysis of NRPSs of the type A1–T1–C1–A2–T2–C2 showsthat the prenylated indole DKP alkaloid NRPSs group, whichdiffered from the group of ETP-related NRPSs, can be furtherdivided into the fumitremorgin cluster and the roquefortine/acetylaszonalenin cluster (García-Estrada et al. 2011).

Fumitremorgin biosynthetic pathway

Fumitremorgin-type alkaloids (Li 2011), derived frombrevianamide F, represent a large group within the prenylatedindole alkaloids (Li 2010, 2011; Williams et al. 2000). This

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group of substances includes tryprostatins, fumitremorgins,and verruculogen (27) (Frisvad et al. 2009; Li 2010, 2011).Some of these compounds have been found to show interest-ing biological activities (Li 2011; Mundt et al. 2012). Forexample, fumitremorgin C (28) and tryprostatin A (29) arespecific inhibitors of the multidrug resistance protein BCRP/ABCG2 (Allen et al. 2002; Rabindran et al. 2000; Woehleckeet al. 2003). Verruculogen and fumitremorgin B (30) wereshown to have genotoxicity (Sabater-Vilar et al. 2003). A genecluster, for the biosynthesis of fumitremorgin-type alkaloids,was identified in A . fumigatus Af293 by comparative studiesof the genome sequences of A . fumigatus Af293, A .fumigatus A1163, and Neosartorya fischeri NRRL181(Mundt et al. 2012) (Fig. 2a). Based on the results obtainedfrom bioinformatic, molecular biological, and biochemicalinvestigations, this cluster was proposed to comprise eightgenes and for which the verruculogen is the end product ofits putative biosynthetic pathway (Mundt et al. 2012)(Fig. 2b). The biosynthesis of verruculogen begins with thecondensation of L -tryptophan and L -proline to form

brevianamide F, which is catalyzed by the NRPS FtmPS(A1–T1–C1–A2–T2–C2). Conversion of brevianamide F totryprostatin B (31 ) is catalyzed by the prenyltransferaseFtmPT1. Hydroxylation of tryprostatin B at C6 by the P450enzyme FtmP450-1 and subsequent methylation by themethyltransferase FtmMT result in the formation oftryprostatin A. The functions of two other P450 enzymes,FtmP450-2 and FtmP450-3, are attributed to the cyclizationof tryprostatin A to fumitremorgin C and its subsequent hy-droxylation to yield 12,13-dihydroxyfumitremorgin C (32).The prenyltransferase FtmPT2, responsible for the N -prenylation of 12,13-dihydroxyfumitremorgin C, results inthe formation of fumitremorgin B, which is then converted toverruculogen by the α-ketoglutarate-dependent dioxygenaseFtmOx1. Recently, a new gene, ftmPT3 , responsible for O-prenylation of an aliphatic hydroxyl group in verruculogenwasfound at a different location in the genome of N . fischeriNRRL181 than the identified cluster (Mundt et al. 2012).Thus, the whole biosynthetic pathway of fumitremorgin A(33) was identified.

Fig. 1 GT biosynthesis. a GT biosynthesis gene cluster of A . fumigatus .The cluster is composed of the following genes: gliZ zinc finger tran-scription factor, gliI 1-aminocyclopropane-1-carboxylate synthase, gliJ adipeptidase, gliP a two-module NRPS, gliC and gliF two P450monooxygenases, gliM O -methyltransferase, gliG a glutathione S -

transferase, gliK a hypothetical protein, gliA a major facilitator typetransporter, gliN a methyltransferase, gliT a GToxidase, gliH a conservedhypothetical protein. b The key steps of the GT biosynthesis pathway. cOrganization of the GT NRPS, GliP

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Roquefortine biosynthetic pathway

Alkaloids of roquefortine nature (such as roquefortine C;glandicolines A (34 ) and B (35 ); and meleagrin (36 )),consisting of residues of tryptophan, histidine, andmevalonate,are produced by Penicillium fungi (Kozlovskii et al. 2013).Among them, the mycotoxin roquefortine C is the most ubiq-uitous and well studied with respect to biological activity. Inaddition, roquefortine C is a well-known neurotoxin (Wageneret al. 1980) and is shown to inhibit the enzymes of the gastro-intestinal tract and P450 activity (Aninat et al. 2001). Genomicmining and microarray data analysis revealed seven genes (anNRPS gene, five modifying enzyme genes, and a transportergene) clustered in the genome of Penicillium chrysogenumwhich are responsible for the biosynthesis of roquefortine/meleagrin metabolites (Ali et al. 2013; García-Estrada et al.

2011) (Fig. 3a). The NRPS PC21g15480 (roqA) (A1–T1–C1–A2–T2–C2) belongs to a group of indole alkaloid type 2module NRPSs and differs in their unique Trp-binding pocketfrom related enzymes involved in the biosyntheses ofacetylaszonalenin and fumitremorgin (García-Estrada et al.2011). The functions of the modifying enzymes have beendeduced from the alkaloid products using mutants deleted ineach gene (Ali et al. 2013). The dimethylallyltryptophansynthase PC21g15430 (roqD ) catalyzes the reverseprenylation of histidyltryptophanyldiketopiperazine (HTD)(37 ) at the C3 position in its indole moiety utilizingdimethylallyl diphosphate (DMAPP) derived from mevalonicacid lactone of the mevalonate pathway to form roquefortine D(38) (Fig. 3b). In addition, it also catalyzes the ring closurebetween C2 and N14 of the DKP moiety. Both conversionsseem to occur simultaneously, similar to the formation of

Fig. 2 Verruculogen biosynthesis. a Known biosynthetic gene clustersof verruculogen in A . fumigatus Af293 and A1163 and N . fischeriNRRL18. Genes with high sequence similarities at the amino acid level

are indicated between dotted lines . Functions of genes with black arrowshave been proven experimentally. b Proposed biosynthetic pathway offumitremorgin A in N . fischeri NRRL181

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aszonalenin (39) from (R)-benzodiazepinedione (40) (Yin et al.2009). Oxidation of HTD at position C12–C15 of the histidinylmoiety to dehydrohistidyltryptophanyldiketopiperazine

(DHTD) (41 ) is carried out by the P450 oxidoreductasePC21g15470 (roqR), leading to a branch in the pathway. Twopossible reactions of HTD lead to a branch of the roquefortine/

Fig. 3 Gene cluster and proposed roquefortine C/meleagrin pathway. a Schematic representation of the open reading frames (ORFs) present in theroquefortine C–meleagrin cluster. b Proposed biosynthetic pathway in P. chrysogenum

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meleagrin pathway: one via oxidation by RoqR to DHTD andfurther to roquefortine C by dimethylallyl addition by RoqD andthe other via dimethylallyl addition by RoqD to roquefortine Dand further to roquefortine C via oxidation carried out by RoqR(Fig. 3b). The MAK 1-like monooxygenase PC21g15460(roqM ) catalyzes the conversion of roquefortine C toglandicoline A. The P450 monooxygenase PC21g15450(roqO) is involved in the oxidation of glandicoline A on its

tryptophan moiety, resulting in the formation of glandicoline B,and the methyltransferase PC21g15440 (roqN ) leads tomeleagrin (Fig. 3b). It is likely that the transporter encoded byPc21g15420 (roqT ) is responsible for the secretion ofroquefortine/meleagrin metabolites. However, its deletion hadlittle effect on the metabolic profile, suggesting that passivetransport, diffusion, or another transporter might be involvedin secretion (Ali et al. 2013).

Fig. 4 a Putative biosynthetic gene cluster of acetylaszonalenin in N . fischeri NRRL181, and b hypothetical biosynthetic pathway foracetylaszonalenin

Fig. 5 Thaxtomin biosynthesis.a Organization of the thaxtominbiosynthetic gene cluster in S .turgidiscabies . The txtR geneencodes a cellobiose-responsivepathway specific activator.Two genes encoding putativetransposases are in light yellow.The txtA and txtB genes encodeNRPSs. The txtC and txtE genesencode P450s. The txtD geneencodes NOS that produces NOfrom L-arginine. b Proposedpathway for thaxtomin Abiosynthesis. c Organizationof the thaxtomin NRPSs, TxtAand TxtB. M N-methylationdomain

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Acetylaszonalenin biosynthetic pathway

Acetylaszonalenin is a mycotoxin derived from tryptophan andanthranilic acid (42 ) and was isolated initially from anunidentified Aspergillus species. Together with its non-acetylated form aszonalenin, it was also identified in variousfungal strains, e.g., A . fumigatus , Aspergillus zonatus , and N .fischeri NRRL181 (Li 2010). A gene cluster, for the biosyn-thesis of acetylaszonalenin, was identified on the chromosomeof N . fischeri NRRL181 by homologous search and sequenceanalysis (Yin et al. 2009) (Fig. 4a). This cluster consists ofthree genes coding for a putative NRPS (AnaPS), aprenyltransferase (AnaPT), and an acetyltransferase (AnaAT).AnaPS is predicted to encode an NRPS (A1–T1–C1–A2–T2–C2), which is expected to be responsible for the condensationof tryptophan and anthranilic acid. AnaPT showed clear

sequence similarity to indole prenyltransferases and was prov-en to be responsible for the reverse prenylation of (R )-benzodiazepinedione at position C3 of the indole moiety inthe presence of DMAPP, resulting in the formation ofaszonalenin. AnaAT was found to catalyze the acetylation ofaszonalenin at position N1 of the indoline moiety, resulting inthe formation of acetylaszonalenin (Fig. 4b). The biosyntheticpathway described for acetylaszonalenin could also be plausi-ble for other dipeptide derivatives with prenyl moieties atposition C3 of the indoline rings.

Thaxtomin biosynthetic pathway

Thaxtomin phytotoxins, produced by plant-pathogenicStreptomyces species, are DKPs formed from the condensa-tion of L-phenylalanine and L-4-nitrotryptophan (43) groups.

Fig. 6 The erythrochelin biosynthetic gene cluster (erc). a The genearrangement of the nrps5 (erc) cluster of S . erythraea . ercA LysRregulator, ercB δ-N-ornithine monooxygenase, ercC ferric-siderophore

lipoprotein receptor, ercD NRPS, ercE MbtH-like protein, ercF andercG ABC-type permease. b The proposed mechanism of erythrochelinbiosynthesis. mcd GCN5-like acetyltransferase, E epimerization domain

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The inclusion of a nitro group that is essential for their phy-totoxicity makes them unique among microbially generatedmetabolites. Individual thaxtomins differ only in the presenceor absence of the N-methyl and hydroxyl groups and theirrespective substitution sites (King and Calhoun 2009).Although the mechanism of action is as yet unknown,thaxtomin A causes the inhibition of cellulose synthesis inplants (Bischoff et al. 2009; Loria et al. 2006; Scheible et al.2003). The genes responsible for thaxtomin biosynthesis areorganized in a cluster within the chromosomes of Streptomycesturgidiscabies , Streptomyces scabies , and Streptomycesacidiscabies (Kers et al. 2005) (Fig. 5a). txtA and txtB encodeNRPSs, as shown in Fig. 5c, that are implicated in the assem-bly of the thaxtominN ,N ′-dimethyldiketopiperazine core fromthe novel nonproteinogenic amino acid L-4-nitrotryptophan,L-phenylalanine, and S-adenosyl-L-methionine (SAM), usingATP and Mg2+ as cofactors (Johnson et al. 2009) (Fig. 5b).txtC encodes a P450 that is required for post-cyclization hy-droxylation of the cyclic dipeptides (Healy et al. 2002). txtDencodes a nitric oxide synthase (NOS) that generates NO fromL-arginine (Kers et al. 2004). Recently, a report that txtE

encodes a unique P450 that catalyzes regiospecific 4-nitrationof L-tryptophan, the first step in thaxtomin A biosynthesis,using NO and O2 as cosubstrates and spinach ferredoxin (Fd)and ferredoxin reductase (Fr) as surrogate electron donors hasbeen published (Barry et al. 2012). This is the first biochem-ically characterized example of direct nitration reaction in abiosynthetic pathway. It also expands the wealth of transfor-mations known to bemediated by P450s andmay prove to be auseful nitration biocatalyst (Barry and Challis 2012). Sincethen, all the enzymes responsible for thaxtomin A biosynthesishave been clarified.

Erythrochelin biosynthetic pathway

Erythrochelin is a bioactive iron-chelating DKP and producedby Saccharopolyspora erythraea . Its structure closely resem-bles foroxymithine (44), which has found clinical applicationin cancer therapy and as an inhibitor of angiotensin-convertingenzyme (Aoyagi et al. 1985; Imoto et al. 1987). In contrast tothe usual DKP biosynthesis mechanism, in which the genesresponsible for the biosynthesis of DKP is located in one gene

Fig. 7 The ergopeptine biosynthesis gene cluster. The gene arrangementof the ergopeptine biosynthesis gene cluster of C . purpurea strain P1.cpox3 a oxidase, cpps2 an NRPS, cpP450-1 a P450 monooxygenase,

cpcat2 a catalase, cpox1 an oxidase, cpd1 a dimethylallyl tryptophansynthase, cpps1 an NRPS. The narrow white boxes in cpps1 and cpps2denote the introns in these genes. Cyc heterocyclization domain

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cluster, the biosynthesis of erythrochelin requires unprece-dented crosstalk between separate nonribosomal peptide geneclusters (nrps5 and nrps1 ) (Lazos et al. 2010). The NRPSgene ercD , within the nrps5 cluster, is implicated in theassembly of erythrochelin (Fig. 6a) and in which theerythrochelin biosynthesis follows an orthodox colinearmechanism, as shown in Fig. 6b. The flavin-dependentmonooxygenase ErcB carries out the δ-N-hydroxylation ofL-ornithine (Lazos et al. 2010). However, a GCN5-like N-acetyltransferase gene (mcd ), known to be required forerythrochelin biosynthesis, is located in nrps1 lying some2 Mbp distant from the nrps5 cluster on the S . erythraeagenome (Lazos et al. 2010). Sequence and biochemical anal-ysis of Mcd has demonstrated that Mcd catalyzes both thedecarboxylation of malonyl-CoA to acetyl-CoA and the sub-sequent acetyl transfer from acetyl-CoA to the δ-amino groupof δ-N -hydroxy-L -ornithine (45 ) to provide the L -δ-N -acetyl-δ-N -hydroxyornithine (46 ) building block forerythrochelin biosynthesis (Fig. 6b) (Gu et al. 2007; Lazoset al. 2010). This is an unprecedented example of functionalcrosstalk between NRPS-dependent biosynthetic pathways.

Ergopeptine biosynthetic pathway

Ergopeptines, which have been isolated as predominant alka-loids from Claviceps purpurea , are D-lysergic acid (47) de-rivatives with tripeptide moieties attached to its carboxylgroup via peptide-like amide bonds. Ergopeptines exert di-verse activities (Wallwey and Li 2011). For example,Ergotamine and dihydroergotamine (48) are clinically usedfor the treatment of diverse diseases, including acute migraineattacks and cluster headache. Ergovaline (49) is implicated inlivestock toxicoses caused by ingestion of endophyte-infectedgrasses. The gene cluster, responsible for the biosynthesis ofergopeptines, includes two distinct NRPS genes, cpps 1(LPS1) and cpps 2 (LPS2), as shown in Fig. 7 (Wallweyand Li 2011). It was proposed that D-lysergic acid is activatedby LPS2 (A–T–C) and bound to the T domain of LPS2. LPS1(A1–T1–C1–A2–T2–C2–A3–T3–Cyc) activates alanine,phenylalanine, and proline by its A1, A2, and A3 domains,respectively, and their active forms are bound to the three Tdomains. D-Lysergic acid is then successively elongated to theD-lysergyl monopeptide, dipeptide, and tripeptide by LPS1.

Fig. 8 Nonribosomal assembly of tyrocidine A. Three NRPSs, TycA, TycB, and TycC, act in concert to synthesize the cyclic decapeptide from theamino acid precursors. TE thioesterase domain

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Finally, enzyme-bound D-lysergyl tripeptide is released asD-lysergyl peptide lactam (Fig. 7). One further heterocyclizationstep results in the formation of the final ergopeptine product.It was speculated that a P450 enzyme is involved in thisreaction (Wallwey and Li 2011). The presence of two distinctNRPS subunits catalyzing the formation of ergot alkaloidpeptides is the first example of a fungal NRPS systemconsisting of different NRPS subunits.

DKPs, as truncated side products, synthesized by NRPSs

In a few cases, DKPs can be formed by NRPSs during thesynthesis of longer peptides, as truncated side products. It isbelieved that, in these cases, the cyclic dipeptide formationresults from the spontaneous intramolecular cyclization of anearly linear peptidyl thioester intermediate bound to a T do-main. For example, cyclo(D-Phe-L-Pro), which is releasedprematurely during the synthesis of the decapeptide tyrocidine

A (50) (Stachelhaus et al. 1998) (Fig. 8), and cyclomarazineA, which is released during the synthesis of the heptapeptidecyclomarin A (51) (Schultz et al. 2008) (Fig. 9). In the firstcase, the D-Phe-Pro intermediate bound to the second moduleis chemically unstable and released as a side product as thecyclo(D-Phe-L-Pro). In the second case, cyclomarazine A isconsistent with this biosynthetic model in which the module 2bond diketide is cleaved from the NRPS CymA to yield theDKP. This premature cleavage reaction may be facilitated bythe thioesterase of type II (TEII) CymQ (Schwarzer et al. 2002).

CDPS-dependent pathways for DKPs biosyntheses

CDPSs belong to a newly defined family of enzymes that areunrelated to NRPSs and use aa-tRNAs as substrates to syn-thesize the two peptide bonds of various cyclodipeptides,which are the precursors of many natural products with vari-ous biological activities. The fact that CDPSs do not need to

Fig. 9 Biosynthetic gene cluster organization of cym and proposedbiosynthesis of cyclomarin A and cyclomarazine A. Each arrow repre-sents the direction of the transcription of an ORF and is color coded tosignify enzyme function. NRPS-related genes are colored blue. Oxidative

genes are colored red , O-methyltransferases in orange , prenylation-related genes in green , 2-amino-3,5-dimethylhex-4-enoic acid (ADH)biosynthetic genes in purple , and regulatory/transport/other genes inblack

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activate the amino acids, but instead use already activatedamino acids in the form of aa-tRNAs, explains how smallenzymes (about 26 kDa) can catalyze the formation of theDKP scaffold. However, the NRPSs involved in DKP biosyn-thesis are large, multienzymatic systems about ten times thesize of CDPSs. Up to now, there are only four CDPS-dependent pathways that have been fully characterized: thosefor albonoursin in Streptomyces noursei , pulcherrimin inBacillus subtilis , mycocyclosin in Mycobacterium tuberculo-sis , and cWW-Me/Me2 in Actinosynnema mirum (Belin et al.2012; Giessen et al. 2013). Albonoursin derived from cyclo(L-Phe-L-Leu) (cFL, 52) has been shown to have antibacterialactivity, pulcherriminic acid derived from cyclo(L-Leu-L-Leu)(cLL, 53) chelates iron in Bacillus species (Cryle et al. 2010),andmycocyclosin derived from cyclo(L-Tyr-L-Tyr) (cYY, 54)may be essential forM . tuberculosis viability, whereas, so far,none of cWW-Me/Me2 derived from cWW (55) has been

shown to have biological activity (Belin et al. 2009; Giessenet al. 2013; McLean et al. 2008).

The albonoursin biosynthetic pathway is the first describedCDPS-dependent pathway, in which AlbC is the CDPS andmainly produces cFL (Gondry et al. 2009; Lautru et al. 2002).albA and albB encode the cyclic dipeptide oxidase that areimplicated in the conversion of cFL to albonoursin (Gondryet al. 2001) (Fig. 10a). In the pulcherrimin biosynthetic path-way, YvmC is the CDPS andmainly produces cLL (Tang et al.2006).CypX encodes a P450, which can oxidate the DKP ringof cLL and convert it to pulcherriminic acid (Cryle et al. 2010)(Fig. 10b). This is the first evidence of a role for a P450 in aCDPS-dependent pathway. In the mycocyclosin biosyntheticpathway, Rv2275 is the CDPS and mainly produces cYY(Gondry et al. 2009). Cyp121 encodes a P450, which cata-lyzes the formation of an intramolecular C–C bond betweentwo tyrosyl carbon atoms of cYY, resulting in a novel

Fig. 10 The CDPS-dependentbiosynthetic pathways of aalbonoursin, b pulcherrimin,c mycocyclosin, andd cWW-Me/Me2

Appl Microbiol Biotechnol (2013) 97:8439–8453 8449

chemical entity (Belin et al. 2009) (Fig. 10c). In the cWW-Me/Me2 biosynthetic pathway, Amir_4627 is the CDPS and ex-clusively produces cWW. Amir_4628 encodes a SAM-dependent N-methyltransferase, which catalyzes two succes-sive methylations at the DKP ring nitrogens of cWW and isthe first identified DKP ring-modifying methyltransferase(Giessen et al. 2013) (Fig. 10d). AlbC, YvmC, and Rv2275share a common architecture, which is highly similar to thecatalytic domain of class-Ic aaRSs, and a common ping-pongmechanism, in which an amino acid is transferred from an aa-tRNA to an active-site serine (Bonnefond et al. 2011; Sauguetet al. 2011; Vetting et al. 2010). Although the structure andmechanism of Amir_4627 have not been studied to date, thehigh substrate specificity, as well as the identity of the cWWproduct, suggests that an as yet unknown mechanism wouldbe involved in the Amir_4627 catalysis (Giessen et al. 2013).So far, all characterized CDPSs are originated from bacteria.However, the Nvec-CDPS2, identified in the sea anemoneNematostella vectensis , is an active CDPS and catalyzes theformation of various cyclodipeptides, preferentially contain-ing tryptophan. This is the first active CDPS identified ineukaryotes (Seguin et al. 2011).

Conclusions

Only a few DKP biosynthetic pathways that belong to eitherNRPS-dependent pathways or CDPS-dependent pathwayshave been fully characterized. For each of these pathways,the nature of the DKP produced has been identified and themechanism of DKP formation has been elucidated. It is con-ceivable that, in some pathogenic fungi and bacteria, DKPs(e.g., thaxtomin phytotoxins and ETP toxins) play an essentialrole in virulence. Therefore, every step of the biosynthesismight be an appropriate drug target for antifungal orantibacterial therapy. Beside this, enzymes involved inDKPs biosyntheses catalyze mechanistically intriguing reac-tions (e.g., nitration of indoles in thaxtomin biosynthesis andC–S or S–S bond formation in GT biosynthesis), and thesebiocatalysts may be of value for biotechnological applications.Once the genome of the producing microbe is sequenced andthe pathways are mined, there will be more opportunities toclone, overexpress, and purify these enzymes to test on sub-strates with the help of synthetic chemists (Zhuo et al. 2010).The characterization of new and versatile enzymes involved inDKP biosynthesis will open up new possibilities for pathwayengineering and combinatorial approaches to further increasethe natural diversity of DKPs, a family of compounds withdiverse biological properties. In the last 10 years, a number ofbiosynthetic gene clusters responsible for the biosyntheses ofimportant natural products have been successfully heterolo-gously expressed in Escherichia coli , which is the mostextensive and successful expression system (Watanabe and

Oikawa 2007). In the future, this system can also be used forthe expression of DKPs gene clusters. By this way, morerational structural modifications through combinatorial bio-synthesis and yield improvements by metabolic engineeringcan be easily implemented.

Acknowledgments This work was financially supported in part bygrants from the 973 program (2013CB734000), 863 Program of China(2013AA092902), Zhejiang Provincial Natural Science Foundation ofChina (LQ13B020004), Zhejiang Marine Biotechnology InnovationTeam (2012R10029-2), Ningbo Marine Algae Biotechnology Team(2011B81007), and K.C. Wong Magna Fund in Ningbo University. L.Z.is an Awardee for the National Distinguished Young Scholar Program inChina.

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