Secondary Metabolite Gene Clusters

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Secondary Metabolite Gene Clusters

YONGQIANG ZHANG, NANCY KELLER, and DIMITRIOSTSITSIGIANNIS

Department of Plant Pathology, Universtiy of Wisconsin–Madison, Madison,Wisconsin, U.S.A.

HEATHER WILKINSON

Department of Plant Pathology, Texas A&M University, College Station,Texas, U.S.A.

1. INTRODUCTION

Filamentous fungi display many unique characteristics that render them of great interestto the research community. Among these characteristics is the production of natural prod-ucts, or secondary metabolites. These compounds often have obscure or unknown functionsin the producing organism but have tremendous importance to humankind. Secondarymetabolites display a broad range of useful antibiotic and immunosuppressant activitiesas well as less desirable phytotoxic and mycotoxic activities. The distribution of naturalproducts is characteristically restricted to certain fungal taxa, particularly the Ascomycetes.Because of the great interest in these compounds, efforts have been expended in the lastdecade to clone and characterize the genes involved in their biosynthesis. Accumulatingdata from these studies support a model of fungal secondary metabolite gene clusterscontaining most if not all of the genes required for product biosynthesis.

A gene cluster can be defined as containing two or more closely linked genes partici-pating in the same functional pathway. Although such a definition could include a descrip-tion of several types of fungal gene clusters—including nutrient utilization clusters [1],pathogenicity islands [2], and mating-type clusters [3,4]—the focus of this chapter is onsecondary metabolite gene clusters. Our goal is to summarize the existing descriptions ofthese clusters as well as to examine models that could explain the clusters’ evolution.

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2. NATURAL PRODUCT PATHWAYS

Based on biological activity, these compounds can be grouped according to their impact onhumankind. In this section, we group these metabolites as either toxins (either mycotoxinsexhibiting toxicity to animals or phytotoxins exhibiting toxicity to plants), pigments,growth hormones, or pharmaceuticals. It is important to keep in mind, however, thatmetabolites may exhibit more than one biological property.

A compilation of available data indicates that all of the gene clusters contain enzy-matic genes and many clusters contain regulatory genes or genes associated with resistanceto the metabolite (Table 1). In some cases, there are also several genes with no apparentrole in production of the metabolite in question. In this chapter, we describe the basicorganization of selected secondary metabolite gene clusters, some of which are examinedin greater detail elsewhere in this book.

2.1. Mycotoxins and Phytotoxins

2.1.1. Ergot Alkaloids

Ergot alkaloids are widely known as fungal neurotropic mycotoxins and as importantpharmaceuticals. They are produced by a wide range of filamentous fungi, primarily bymembers of the family Clavicipitaceae, including the ergot fungus Claviceps purpurea

Table 1 Physical Characteristics of Selected Fungal Secondary Gene Clustersa

Function of Genesb

Cluster Number of Transport/Cluster Size (kb) Genes Regulatory Enzyme Resistance Unknown

Aflatoxinc ∼75 24 2 17 1 4AK-Toxin ? 6d 2 4 0 0Cephalosporine ∼17 4 0 2 1 1

∼3.5 2 0 2 0 0Compactinc ∼72 20 1 6 2 11Ergot alkaloidc ∼50 12 0 9 0 3Fumonisinc ∼75 ∼23 4 16 3 0Gibberellins 17.2 7 0 7 0 0HC toxinc ∼600 >17d 1 14 2 0Lovastatinc ∼64 ∼18 2 6 5 5Melanin ∼19 6 0 6 0 0Paxillinec ∼50 17 2 9 1 5Penicillinc ∼20 3 0 3 0 0Sterigmatocystinc ∼60 26 2 20 0 4Trichothecenec ∼29 12 2 7 1 2

a References can be found in the text.b The function of genes was annotated based on their amino acid similarity with known proteins in theGenBank.c Cluster size (kb), number of genes/cluster, and function of each gene are not completely characterized.d Duplication of some of these genes in the cluster.e Genes are located on two clusters for cephalosporin production.

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and the grass endophytes of the genera Epichloe, Neotyphodium, Balansia responsible forsevere livestock intoxications [5,6]. Ergot alkaloids are also produced from the higherplants Ipomoea, Rivea, and related genera of the Convolvulaceae. The broad physiologicaleffects of ergot alkaloids are mostly based on their interactions with neurotransmitterreceptors on the cells [5,6].

The characteristic structural feature of most of the natural ergot alkaloids is thetetracyclic ergoline ring. The ring structure is derived from a hemiterpene unit, dimethylal-lyl diphosphate (DMAPP). The biosynthesis of ergot alkaloids begins with the condensa-tion of L-tryptophan with DMAPP by the enzyme DMAT synthetase yielding 4-dimethyl-allyltryptophane (DMAT). Mixed function oxidases convert the DMAT to thecorresponding hydroxy derivative. Several cyclase enzymes are involved in convertingthe intermediates into chanoclavine I, agroclavine, elymoclavine, and finally lysergic acid.The peptide alkaloid ergotamine is synthesized by the addition of amino acids (alanine,phenylalanine, and proline) to a lysergic acid precursor [5].

Tsai et al. [7] were the first to clone a gene of the ergot alkaloid pathway, dmaW,which encodes DMAT synthase, the first enzyme of the pathway in C. fusiformis. Thegene is induced under ergot alkaloid production conditions [8]. Using the dmaW gene ofC. fusiformis as a probe, a putative DMAT synthase gene (termed cpd1) was isolatedfrom the strain P1 of C. purpurea, a strain capable of producing ergot alkaloids (mainlyergotamine) in axenic cultures. The cpd1 gene served as a starting point leading to thedetection of a putative ergot alkaloid gene cluster (Fig. 1) [9]. Another gene, cpps1, waslocalized downstream of cpd1 and appears to encode a peptide synthetase required for the

Figure 1 Secondary metabolite gene clusters. Size of genes is not representative. Genes weregrouped according to putative function; however some of the genes have been disrupted with noapparent phenotype on metabolite production as discussed in text. (continues)

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Figure 1 (continued)

penultimate step in alkaloid biosynthesis, that is the activation of the three amino acidsof the peptide part of ergotamine linking them to the activated lysergic acid [6]. Thecpps1 encodes the previously characterized alkaloid biosynthetic enzyme LPS1 that wasdescribed by Riederer et al. [10]. Further sequencing of the upstream region of the cpd1gene in C. purpurea led to the identification of several genes that may be involved inalkaloid biosynthesis: (1) two putative monomodular peptide synthetase genes (cpps2 andcpps3) that might encode the lysergic acid–activating enzyme; (2) one P-450-monooxygen-ase gene (cp450-1), which could catalyze the last steps of the lysergic acid biosynthesisand the last step of ergopeptine biosynthesis; and (3) several oxidases (cpox1, cpox2,cpox3) that are good candidates for the early steps of biosynthesis. The presence of ahousekeeping gene (isopropylmalate–dehydratase involved in amino acid biosynthesis) atthe far left region of the available sequencing data may indicate the end of the cluster.Preliminary data show that all these genes of the cluster are induced in alkaloid-producingcultures (low phosphate) of strain P1 and repressed under high phosphate, conditions thatdo not favor the alkaloid production [6]. Progress has also been made in identifying ergotalkaloid genes in Neotyphodium spp. A peptide synthetase gene (lpsA) was cloned fromNeotyphodium lolii and was insertionally mutated in Neotyphodium sp. Lp1. The lpsAloss-of-function endophyte did not produce any detectable quantities of the alkaloid ergo-valine and retained full compatibility with its perennial ryegrass host plant [11].

Ergot alkaloid biosynthesis in axenic culture is strictly regulated in most strains.Tryptophan acts as both precursor and inducer, whereas phosphate, glucose, and ammo-nium repress synthesis. The presence of putative CreA (global regulator of carbon catabo-lite repression) and AreA (global regulator of nitrogen derepression) binding sites in thepromoters of some of the alkaloid clustered genes may be indicative of alkaloid biosyn-


thesis regulation by C and N sources [12]. The involvement of these global regulators ingene cluster regulation is a recurring theme addressed in section 3 of this chapter.

2.1.2. Indole-Diterpene Alkaloids

Paxilline is representative of a type of alkaloid secondary metabolite sharing a commoncore structure composed of an indole and a diterpene skeleton [13]. Many of these metabo-lites exhibit mammalian tremorgenic or insecticidal activities [14–16]. The tremorgenicpaxilline is synthesized by Penicillium paxilli [17] and is proposed to be an intermediatein the biosynthetic pathways of other indole-diterpenes [18]. The pax gene cluster hasbeen identified in P. paxilli. The cluster is located within a 50-kb region and contains 17genes. Twelve genes have significant similarity to genes of known function, four to genesof unknown function, and one gene has no significant similarity to genes in the databases(Fig. 1) [13]. The 12 genes are predicted to encode a geranylgeranyl pyrophosphate (GGPP)synthase (paxG), a prenyltransferase (paxC), a dehydrogenase (paxH), a metabolite trans-porter (paxT), an oxidoreductase (paxO), two FAD-dependent monooxygenases (paxMand paxN), two cytochrome P450 monooxygenases (paxP and paxQ), a dimethylallyltryp-tophan (DMAT) synthase (paxD), and two possible transcription factors (paxR and paxS),which contain a Cys6 DNA-binding motif [13]. Gene deletion analysis has confirmed therequirement of paxG for paxilline biosynthesis.

Although the involvement of the other 16 genes in paxilline production remains tobe verified, gene expression studies have shown that paxU, paxV, paxY, paxM, paxW,and paxP are transcribed during the onset of paxilline production [13]. Furthermore, theexpression profiles of paxM, paxW, and paxP correlate well with the initiation of paxillinebiosynthesis [13].

Most recently a putative lolitrem B gene cluster has been identified in Neotyphodiumlolii [19]. Lolitrem B is an analogue of paxilline and the cognate gene cluster appears tocontain putative Pax orthologs which are expressed in planta.

2.1.3. Trichothecenes

The trichothecenes comprise a large family of sesquiterpenoid metabolites produced bya number of fungal genera, including Fusarium, Myrothecium, Stachybotrys, Cephalospor-ium, Trichoderma, and Trichothecium [20–22]. These compounds not only exhibit toxicityto vertebrates and plants but also are associated with virulence in specific plant–pathogeninteractions [23–25]. The structurally diverse trichothecenes are classified as macrocylicor nonmacrocyclic, depending on the presence of a macrocycle formed by esterificationbetween C4 and C15 hydroxyl groups. Diacetoxyscirpenol (DAS), deoxynivalenol (DON),and T-2 toxin are the best studied nonmacrocyclic trichothecenes produced by Fusariumspp. Biochemical and genetic analyses of the T-2 toxin producer F. sporotrichioides ledto the identification of the first trichothecene biosynthesis gene cluster. The gene clusterfor DON production has also been identified in F. graminearum. The two clusters contain10 to 12 ORFs and span about 29 kb (Fig. 1) [26–28]. The functions of 10 genes havebeen determined. Seven of them encode biosynthetic enzymes, including Tri3 (a 15-O-acetyltransferase), Tri5 (a trichodiene synthase), Tri8 (a c-3 esterase), Tri7 (required foracetylation of the oxygene on C-4 in T-2 toxin), as well as three cytochrome p450 monoox-ygenases, including Tri4, Tri11, and Tri 13 [1,27–30]. Tri6 and Tri10 are regulatoryproteins and Tri12 is the efflux pump that is implicated to play a self-protection role[31,32]. The organization and transcription orientation of the genes in the two clusters areidentical; however, Tri7 in F. graminearum is nonfunctional, consistent with the structuraldifference between T-2 toxin and DON [27]. Interestingly, one gene required for trichothe-

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cene production, Tri101 encoding a 3-O-acetyltransferase, resides outside of the clusterin both F. graminearum and F. sporotrichioides [33]. Most recently, a second mini-clusterhas been described in F. sporotrichioides that contains two additional genes required forT-2 toxin formation [34].

Macrocyclic trichothecenes (e.g., roridin E, verrucarin A, and baccharinoid B7),which have similar toxic affects on vertebrates, are associated mostly with Myrotheciumspp. Elucidation of the macrocyclic trichothecene biosynthetic pathway is less completecompared with that of the nonmacrocyclic trichothecenes. Genetic studies of M. roridumhave identified three genes (e.g.,MRTRI4, MRTRI5, andMRTRI6) involved in the biosyn-thesis of macrocyclic trichothecene [35]. MRTRI5 encodes the trichodiene synthase andMRTRI6 encodes a pathway specific transcription factor. The predicted MRTRI4 productis a cytochrome P450 monooxygenase. Mapping data show that these genes are clusteredwithin a 40-kb region, but their organization and orientation differ significantly from thoseof the cluster inF. sporotrichioides [35]. These data suggest that significant rearrangementshave occurred during the evolution of gene clusters for the biosynthesis of these metabo-lites.

2.1.4. Fumonisins

Fumonisins are a group of polyketide mycotoxins that are produced by the maize pathogenFusarium verticillioides (teleomorph Gibberella moniliformis) and several other Fusariumspp. These toxins can cause fatal animal diseases, including kidney and liver cancer inlaboratory rodents [36,37]. Fumonisins resemble the sphingolipid intermediates sphingan-ine and sphingosine in structure, and they disrupt sphingolipid metabolism via inhibitionof the enzyme ceramide synthase (sphinganineN-acyltransferase) [38]. Fusarium verticilli-oides is an economically important plant pathogen of maize and sorghum [39] and oftencontaminates maize kernels with fumonisins. B-series fumonisins (FB1, FB2, FB3, andFB4), which are generally the most abundant fumonisins in naturally contaminated corn[39], consist of a linear 20-carbon backbone with an amine, one to three hydroxyl, twomethyl, and two tricarboxylic acid moieties substituted at various carbon positions. Radio-labeling experiments suggest that the backbone is produced by a polyketide synthase [40].The order in which the functional groups are attached to the polyketide backbone is obscure[41].

The genes involved in fumonisin biosynthesis are clustered (Fig. 1). Initially, Desjar-dins et al. [42] identified the tight linkage of three genetically defined G. moniliformisloci (Fum1, Fum2, and Fum3) required for fumonisin biosynthesis. Subsequent studies inthe same fungus led to the discovery of a 75-kb region of DNA that consists of 23 genesthought to include the Fum1–3 loci [41]. The predicted functions of most of these proteinswere consistent with enzyme activities expected to be required for fumonisin biosynthesisor self-protection [41,43]. Expression analysis indicated that 15 of these genes (ORF1 andORF6–19) are coregulated and exhibited patterns of expression that were correlated withfumonisin production. These ORFs are designated as FUM genes (FUM1 and FUM6–19)and consist of an approzimately 45-kb cluster of genes that is part of the initially character-ized circa 75-kb region [41]. FUM5 encodes the polyketide synthase gene that was shownto be required for fumonisin biosynthesis [44]. Disruption of FUM6 and FUM8 blockedproduction but did not lead to accumulation of detectable intermediates [43]. Complemen-tation analysis results revealed that the Fum1 locus is equivalent to the FUM1 gene [41].Most recently, disruption of FUM9, which is predicted to encode a dioxygenase, produceda phenotype equal to the Fum3 locus, and sequence of FUM9 in the Fum3 mutant showeda mutation in the coding region [45].


Considering the nonspecific toxicity of fumonisin to other organisms, researchershave speculated that Fusarium might contain a self-protection mechanism. Therefore, itwas particularly interesting when two of the cluster genes, FUM17 and FUM18, showedsimilarity to the tomato longevity assurance (LA) factor gene, Asc-1, which confers resis-tance to fumonisin B1 and the structurally similar AAL toxins [41,46]. The FUM19 proteinis similar to ABC transporters that act as efflux pumps transporting compounds frominside cells to the surrounding environment. However, disruption of FUM17–19 did notlead to any obvious phenotype in F. verticillioides, and it is not known whether the fungusrequires a self-protection mechanism against fumonisins [41].

Studies regarding the regulation of the fumonisin biosynthetic pathway are limitedto evidence indicating that fumonisins are synthesized under nitrogen stress and acidicpH conditions [47,48]. None of the 15 genes within the cluster appears to be a regulatorygene. However, two ORFs upstream of FUM1 (the left far end gene in the FUM cluster)appear to encode regulatory proteins: (1) a predicted WDR1 protein (FUM2) similar toseveral regulatory proteins with tryptophan-aspartic acid repeats and (2) a ZNF1 protein(FUM4) including regions similar to the cysteine-rich zinc finger domains of some tran-scription factors and kinases. Characterization of these genes has not been reported. Fur-thermore, another gene (FCC1) in F. verticillioides that does not seem to be clusteredwith the FUM genes plays a role in a putative signal transduction pathway that regulatesfumonisin biosynthesis. FCC1 is closely related to UME3, the cyclin C of S. cerevisiae(cyclins are essential activating subunits of cyclin-dependent kinases [CDKs]) and regu-lates the expression of genes involved in conidiation and FB1 biosynthesis when grownon cracked corn [48].

2.1.5. Aflatoxins and Sterigmatocystin

Aflatoxins and sterigmatocystin are polyketides derived from the same biosynthetic path-way. They are produced by several fungal genera, primarily by Aspergillus spp. Aflatoxinsare the end-products in two agronomically significant fungi, A. parasiticus and A. flavus.Sterigmatocystin, the penultimate precursor to aflatoxin B1, is the final product in thegenetic model organism A. nidulans [49] and the building mold A. versicolor [50]. Bothcompounds are potent carcinogens and also exhibit mutagenic, teratogenic, and immuno-suppresive properties. The aflatoxin cluster in A. parasiticus and A. flavus contains 25genes that constitute a cluster spanning more than 70 kb (Fig. 2). Among the genes, 21genes have been verified or appear to encode biosynthetic enzymes, including fatty acidsynthases, a polyketide synthase, mono-oxygenases, reductases, dehydrogenases, methyl-transferases, an esterase, a desaturase, and an oxidase [51,52]. One of the genes in thecluster, aflR, encodes a binuclear zinc cluster transcription factor regulating transcriptionof the aflatoxin biosynthetic genes [53]. Another cluster gene, aflJ, also seems to have arole in regulating aflatoxin production in A. flavus [54] by binding with AflR protein asa coactivator for biosynthetic gene expression [55]. In A. nidulans, the 60-kb sterigmato-cystin cluster consists of 26 genes also regulated by aflR [56,57] (Fig.1). The function ofmost of the sterigmatocystin cluster genes has been determined: they are orthologs ofaflatoxin cluster genes [58]. The roles of some genes remain elusive, however. For instance,disruption of stcT, stcC, stcQ, stcI, and stcV showed no effect on ST production (N. Kelleret al., unpublished data). Deletion of stcN abolished ST biosynthesis but its function hasnot been assigned. Althoughmost of the genes in the aflatoxin and sterigmatocystin clustershave the same functions, their order and transcription orientation are not well conservedbetween the two clusters (Fig. 2) [1]. In East Asian countries, A. oryzae, a nontoxic clade

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Figure 2 Order and direction of transcription of homologous genes in the sterigmatocystin (ST),aflatoxin (AF), and dothistromin (DOT) gene clusters. Homologous genes in the three clusters areindicated by the same bar pattern and the same letter that corresponds to the ST genes. Solid blackbars represent ST, AF, DOT genes with no known homology among them. Size of genes is notrepresentative.

of A. flavus [59], is traditionally used for fermented food and beverage production and doesnot produce aflatoxin or sterigmatocystin. Interestingly, studies have shown the presence ofthe entire or partial aflatoxin biosynthetic cluster in all of the A. oryzae strains tested[60–64]. The order of the genes in the cluster is identical to that of the cluster in A.parasiticus. Lack of aflR transcript is implicated as the reason for loss of toxin productionin those strains containing an intact AF biosynthetic cluster [60].

Regulation of aflatoxin and sterigmatocystin biosynthesis has probably receivedmore attention than any other mycotoxin. Several recent reviews address this topic[58,65,66] and show that regulation is complex involving pH, nitrogen, carbon, and signaltransduction regulatory circuits. This is addressed in more detail in section 3 of this chapter.

2.1.6. Dothistromin

The difuranoanthraquinone polyketide dothistromin is produced by several plant patho-gens, including Dothistroma pini and Cercospora arachidicola [67,68]. Studies haveshown that dothistromin has a broad-spectrum toxicity to plant, animal, and microbialcells [69,70], and the compound is considered a virulence factor in needle blight of pinescaused by Dothistroma pini [69]. 13C-NMR analysis has shown that dothistromin andaflatoxin share the same biosynthetic steps, in agreement with the substantial structuralsimilarity between dothistromin and versicolorin B, a precursor of aflatoxin [71]. One gene,dotA, has been identified to be required for dothistromin biosynthesis. The accumulation ofverisicolorin A in the dotA mutants and significant sequence similarity between DotA andA. parasiticus Ver-1 (� A. nidulans StcU) required for aflatoxin and sterigmatocystinbiosynthesis indicates a ketoreducatase function for DotA [72]. Analysis of the genomicregion beside dotA has identified three ORFs—dotB, dotC, and dotD—homologous to anoxidase, a toxin pump, and a thioesterase domain of a polyketide synthase associated withaflatoxin and sterigmatocystin production, respectively (Fig. 2) [72]. Unpublished dataindicate the presence of additional biosynthetic genes exhibiting similarity to aflatoxinand sterigmatocystin cluster genes (Fig. 2) (R. Bradshaw, personal communication, 2003).Therefore, it appears that the genes for dothiostromin biosynthesis also constitute a cluster.The identification of this cluster suggests that variations of the aflatoxin/sterigmatocystin


cluster exist in a wide distribution of genera and may provide clues toward the evolutionof a gene cluster.

2.1.7. Alternaria Host-Specific Toxins

A number of plant pathogenic fungi produce a class of low-molecular-weight metabolitestermed host-specific toxins. They are so-called because these toxins are the crucial determi-nants for the outcomes of specific host–pathogen interactions. Pathotypes of the fungusAlternaria alternata produce several structurally diverse host-specific toxins. Among themare AF-toxin produced by the strawberry pathotype, AK-toxin produced by the Japanesepear pathotype, and ACT-toxin produced by the tangerine pathotype. All contain a common9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid structural moiety [73–77]. Amutagene-sis study has led to identification of the first two genes, AKT1 and AKT2 essential for AK-toxin biosynthesis in the Japanese pear pathotype [78]. The product of AKT1 is predicted tobe a member of the carboxyl-activating enzyme superfamily. The predicted AKT2 producthas no homology to any proteins in the database. Both of the two genes have multiplecopies not functional for AK-toxin production. Downstream of AKT2 are two ORFs desig-nated AKTR-1 and AKT3-1. Attempts to disrupt these two ORFs revealed two other genes,AKTR-2 and AKT3-2, which were shown to be essential for AK-toxin biosynthesis. Se-quence comparison indicated thatAKTR-1 and AKT3-1 share high similarity to AKTR-2 andAKT3-2, respectively. AKTR-1 and AKTR-2 are predicted to encode a protein containing azinc binuclear cluster DNA-binding domain, typical of a fungal transcription factor. Thepredicted products of AKT3-1 and AKT3-2 have similarity to members of the hydratase/isomerase enzyme superfamily. Both AKT3-1 and AKTR-1 are transcribed; however, theirroles in AK-toxin production remain to be determined since neither has been successfullydisrupted.Mapping analyses showed thatAKT1, AKT2, AKT3, andAKTR and their paralogsare on a single chromosome [79] (Fig. 1).

The AKT paralogs have also been detected in the strawberry and tangerine patho-types, in keeping with the fact that AK-toxin, ACT-toxin, and AF-toxin share a commoncore moiety. In one strain of the strawberry pathotype, three AKT homologs (AFT1-1,AFR-1, and AFT3-1) are present in multiple copies on a 1.05-Mb chromosome. Deletionof this chromosome resulted in loss of AF-toxin production and pathogenicity but did notaffect saprophytic growth, suggesting that the chromosome is conditionally dispensable[80]. This is reminiscent of the discovery of a pathogenicity island located on a dispensablechromosome in the pea pathogen Nectria haematococca [2].

2.1.8. HC-Toxin

The cyclic tetrapeptide HC-toxin exhibits a cytostatic effect on plant and animal cells byinhibiting histone deacetylase [81]. Being a host-selective toxin, it is a critical virulenceand specificity determinant for the interaction between maize and the toxin producerCochliobolus carbonum race 1 [82]. An initial genetic study showed that HC-toxin produc-tion appeared to be controlled by a single locus, TOX2 [83]. Recent molecular analysesindicated that TOX2 consists of at least seven different types of genes that have beenduplicated one or more times (Fig. 1). HTS1 encodes a nonribosomal peptide synthetase,TOXA encodes a putative HC-toxin efflux carrier, TOXC encodes a fatty acid synthasebeta subunit, TOXD encodes a putative dehydrogenase (its role in HC-toxin biosynthesishas not yet experimentally confirmed), TOXE encodes a pathway-specific transcriptionfactor, TOXF encodes a putative branched-chain amino acid transaminase, and TOXGencodes an alanine racemase [84–88]. Arrangement of these genes in different isolates

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of C. carbonum race 1 is slightly different and can be divided into two types. In the so-called type 1 pattern, one copy of TOXE is on a 0.7-Mb chromosome, whereas the secondcopy of TOXE and all copies of the other six genes are on a 3.5-Mb chromosome. In thetype 2 pattern, the entire cluster, spanning about 600 kb, is on a 2.2-Mb chromosome. Amechanism of reciprocal translocation has been proposed to explain the phenomenon [89].

Gene disruption analyses indicated that most, if not all, copies of the TOX2 genesare functional. One gene (encoding an exo-beta 1,3-glucanase) that has no role in HC-toxinbiosynthesis has been located within the cluster [89]. The TOX2 cluster inC. carbonum hasseveral unique characteristics. For example, genetic analyses showed that the TOX2 clusteris genetically unstable since about 5% of sexual progeny undergo spontaneous loss of oneor more of their TOX2 genes [90]. In type 1 strains, a large part (up to 1.4 Mb) of the3.5-Mb chromosome is dispensable since deletion of this part affects only virulence andHC-toxin production but not fungal growth [90].

2.2. Pigments

2.2.1. Melanins

Melanin is a high-molecular-weight pigment produced by a wide range of fungi. Ascomy-cota and related Deuteromycota generally synthesize DHN melanin by oxidative polymer-ization of phenolic compounds via polyketide biosynthesis. 1,3,6,8-tetrahydroxynapththa-lene (1,3,6,8-THN) is the first polyketide intermediate, which is subsequently reduced toform scytalone. Scytalone is then dehydrated to produce 1,3,8-trihydroxynapththalene(1,3,8-THN), which is then converted to 1,8-dihydroxynapththalene (1,8-DHN) after addi-tional reduction and dehydration cycles [91]. Finally, 1,8-DHN is polymerized to formDHN-melanin [91]. Melanin plays a crucial role in the survival and longevity of fungalpropagules [91]. Particularly, DHN melanin is essential for the function of the rigidity ofappressorium in penetration of host plants by Colletotrichum and Magnaporthe spp. [92]Furthermore, melanin has been shown to be important for virulence in human pathogenicfungi, includingCryptococcus neoformans [93], Aspergillus fumigatus [94], andWangielladermatitidis [95].

The melanin biosynthesis genes are organized in clusters in some fungi and not inothers. In A. alternata, a melanin pathway gene cluster contains at least three genes withina 30-kb region [96]. Characterization of the 30-kb region by complementation and genedisruption analysis led to the identification of genes encoding the polyketide synthase,1,3,6,8-tetrahydroxynaphthalene synthase (ALM), the scytalone dehydratase (BRM1), andthe 1,3,8-trihydroxynaphthalene reductase (BRM2). The three mRNA species accumulatein cultured mycelia of the wildtype strain synchronously with mycelial melanization [96].Another developmentally regulated six-gene cluster spanning a region of 19 kb was identi-fied in A. fumigatus and is involved in conidial pigment biosynthesis [97] (Fig. 1). DNAsequencing, gene disruption, expression, and biochemical analyses indicated that A. fumi-gatus synthesizes its conidial pigment through a pathway similar to the DHN-melaninpathway found in many brown and black fungi. The gene products of alb1, arp1, andarp2 have high similarity to polyketide synthases, scytalone dehydratases, and hydroxy-naphthalene reductases, respectively. The abr1 gene encodes a putative protein possessingtwo signatures of multicopper oxidases. The abr2 gene protein has homology to the laccaseencoded by the yA gene of A. nidulans. Abr2 and abr1might polymerize and oxidize DHNinto melanin. Ayg1 catalyzes a novel biosynthetic step downstream of Alb1 (heptaketidesynthase) and upstream of Arp2 (1,3,6,8-THN reductase). The protein Ayg1 shortens the


heptaketide product of Alb1 to 1,3,6,8-THN, facilitating the participation of a heptaketidesynthase in a pentaketide pathway via a novel polyketide-shortening mechanism in A.fumigatus. Involvement of the six genes in conidial pigmentation was confirmed by thealtered conidial color phenotypes that resulted from disruption of each gene in A. fumigatusand the presence of a DHN-melanin pathway intermediate in A. fumigatus [97].

Conventional genetic analysis of melanin biosynthesis has also been performed withseveral other plant pathogenic fungi, such as Cochliobolus heterostrophus [98],Cochliobo-lus miyabeanus [99], M. grisea [100], and Colletotrichum lagenarium [101–103]. In C.heterostrophus and C. miyabeanus, the 1,3,6,8-tetrahydroxynaphthalene synthase and the1,3,8-trihydroxynaphthalene reductase genes are closely linked but the scytalone dehydra-tase gene is segregated independently of the these two genes [104]. In contrast, the melaninbiosynthetic genes are dispersed in genome of M. grisea [100,105] and C. lagenarium[104].

2.2.2. Carotenoids

Carotenoids are a class of fat-soluble terpenoid pigments found principally in plants, algae,and photosynthetic bacteria, where they play a critical role in the photosynthetic processes.They also occur in some nonphotosynthetic bacteria, yeasts, and filamentous fungi, wherethey may carry out a protective function against damage by light and oxygen or play arole in cell signaling [106]. Phytoene is the precursor in carotenoid biosynthesis and isproduced from geranyl-geranyl pyrophosphate (GGPP) by the enzyme phytoene synthase.Further modifications of phytoene yield a variety of carotenoids accumulated by fungi.The synthesis of �-carotene from phytoene requires four consecutive dehydrogenationsand two cyclizations. Oxygenated carotenoids (xanthophylls), such as neurosporaxanthinin Neurospora crassa and F. fujikuroi (formerly G. fujikuroi [107]) or asthaxanthin inXanthophyllomyces dendrorhous, require the activity of additional enzymes [106].

Genes that encode phytoene synthase and carotene cyclase are catalyzed by proteinsencoded by different genes in plants and bacteria and by a single bifunctional gene infungi [108]. Another structural gene of the pathway, phytoene dehydrogenase, has beencloned from many plants, bacteria, and fungi. In contrast to plants and bacteria, wherethe sequential dehydrogenations are performed by two enzymes, a single dehydrogenaseis responsible for all the dehydrogenation reactions in fungi [109]. The linkage distancebetween the dehydrogenase and the phytoene synthase differs in the three fungi investi-gated. In the zygomycetes P. blakesleeanus [110] and M. circinelloides [111], the genesare organized in a gene cluster, whereas in the ascomycete N. crassa, they are located onthe same chromosome but are not genetically linked. In F. fujikuroi, Linnemannstons etal. [112] reported the existence of a carotenoid biosynthesis gene cluster containing atleast four genes. The gene carB is very similar to the genes that encode for phytoenedehydrogenases in other fungi [110,113,114], and its function was verified by mutationalanalysis [115]. The gene carRA encodes the bifunctional protein with phytoene synthaseand carotene cyclase activities [112]. The expression level of carRA and carB is inducedby light, and deletion of carB led to the enhanced expression of the carRA gene, suggestingthe existence of a feedback regulatory mechanism.

2.3. Growth Hormones

2.3.1. Gibberellins

Gibberellins belong to a large family of tetracyclic diterpenoid carboxylic acids that occurin green plants, fungi, and bacteria. A total of 121 gibberellins have been identified from

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these natural sources [116]. They were first identified as secondary metabolites of the ricepathogenic fungus Gibberella fujikuroi (mating population C) and some other fungal spe-cies [117]. Some members of gibberellins function as natural growth hormones in higherplants able to promote processes such as seed germination, stem elongation, leaf growth,flower development, and seed and pericarp growth [118]. Three groups of enzymes areinvolved in the gibberellin biosynthesis: terpene cyclases (ent-kaurene synthesis), P-450monooxygenases (oxidation of ent-kaurene), and dioxygenases. The initial steps of thegibberellin biosynthetic pathway from transgeranylgeranyl diphosphate to GA12-aldehydeare identical for plants and fungi, but the following steps diverge. Gibberellic acid (GA3)is the major end-product of the pathway in G. fujikuroi, whereas it is a minor gibberellincomponent in most plant species. Gibberellins do not have a defined role in fungi, andstrains of G. fujikuroi that lack the gibberellin biosynthetic genes grow normally in culture.However, their pathogenicity has not been assessed to date [119]. In contrast to plants,genes involved in gibberellin biosynthesis in G. fujikuroi were discovered to be organizedin a cluster containing seven genes in a 17.2-kb DNA region (Fig. 1) [119]. Because about18 steps are required for the formation of GA3 from transgeranylgeranyl diphosphate(GGPP), it appears that not all of the genes have been identified or some genes encodemultifunctional enzymes (Table 1). In G. fujikuroi, GGPP synthase is encoded by twogenes, and one of these, ggs2, is specific for gibberellin biosynthesis [117]. Cyclizationof GGPP is catalyzed by the bifunctional copalyl pyrophosphate (CPP)/ent–kaurene syn-thase (KS) enzyme [120]. The ggs2 and cps/ks genes are clustered together in the giberellincluster. Four genes in the gibberellin biosynthesis cluster inG. fujikuroi encode cytochromeP450 monooxygenases. These genes are designated P450-1, P450-2, P450-3, and P450-4.The P450-1 gene is closely linked to P450-4 in the gene cluster, sharing the samepromoter sequence but being transcribed in the opposite direction. P450-4 encodes for amultifunctional ent-kaurene oxidase that catalyzes all three early oxidation steps betweenent-kaurene and ent-kaurenoic acid [121]. P450-1 catalyzes the next four oxidation stepsin the main pathway from ent-kaurenoic acid to GA14 via GA12 aldehyde. P450-1 andP450-2 are classified as part of the CYP68 family [122]. P450-2 encodes a 20-oxidase,and its product oxidizes the 3�-hydroxylated intermediate, GA14, and its nonhydroxylatedanalogue GA12 to GA4 and GA9, respectively [123]. This reaction (20-oxidation) in plantsis catalyzed by dioxygenases and not monooxygenases as in G. fujikuroi. The characteriza-tion of the last two genes in the cluster, a fourth P450 monooxygenase (P450-3) and adesaturase gene that is thought to introduce the 1,2-double bond in the conversion of GA4to GA7, is currently in progress (B. Tudzynski, personal communication, 2003).

Six of the seven genes of the gibberellin cluster are strongly induced under gibberel-lin production conditions (low nitrogen) indicating they may be under the control of thesame regulatory gene(s) to ensure that gibberellin production occurs only at low nitrogenlevels [117,123] (B. Tudzynski, personal communication, 2003). High nitrogen concentra-tions and specifically ammonium and glutamine repress gibberellin biosynthesis inG. fujikuroi. Disruption of the positive-acting nitrogen regulatory areA-GF gene inG. fujikuroi led to a 10% to 20% reduction of gibberellin production in giberellin inductionmedium. In addition, the loss-of-function areA-GF strains were insensitive to ammonium-mediated gibberellin repression, supporting the conclusion that gibberellin biosynthesis isunder the control of AreA-GF [124]

As is covered in section 4 of this chapter, the profound differences in gibberellinbiosynthesis between G. fujikuroi and plants at the chemical, biochemical, and genetic


levels indicate that higher plants and fungi have evolved the gibberellin biosynthetic path-way independently and not by horizontal gene transfer [119].

2.4. Pharmaceuticals

2.4.1. Lovastatin

Lovastatin is an inhibitor of the enzyme (3S)-hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase that catalyzes the reduction of HMG-CoA tomevalonate during cholesterolbiosynthesis. The compound is also toxic to fungi by inhibiting the same enzyme requiredfor ergosterol biosynthesis [125]. This activity makes lovastatin a medicinally importantcompound with antihypercholesterolenic attributes [126] and antifungal properties [125].Lovastatin is a secondary metabolite produced by Aspergillus terreus and is biosyntheti-cally composed of two distinct polyketide chains joined through an ester linkage. One chainis the diketide 2-methylbutyrate and the other is a nonaketide that includes a distinctiveconjugated hexahydronaphthalene ring system [127].

Kennedy et al. [126] recently sequenced the lovastatin biosynthetic cluster of A.terreus and identified 18 potential genes over a 64-kb genomic region, the functions ofwhich were predicted by sequence comparisons and disruption analysis experiments (Table1; Fig. 1). Of these genes, lovB and lovF encode two type I polyketide synthases (PKS).The lovB gene encodes the previously described lovastatin nonaketide synthase (LNKS)[128], which is required for the synthesis of the main nonaketide-derived skeleton. ThelovF gene encodes the lovastatin diketide synthase (LDKS) that is probably responsiblefor the biosynthesis of the (2R)-2-methylbutyryl side chain of lovastatin. Lovastatin hastwo methyl groups derived from S-adenosyl-L-methionine (SAM), one on the nonaketideand the other on the diketide side chain. The presence of methyltransferase domains inLovB and the LovF protein indicates that in both cases, the methyl groups are likely tobe added while the polyketide is being synthesized. The lovC gene is located adjacent tolovB and encodes a protein with high similarity to the product of the Cochliobolus car-bonum toxD gene of unknown function from the HC-toxin biosynthesis cluster, to hormoneand ripening-induced proteins from plants, and to ER domains of PKSs. Gene disruptionanalysis of lovC led to the conclusion that LovB and LovC proteins interact with eachother to produce a polyketide of the correct length and with the correct reduction andcyclization pattern. The cooperation of lovB and lovC genes accomplish the approximately35 steps necessary to generate dihydromonacolin L from acetyl-CoA, malonyl-CoA,NADPH, and SAM. Oxidative transformation of the PKS product dihydromonacolin Lled to the formation of monacolin J. The 2-methylbutyryl side chain is produced by lovFand is further used by LovD to directly acylate monacolin J and yield lovastatin. The lovDgene is another gene of the cluster that is functionally associated with LovF and hassimilarity to �-lactamases, carboxypeptidases, lipases, and esterases. Disruption of lovDled to a strain that accumulated monacolin J, the immediate precursor to lovastatin. Ken-nedy et al. [126] proposed that lovD is responsible for the last step, the biosynthesisof the 2-methylbutyryl/monacolin J transesterase that joins together the two polyketidecomponents of lovastatin. The function of LovA, a protein essential for formation oflovastatin, is not fully understood, but it has sequence homology to P-450 enzymes andits disruption leads to a very active �-oxidation system in A. terreus [129]. Among therest of the ORFs in the biosynthetic gene cluster, two were annotated to encode regulatoryproteins (LovE and ORF13), two belong to potential resistance genes, three encode putativetransporter genes, and five genes have no known functions [126]. Interestingly, one of

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the resistance genes encodes a putative HMG-CoA reductase [126], which is speculatedto provide resistance to the fungal species containing the lovastatin cluster [130].

Despite the knowledge of the genes and the enzymes involved in the biosyntheticpathway, little is known about the regulation and the physiology of lovastatin biosynthesis.Some recent experiments [131] showed that lovastatin synthesis is dependent on the nitro-gen source. Ammonium, nitrate, and urea inhibited the production of lovastatin, and onlyglutamate, histidine, and, to a lesser extent, glycine supported lovastatin biosynthesis.Experimental results from the same studies indicate also that carbon source starvation isrequired for the onset of lovastatin biosynthesis [131]. Analysis of the lovastatin biosyn-thetic cluster revealed that the motif of functional CreA (involved in carbon cataboliterepression in A. nidulans) binding site in vivo is present in the putative promoters ofORF13 and in the putative promoter of the divergently transcribed ORF8 and lovE. Thus,the presence of putative functional CreA binding sites in two putative regulatory genessuggests that repression of lovastatin biosynthesis by glucose could be mediated by CreA[131].

2.4.2. Compactin

Several other fungi produce lovastatin-related structures, includingMonascus ruber, whichproduces lovastatin, and Penicillium citrinum and P. brevicompactum, which producecompactin (ML-236B) [132]. Compactin is identical to lovastatin except that it is missingthe methionine-derived methyl group on the nonaketide. Compactin, like lovastatin, inhib-its the enzyme HMG-CoA reductase and is used as a substrate for microbial conversionto pravastatin sodium, a compound that has been widely used as a pharmaceutical drugin the treatment of hypercholesterolemia [133].

Genetic analyses in P. citrinum led to the discovery of an entire gene cluster relatedto compactin biosynthetic genes, spanning a 72-kb region that revealed the existence of20 open reading frames (Table 1; Fig. 1) [133]. Nine genes were localized within a 38-kb region and were transcribed when compactin was produced. Nine genes, designatedas mlcA–mlcH and mlcR, have predicted amino-acid sequences similar to those encodedby the genes for lovastatin biosynthesis. Two genes, mlcA and mlcB, encode putativenovel multifunctional type I PKSs and share 59% and 61% identity with LovB and LovF,respectively. Disruption experiments provided evidence that mlcA and mlcB are requiredfor the biosynthesis of the nonaketide and the diketide chains. mlcC encodes a putativeP450 monooxygenase and shares 72% identity with LovA. mlcF encodes a putative oxido-reductase and shows some similarity to dihydrofolate reductases and also shares 57%identity with a putative polypeptide encoded by ORF5 in the lovastatin gene cluster. mlcGencodes a putative oxidoreductase and shows 70% identity to LovC, which has an enoylreductase activity required for lovastatin biosynthesis. mlcH encodes a putative transester-ase and displays 75% identity with LovD. Two other genes, mlcD and mlcE, encodeputative polypeptides that may be involved in conferring resistance to compactin and inmetabolite secretion [133].

Sequence of the mlcR gene suggests it is a Cys6 zinc binuclear cluster protein, andit exhibits 34% identity with LovE, indicating that it may be involved in the regulationof compactin biosynthesis in P. citrinum [134]. The induction of compactin productionis correlating with the expression of mlcR and the biosynthetic genes mlcA–H, and itoccurs mainly during the stationary phase. Introduction of additional copies of mlcR inP. citrinum showed increased transcription of mlcR and produced higher amounts of com-pactin. Constitutive expression of mlcR led to the production of compactin during the


exponential growth phase. Alterations in mlcR expression resulted in concomitant altera-tions in expression of some of the compactin biosynthetic genes, suggesting that mlcRmay indeed be a transcriptional activator of some of the pathway-specific genes requiredfor compactin biosynthesis. ORF1 is located next to mlcR and also encodes a putativeCys6 polypeptide, but its function still remains unknown [134].

2.4.3. �-lactams

The most commonly used �-lactams (�-cyclic amides) antibiotics are penicillins and ceph-alosporins. Their biosynthesis begins with nonribosomal condensation of three precursoramino acids to yield a tripeptide by ACV [delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine] synthetase. An IPN (isopenicillin N) synthetase catalyzes the cyclization of ACVto produce IPN. From IPN, different reactions lead to various penicillin and cephalosporinfinal products [135]. Cephalosporins are produced by both bacteria and fungi (e.g.,Acremo-nium chrysogenum), whereas penicillins are produced only in several filamentous fungi(most notably Aspergillus nidulans and several Penicillium spp.). The three genes forpenicillin biosynthesis—pcbAB (encoding ACV synthetase), pcbC (encoding IPN syn-thase), and penDE (encoding acyltransferase)—form a cluster that spans approximately20 kb identified in A. nidulans, P. chrysogenum, P. nalgiovernse, P. notatum and P.griseofulvum [136,137]. In these organisms, the three genes maintain the same order andtranscription orientation (Fig. 1). In many industrial strains with high penicillin yield, thecluster is often amplified many times in tandem repeats [138]. A putative multidrug effluxpump encoded by cefT has been recently identified in Acremonium chrysogenum, alongwith a putative D-hydroxyacid dehydrogenase gene (orf 3) that is not required for cephalo-sporin biosynthesis [139]. pcbAB, pcbC, orf 3, and cefT form a so-called early cluster ofabout 17 kb on chromosome VI, while cefEF (encoding deacetylcephalosporin Csynthetase/hydroxylase) and cefG (encoding an acetyl transferase) form a late cluster ofabout 3.5 kb on chromosome II (Fig. 1) [140].

Like several other gene clusters, expression of penicillin biosynthesis genes is undercomplex control by carbon and nitrogen source and ambient pH. It has been shown thatpH effect is mediated by the global transcription factor PacC [141,142]. The major nitrogenregulatory protein AreA is implicated to regulate expression of penicillin biosyntheticgenes, but it has not been verified by in vivo analysis [143]. The molecular basis of C-source regulation of ipnA expression remains to be elucidated [141,144] although datasuggest carbon regulation of penicillin production differs to some degree between Aspergil-lus and Penicillium spp. [142]. No pathway-specific regulator has yet been identified toregulate the penicillin gene clusters. However, PENR1, a HAP-like transcriptional com-plex, has been shown to positively regulate penicillin cluster expression in A. nidulans[145]. For further discussion of regulation of penicillin gene expression, several reviewpapers are available [135,146,147].

3. REGULATION OF GENE CLUSTERS

3.1. Transcriptional Regulation

In the preceding, mention was made of several modes of regulation of some of the geneclusters. Examination of several of the clusters shows the presence of genes encodingzinc-binding proteins [13,24,35,79,126,130,148], a major class of transcription factorsthat fungi employ to regulate secondary metabolism as well as development and nutrient

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utilization. These proteins bind to the promotors of the target genes and control theirtranscription. Cys2His2 zinc finger proteins and Cys6 zinc binuclear cluster proteins arethe most common types of zinc-requiring transcription factors, with the former proteinfound in many organisms but the latter found only in fungi. Evidence to date suggeststhat these transcription factors act positively to regulate the biosynthetic pathway genesin the cluster [1,24,56,57,134]. PAXR and PAXS for paxilline production, AKTR-1 andAKTR-2 for AK-toxin production, MlcR for compactin production, LovE for lovastatinproduction and AflR for AF/ST production are Cys6 zinc binuclear proteins, whereas Tri6and MRTRI6 for trichothecene production are Cys2His2 zinc finger proteins. Anothertype of transcription factor found to specifically regulate secondary metabolism is the C.carbonum ToxE, which regulates HC-toxin biosynthesis. This protein has four ankyrinrepeats and a basic region similar to those found in basic leucine zipper (bZIP) proteins, butit lacks any apparent leucine zipper [149]. PENR1 is a HAP-like transcriptional complexinvolved in regulating penicillin production in A. nidulans [145]. It is unknown, however,whether it is pathway specific.

Also novel is Tri10, a regulatory gene required for T-2 toxin production. The Tri10protein does not contain any known DNA-binding motif, and as of yet, its precise functionremains unknown [31]. AflJ, as mentioned in the preceding section, is a aflatoxin/sterig-matocystin cluster protein that appears to act as a coactivator by binding to aflR; however,the precise mechanism of how such binding influences aflR activation is unknown [55].

Numerous reports in the literature have shown that secondary metabolite biosynthesisis responsive to environmental cues like carbon and nitrogen source, ambient temperature,and pH. As mentioned earlier, the effect of these environmental signals is mediated throughthe global transcription factors CreA, AreA, and PacC, respectively, in A. nidulans[150–152]. Molecular studies have shown that these Cys2His2-type zinc finger proteinsare important in the regulation of aflatoxin, sterigmatocystin, gibberellin, and penicillin[49,116,153,154]. It is likely they also play a role in the regulation of other, if not all,secondary metabolites. A thorough examination of the intergeneic region between aflRand aflJ in several A. flavus isolates suggests that increasing numbers of AreA bindingsites is positively correlated with degree of nitrate required to repress aflatoxin geneexpression. This may indicate that AreA is a negative regulator of aflatoxin biosynthesis[154]. Interestingly, there are no AreA sites in the corresponding aflR/aflJ intergenic regionin A. nidulans and sterigmatocystin biosynthesis is not repressed by nitrate.

3.2. Signal Transduction Regulation

It is known that secondary metabolism is linked with fungal development [155]. However,only recent molecular analyses (mostly conducted in A. nidulans) have begun to unravelthe underlying mechanism connecting these two processes. This linkage of secondarymetabolism and fungal development has been the topic of a recent review [65]. Throughcomplementation of aconidial, nonsterigmatocystin-producing A. nidulans strains, it wasdetermined that a G-protein signaling pathway negatively regulated both conidiation andsterigmatocystin biosynthesis [156]. The same study showed this regulation to be con-served in A. parasiticus. Further studies identified a protein kinase A catalytic subunit aspartially mediating this repression [157]. The target of regulation in the sterigmatocystin/aflatoxin gene cluster is aflR, which is both transcriptionally and posttranscriptionallyregulated by protein kinase A. Recent experimentation has identified a putative proteinmethyltransferase, LaeA, which mediates protein kinase A transcriptional regulation of


Figure 3 Proposed model of G-protein signal transduction regulating sterigmatocystin productionand sporulation. Arrows indicate positive regulation, and blocked arrows indicate negative regulation.Solid lines indicate known pathways. Dashed lines indicate postulated pathways. FluG, early actingdevelopmental regulator; FlbA, regulator of G-protein signaling; FadA, � subunit of G-protein;PkaA, catalytic subunit of PKA; LaeA, AflR regulator; AflR, sterigmatocystin-aflatoxin-specifictranscription factor; BrlA, conidiation-specific transcription factor (From Ref. 65; copyright 2002,with kind permission from American Society for Microbiology.)

aflR (Fig. 3) [158]. LaeA also transcriptionally regulates penicillin and lovastatin clustergenes [158] raising the possibility of its global involvement in gene cluster regulation.This protein appears to be conserved in filamentous fungi.

Expanding studies on the role of G-protein signaling on fungal development suggeststhat signal tranduction pathways will likely have significant impact on secondary metabo-lism as either a positive or negative regulator [65]. For example, while the A. nidulans Gprotein negatively regulates AF/ST biosynthesis, it positively regulates penicillin produc-tion in the same fungus and positively regulates trichothecene production in Fusariumsporotrichiodes [159].

4. EVOLUTION OF GENE CLUSTERS

The evolution and maintenance of gene clusters have received a great deal of attention forboth bacterial systems [160–162] andmore recently for fungal gene clusters [1,163,164]. Inthis section, we discuss four models and how they may be used to explain either formationor maintenance of clusters [160,164] with consideration of current data.

4.1. Extant Models for Gene-Cluster Evolution

4.1.1. Natal Model

In the natal model, the cluster is a product of history. Duplication and subsequent diver-gence of genes result in clusters of genes within the same gene family. This model origi-nated prior to a more modern understanding of molecular genetics and biochemistry. Thereasoning was that because enzymes involved in a pathway would be making minor

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changes to very similar substrates, the pathway would evolve based on gene duplicationsand minor changes in substrate specificity and enzymatic function [160]. Clearly thiswould be an adequate explanation for a limited set of fungal gene-cluster types, such asenzymatic genes involved in as wood degradation [165]. However, because most function-ally related gene clusters considered in this review represent assemblages of a variety ofgene types (Table 1; Figs. 1, 2) and because the most closely related members of each ofthese gene families represented are frequently located in other unlinked gene clusters, thiswould argue against the natal model for the origin of most clusters considered here. Further,the natal model provides no mechanism for maintenance of a gene cluster once it is formed[160].

4.1.2. Coregulation Model

The coregulation model predicts that there is a selective benefit to clustering of functionallyrelated genes due to more efficient regulation mechanisms associated with proximity ofthe genes. Presumably, this would either involve some sort of cis-acting element (as inbacterial operons) or an effect of the chromatin environment. Although Walton [164]dismissed the role of a common chromatin environment due to the idea that nonclusteredfungal primary metabolism pathway genes are effective without a common chromatinenvironment, recent data hint that chromatin environment or gene proximity could beimportant in regulation of secondary metabolism. Chiou et al. [166] showed that aflatoxinbiosynthetic genes are not transcribed normally when removed from the gene cluster, andthe same has been observed for sterigmatocystin genes (N. Keller et al., unpublished data).It is intriguing to speculate that the recently described LaeA protein [158] could be involvedin cluster regulation. The function of LaeA is not fully delineated but sequence analysissuggests it to be a protein methyltransferase with most similarity to the histone and argininemethyltransferases that play important roles in regulating gene expression. An interestingaspect of histone methyltransferases in regulating gene expression has been the recentdiscovery that histone methylation plays a role in defining boundaries of euchromatic andheterochromatic chromosomal domains, such as in the mating locus of yeast and the beta-globin locus in mice [167–170]. These findings suggest that histone methylation may beimportant in the regulation of gene cluster boundaries and may support a relationship withLaeA and histones. Also, studies of gene regulation in the A. nidulans penicillin genecluster and the A. nidulans nitrate utilization gene cluster have shown that chromatinremodeling or DNA conformational changes are required for expression of genes in theseclusters [145,171]. Clearly, coregulation would provide the selection pressure necessaryto maintain a gene cluster; it is not clear, however, that it would be able to act to causethe formation of clusters [161].

4.1.3. Selfish Cluster Model

The selfish cluster model proposed recently byWalton [164] is an extension of the ‘‘selfishgene’’ [172] and the ‘‘selfish operon’’ [160–162] concepts. Basically, the idea is that thecluster is the unit on which selection is acting. The fitness of the cluster is enhanced bytight linkage of the genes because the probability of transfer via horizontal transmissionis quite high. In the selfish operon model for bacteria, a great deal of emphasis is placedon the importance of horizontal gene transfer as a mechanism for the formation of clustersof genes. The horizontal transmission of an incipient cluster (fortuitous preexisting looselinkage between genes with related function separated by intervening sequences) wouldthen provide for selection of the trait in absence of the need for the intervening sequences.


Thus, the horizontal transfer aspect of this model acts to make intervening sequencesnonessential so that these sequences would experience strong selection to be lost. It couldbe argued that identification of secondary metabolite gene clusters located on dispensablechromosomes such as the Alternaria host-specific toxins [80] and the Nectria haemato-cocca pathogenicity island [2] may support this model.

Horizontal transmission has also been proposed as an explanation for why regulatorygenes are present in many clusters of genes for dispensible functions in fungi. Thesetransacting regulatory genes need not be proximate to influence the cluster function. How-ever, were the cluster to be transferred to a recipient without the regulatory gene, thecluster would not provide a selectable phenotype.

The mechanisms and conclusive evidence for horizontal transfer in fungi is notnearly as well-established as in bacterial systems [163]. However, the advent of sufficientgenome sequence data for fungi, bacteria, and plants will likely expand our understandingof the prevalence of horizontal gene transfer in fungi. To address gene cluster specifically,an obvious place to begin would be clusters with functions that are also present outsidethe fungal kingdom (e.g., penicillin or gibberellins).

Evidence from the penicillin cluster in bacteria and fungi provides the best supportfor horizontal transfer [163,173–175]. Many prokaryotes are capable of �-lactam produc-tion, whereas among eukaryotes, only a limited number of filamentous fungi possess thistrait. Despite the length of time since divergence between bacteria and fungi, both thebiochemical pathway and sequence of genes involved (e.g., IPNS) are similar [174,176],with the fungal genes showing codon usage more like prokaryotes than eukaryotes [176].Buades and Moya [174] applied maximum likelihood statistics to examine the molecularclocks of the IPNS genes. This analysis revealed that fungal IPNS genes appear 1400million years closer than expected to the bacterial genes [174].

Alternatively, evidence from gene cluster in Gibberella fujikuroi does not supporthorizontal gene transfer between plants and fungi (119). Unlike G. fujikuroi, genes forgibberellin biosynthesis in plants are dispersed, not clustered. Furthermore, different typesof enzymes perform the same function in the two different kingdoms. For example, thegibberellin 20-oxidase is a cytochrome P450 monooxygenase in G. fujikuroi and not a2-oxo-glutarate-dependent dioxygenase like in plants. Finally, the difference in this gibber-ellin 20-oxidase has broader implications relative to gibberellin biosynthesis gene regula-tion. In plants, expression of this enzyme is regulated by negative feedback in the presenceof biologically active gibberellins, thus maintaining homeostasis. This sort of negativefeedback regulation does not seem to occur at this step in fungi [119].

4.1.4. Fisher Model

The Fisher model is an extension of the theory associated with linkage disequilibriumamong coadapted gene complexes (177). Fisher’s original idea was that fitness differencesassociated with variation in effectiveness of particular allele combinations from distinctgenetic loci results in linkage of these alleles in the genotypes that persist in the population,despite the lack of physical linkage in the genome (linkage disequilibrium). Applicationof this idea to gene clustering requires the additional assumption that the selection is strongenough to favor actual physical clustering of these loci, thus, further ensuring decreasedprobability of breaking up the coadapted gene complexes. Both frequent recombinationand the prerequisite genetic and phenotypic variation (polymorphism) among genotypesin the population would be required for the assembly of clusters. Once formed, the clusterswould be more resilient to breakup by recombination. Thus, clustering would be both

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selected for and presumably maintained in this model. This model is distinct from theidea of a selfish cluster in that selection acts at the level of individuals within the population,not on the cluster itself. The clusters form based on selection favoring gene conversionevents that occur within the genome as opposed to invoking horizontal gene transfer.

One could argue that there is evidence to support this model available from possiblythe most extensive study of secondary metabolite gene cluster diversity across a geographicsample of a fungus [178]. Initially, O’Donnell et al. [107] established seven biogeographiclineages within a worldwide collection of isolates of Fusarium graminearum. This robustresult was based on concordance (reciprocal monophyly) across gene genealogies for sixsingle-copy genes, including one gene trichothecene gene (TRI101; 3-O-acetyltransferase)that happens to be separate from the trichothecene biosynthesis cluster [179]. Since thethree known trichothecene chemotypes (NIV, 3ADON, and 15ADON) proved not to belineage specific, these researchers chose to investigate the phylogenies revealed by 19 kbof sequence from the trichothecene gene cluster of 39 isolates [178]. The combined genegenealogies from analysis of the TRI genes grouped isolates into chemotype-specificclades. Thus, the authors interpreted this to mean that the TRI-cluster haplotypes eachhave a single evolutionary origin. This was further supported by the nearly identical pat-terns exhibited between phylogenetic trees generated based on variation at synonymoussites and both the respective noncluster and TRI-cluster trees, indicating that the differencesin the cluster and noncluster trees were not due to convergent evolution of the clusters.The authors’ interpretation of these data was that the polymorphisms associated withchemotype were maintained by balancing selection despite divergence into separate spe-cies. These data are particularly consistent with an idea of coadapted gene complexes. Inthe absence of this sort of extensive study of genetic and phenotypic polymorphism amongintraspecific or closely related interspecific lineages, it is difficult to know how prevalentthis pattern may be.

4.2. Adopting a Unified Model for the Evolution of Gene Clusters

Because a variety of selection pressures likely interact to promote formation and mainte-nance of complex traits, a unified model may be the best explanation for evolution offungal secondary metabolite gene clusters.

4.2.1. Formation of Gene Clusters

Selection acting on the function of the cluster (thus, its contribution to the organism) willfavor combinations of alleles that provide the most benefit (or least cost) to the organism.In the presence of sufficient allelic variation and frequent recombination, selection willfavor increased linkage among these coadapted gene complexes, thus favoring formation of(at least loosely) clustered functionally related genes. Because genes for essential functionsexperience strong purifying selection that will act to reduce allelic polymorphism [180],clusters of genes associated with essential functions will be rare.

At the level of the cluster itself, any factors that select against or obviate the needfor intervening sequences (i.e., promote loss) within these loosely linked genes will alsofavor cluster formation. Although horizontal gene transfer is cited as the mechanism forthis in selfish operon/cluster models, it is not the sole mechanism likely to promote selec-tion against intervening sequences. Operating under the same idea that clusters are selfish(i.e., promote their own dissemination/fitness irrespective of organism’s fitness), any fac-tors that promote duplication of all or part of the incipient cluster (sensu the natal model)


within that genome would provide the same opportunity to select against the interveningsequences (which would then be redundant in the genome).

Furthermore, much like models for the evolution of novel traits after duplicationand subfunctionalization of single genes [181], selection would act on duplicated clusters(or on partial clusters or incipient clusters) to favor new traits or they would be lost.Clearly, formation of clusters in this manner would not represent the same ‘‘ready-made’’adaptation one might envision under a horizontal transfer scenario. However, becausethere is arguably a much higher probability of duplication of DNA within a fungal genomethan horizontal transfer of DNA between genomes, seeding of new clusters from duplicatedsequences seems an even more probable mechanism for these selfish units to proliferate.

4.2.2. Maintenance of Gene Clusters

Once formed, selection must act to favor maintenance of a gene cluster; otherwise, avariety of processes acts to disperse the cluster. Here again, selection favoring the functionof the cluster acts to maintain (or continue to improve) particular coadapted gene com-plexes. Since any recombination at the locus that brings together nonnative combinationsof genes results in reduced fitness for the organism, those lineages would not persist. Infact, under strong enough selection this might lead to evolution of reduced recombination inthe region. Furthermore, if the trait encoded by the cluster was sufficiently phenotypicallypolymorphic relative to different native combinations of genes, then one might expect thatecological differentiation promoted might reduce the probability of recombination amonglineages with different clusters (e.g., colonizing different niches within a heterogeneousenvironment resulting in fewer opportunities to mate).

Improvement of cluster function once clustered would be reasonable based on evolu-tion of favorable protein–protein interactions or on substrate channeling associated withcolocalization of the genes. Thus, selection would act against random factors acting todisperse genes over time. Furthermore, if there was any advantage to a common chromatinenvironment or via coregulation, these mechanisms could also help to maintain the cluster.Selection acting at the level of the selfish cluster will maintain the cluster so long as thefactors that promoted its formation as a selfish unit are present (e.g., transposons). Thatis, factors that act to promote cluster duplication and/or horizontal transfer of clusters willmaintain a prevalent pattern of functionally related dispensable gene clusters.

5. CONCLUSIONS AND FUTURE RESEARCH

Comparative and functional analyses of fungal genomes promise to be a tremendousresource for discerning the relative importance of these different factors (horizontal genetransfer, duplication, coadaptation of genes, coregulation) contributing to formation andmaintenance of fungal gene clusters. Evidence for coadapted gene complexes will requirecomparisons within and across closely related species and subsequent tests for superiorfunction. Ultimately, it will be of great interest to determine to what degree ecology andlife history traits are associated with differences in the relative importance of all thesefactors. For example, will there be differences across asexual vs. sexual fungal lineages,or across pathogens vs. saprophytes.

In the rare cases in which genes for an essential function are associated with a cluster,it will be of interest to determine whether there is evidence for greater polymorphism orco-adaptation among the genes involved. Also, of interest will be the cases in which genesare clustered in some lineages but not others (e.g., melanin and carotenoid biosynthesis).

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If such genes are known to be orthologous, then it will be of interest to determine whetherthere has been dispersal of a cluster or clustering of dispersed genes. Clearly, addressingthis question will require ample access to data from many species with the trait. If thereis evidence that it was a cluster that dispersed, it will be interesting to determine whatsort of selection was relaxed.

Generally, across clusters, it will be of interest to assess whether genes in dispensablefunction pathways/clusters are more polymorphic than the paralogous members of thegene family involved in nondispensable functions. Is there phylogenetic evidence forcoevolution of coadapted gene complexes? Is there biochemical/functional evidence forcoadapted gene clusters? Are clusters associated with genome regions that are particularilylikely or unlikely to recombine? Are clusters more likely than other pieces of DNA to beassociated with genetic elements that promote movement of DNA segments?

Clustering of genes involved in fungal secondary metabolism is a clear trend uncov-ered during the decades of research to identify genes associated with particular naturalproducts. On the horizon, we see a more reverse genetic approach in which predicted openreading frames identified in sequenced genomes might be targeted to determine whetheror not they play a role in secondary metabolism. Furthermore, there will be a tremendousamount of data available to search for clusters of genes surrounding members of genefamilies associated with secondary metabolism. This will be the new frontier in prospectingfor new natural products, based on predicting the products produced by this arrangementof genes. Also looming on the horizon is the need to determine the roles of the productsfor the producing organisms. This will assist in both applied use of the products and indeducing the ecological niches and evolutionary history of the organisms.

ACKNOWLEDGMENTS

The authors thankDeepak Bhatnagar, Rosie Bradshaw, Richard Hutchinson, Chris Schardl,Bettina Tudzynski, Paul Tudzynski, and Jiujiang Yu for sharing unpublished data.

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