Advances in cloning, functional analysis and heterologous expression of fungal polyketide synthase genes

Journal of Biotechnology 124 (2006) 690–703

Review

Advances in cloning, functional analysis and heterologous expression of fungal polyketide synthase genes Julia Sch¨umann, Christian Hertweck ∗ Leibniz-Institute for Natural Product Research and Infection Biology, HKI, Beutenbergstr. 11a, 07745 Jena, Germany Received 28 October 2005; received in revised form 20 January 2006; accepted 29 March 2006

Abstract Fungal polyketides comprise a diverse group of secondary metabolites that play an important role for drug discovery, as pigments, and as mycotoxins. Their biosynthesis is governed by multidomain enzymes, so-called fungal type I polyketide synthases (PKS). Investigating the molecular basis of polyketide biosynthesis in fungi is of great importance for ecological and pharmacological reasons. In addition, cloning, functional analysis and expression of fungal PKS genes also set the basis for engineering the yet largely untapped biosynthetic potential. © 2006 Elsevier B.V. All rights reserved. Keywords: Fungi; Metabolic engineering; Natural products; Polyketides

Contents 1. 2. 3. 4. 5.

Fungal polyketide biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Locating and cloning fungal PKS genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional analyses of fungal PKS genes (transformation and inactivation) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Heterologous expression of fungal PKS genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

690 693 696 698 701 701 701

1. Fungal polyketide biosynthesis ∗

Corresponding author. Fax: +49 3641 656705. E-mail address: [email protected] (C. Hertweck). 0168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2006.03.046

Fungal polyketides constitute a large family of secondary metabolites endowed with a high degree of structural diversity and various biological activities

J. Sch¨umann, C. Hertweck / Journal of Biotechnology 124 (2006) 690–703

(O’Hagan, 1991). Many of these compounds, or derivatives thereof, are used as pharmaceuticals, for example the cholesterol-lowering agent lovastatin. On the other hand, many fungal polyketides are infamous for causing severe damage in food industry, agriculture and human health. Important examples are T-toxin, the fumonisins and the aflatoxins (Leistner, 1984; Chu, 1991; Sweeney and Dobson, 1999). Furthermore, various (poly)phenolics, such as 6-methyl salicylic acid (6-MSA), orsellinic acid, tetrahydroxynaphthalene (THN), YwA1 and norsolorinic acid, are components of fungal pigments (Fig. 1b). Common to all fungal polyketides is their biosynthetic origin. They are produced by repetitive Claisen condensations of an acyl-coenzyme A (CoA) starter with malonyl-CoA elongation units in a fashion reminiscent of fatty acid biosynthesis (Fig. 1a). One of the major differences between both metabolic pathways is that polyketides can differ in the degree of ␤-keto processing. Some are not or only partially reduced, resulting in the formation of (poly-)cyclic aromatic compounds. Alternatively, they may be partially or largely reduced, which usually gives rise to linear or macrocyclic, non-aromatic carbon frameworks (Rawlings, 1999; Staunton and Weissman, 2001). The biosynthesis of fungal polyketides is governed by multidomain enzymes, which are termed type I polyketide synthases (PKS) in analogy to multidomain fatty acid synthases. Fungal polyketide synthases consist of a minimal set of ketosynthase (KS), acyl transferase (AT), and acyl carier protein (ACP) domains. In addition, optional ␤-keto processing reactions may be catalyzed by keto reductase (KR), dehydratase (DH) and enoyl reductase (ER) domains. Further optional accessory domains are represented by cyclase (CYC) (Fujii et al., 2001) and methyl transferase (MT) activities (Hutchinson et al., 2000) (Fig. 2). In contrast to ‘post-PKS’ O- and N-methylation reactions, which are catalyzed by distinct enzymes after polyketide assembly, methylation of the polyketide carbon backbone takes place during chain formation by means of the intrinsic fungal C-MT domains (Nicholson et al., 2001). It should be noted that in fungal polyketide biosynthesis no methylmalonyl elongation units are employed, as opposed to numerous examples in bacterial polyketide biosynthesis involving modular type I PKS. Fungal iterative PKS can use each active site in an iterative way during chain assembly, and determine

691

the degree of reduction and C-methylation within each elongation round (Fujii et al., 1998; Rawlings, 1999). To date it has remained a mystery how a single set of catalytic domains determines chain length, degree of reduction, and timing of C-methylation at a particular step in the pathway (see below). It should be mentioned that although most fungal PKS work in an iterative fashion, some only catalyze a single round of elongation. Thus it is not appropriate to classify them as ‘iterative’ type I PKS (M¨uller, 2004). According to their architecture and the presence or absence of additional ␤-keto processing domains, fungal PKS are grouped into non-reducing or aromatic (NR-PKS), partially reducing (PR-PKS), and highly reducing PKS (HR-PKS). Recent phylogenetic studies on the basis of KS amino acid sequences provided valuable insights into the evolutionary relationship between different types of fungal PKS (Bingle et al., 1999; Nicholson et al., 2001). Kroken et al. showed that amino acid sequences of fungal KS domains cluster according to the degree of reduction of their products in reducing PKS (additional reductive domains: KR, ER, DH) and non-reducing PKS (no reductive domains), each type further divided into four subclades (Kroken et al., 2003). More recently, phylogenetic studies were also performed using KS sequences of non-reducing PKS genes of the Pertusariales (lichenized Ascomycota) including further non-reducing PKS sequences found in GenBank. Sequences clustered in eighteen clades including only lichenized taxa, only non-lichenized taxa or both. These findings are, at least partially, in accord with the groupings found in the studies of Kroken et al. for non-reducing PKS, but no clear linkage was found between groupings of PKS genes and their potential roles in secondary metabolism (Schmitt et al., 2005). Two clades of this study correspond to the phylogenetic studies on mainly Lecanora species by Grube and Blaha in 2003. Grube and Blaha carried out phylogenetic studies on the amino acid sequence of KS domains of putative PKS from 15 lichenized Lecanora species, three other lichen genera and other non-lichenized fungi. The Lecanora sequences all clustered with PKS that produce complex aromatic compounds. Lecanora PKS sequences are also found in two distinct clades, one with PKS producing precursors of dihydroxynaphthalene melanins from nonlichenized spezies, and another clade, which has no

692

J. Sch¨umann, C. Hertweck / Journal of Biotechnology 124 (2006) 690–703

Fig. 1. (a) Basic mechanisms of fungal polyketide biosynthesis. (b) Structures of selected fungal polyketide metabolites.

J. Sch¨umann, C. Hertweck / Journal of Biotechnology 124 (2006) 690–703

693

Fig. 2. Examples for non-reducing (NR: PksA, Pks1, WA), partially reducing (PR: 6-MSAS), and highly reducing (HR: LDKS, MlcB, SQTKS, LNKS, MlcA) PKS, and their domain architectures (for individual functions see text).

close relationships with known PKS from other fungi (Grube and Blaha, 2003). In addition to the chain length, the degree of ␤-keto processing and cyclization, the large structural diversity of polyketides derives from derivatization of the polyketide carbon skeleton by alkylation, acylation, and oxygenation, by so-called post-PKS or tailoring reactions. From analyses of the few PKS gene loci that have been investigated to date it appears that all genes necessary for fungal polyketide biosynthesis are clustered, i.e. PKS genes, genes encoding enzymes involved in tailoring reactions, as well as genes required for regulation and resistance, as exemplified for the lovastatin (Kennedy et al., 1999), compactin (Abe et al., 2002) and aflatoxin (Yu et al., 1995; Bhatnagar et al., 2003) biosynthesis gene clusters (Fig. 3). Knowledge on the biosynthesis genes and their regulation could provide important information of the ecological role of fungal polyketide metabolites. Furthermore, investigations of fungal PKS genes and their expression could set the basis for improving metabolite production and engineering the biosynthetic machinery with the aim to synthesize new compounds (Burkart, 2003; Reeves, 2003). While such pathway engineering

or combinatorial biosynthesis approaches have been impressively demonstrated using bacterial PKS systems (Cane et al., 1998; Walsh, 2002; Weber et al., 2003), analogous experiments in fungi have been hampered for several reasons. First, apart from the large size of the fungal genomes, which renders screening a cumbersome task, the presence of introns can be a major challenge for screening, heterologous expression, and functional studies of genes. Furthermore, inactivation and complementation of genes can be demanding. Finally, pathway engineering approaches are also handicapped by the lack of universal expression systems. In the following overview, the status quo of the locating, cloning and expression of fungal polyketide biosynthesis is outlined, highlighting currently available molecular techniques and approaches.

2. Locating and cloning fungal PKS genes As a consequence of recent fungal genome sequencing projects, a large body of data is available for yet unexplored putative PKS gene clusters (Galagan et al., 2003; Kroken et al., 2003; Nierman et al., 2005). If

694

J. Sch¨umann, C. Hertweck / Journal of Biotechnology 124 (2006) 690–703

Fig. 3. Organization of selected fungal PKS gene clusters (lovastatin, compactin, aflatoxin).

targeting a specific PKS gene from a particular producer strain is desired, in most cases genomic DNA (cosmid, BAC), as well as cDNA (phage) libraries are created and screened by Southern hybridisation with heterologous or homologous probes and/or by PCR with degenerated primers. Initially, PKS gene fragments from bacterial genomes were tested for targeting related genes in fungi. However, bacterial heterologous probes did not hybridize well with fungal PKS genes. As a consequence, in 1990 the first fungal PKS gene, encoding 6-methylsalicylic acid synthase (6-MSAS), was cloned and sequenced by an alternative approach. Beck et al. successfully screened an Escherichia coli expression library of Penicillium patulum DNA with antibodies raised against the purified 6-MSAS protein (Beck et al., 1990; Spencer and Jordan, 1992). The cloned MSAS gene could then be used as a probe to search for MSAS and related genes in various other fungal genomes (Fujii et al., 1996; Feng and Leonard,

1998). This approach, however, has not always been successful, e.g. in cloning the PKS genes involved in lovastatin biosynthesis in Aspergillus terreus. Lovastatin, which consists of two polyketide chains, possesses antihypercholesterolemic activity by inhibiting the enzyme (3S)-hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, a key enzyme required for cholesterol biosynthesis (Wagschal et al., 1996). Locating the lovastatin biosynthesis genes by hybridizing to the KS domain of the P. patulum 6-MSAS gene failed as only A. terreus 6-MSAS homologs were cloned (Hendrickson et al., 1999). The biosynthesis genes for lovastatin biosynthesis were identified in 1999 by Hendrickson et al. by screening mutants of the lovastatinproducing strain A. terreus. Several mutants were found that were either unable to produce the nonaketide part of lovastatin or defective in synthesizing the methylbutyryl group. This result indicated that these two portions are independent and use separate enzymes, named

J. Sch¨umann, C. Hertweck / Journal of Biotechnology 124 (2006) 690–703

lovastatin nonaketide synthase (LNKS) and lovastatin diketide synthase (LDKS). A genomic cosmid library was prepared in an Aspergillus/E. coli shuttle cosmid (pLO9) and used to complement the LNKS mutant, which was incapable of producing lovastatin. One lovastatin producing transformant was identified and the cosmid was recovered. In addition, cDNA was prepared from a lovastatin-producing culture and screened with antiserum raised against a polypeptide shown to represent a PKS involved in lovastatin synthesis. One of the isolated clones that even hybridized with the complementing cosmid insert contained a 3 -untranslated region and the carboxyl terminus of a protein with an ACP motif. The cDNA clone was used to isolate overlapping clones from the genomic library yielding an 11.6 kb region coding for the LNKS. Specific primers were then used to obtain the cDNA sequence by polymerase chain reaction (Hendrickson et al., 1999). By mutant complementation, also a PKS gene involved in the formation of a red perithecial pigment of Nectria haematococca was identified more recently (Graziani et al., 2004). A gene encoding the PKS involved in T-toxin biosynthesis was detected by tagged mutations causing a T-toxin negative phenotype. By the restriction enzyme-mediated integration (REMI) procedure, a PKS gene locus flanking the tag was identified, which is required for T-toxin production and virulence (Yang et al., 1996). Already in 1995 Keller et al. designed degenerated ␤-ketoacyl synthase domain primers (KS1 and KS2) based on two conserved amino acid regions in the ␤-ketoacyl synthase domains of type I fungal and bacterial PKS (Keller et al., 1995). These primers were used to amplify the KS region of the fumonisin polyketide synthase gene fum5 from cDNA of Giberella fujikuroi. Fumonisins are important mycotoxins produced from

695

G. fujikuroi associated with several mycotoxicoses (Proctor et al., 1999). A breakthrough in specifically targeting fungal PKS genes by PCR was achieved by Bingle et al. in 1999. Two pairs of degenerate primers (LC1 and LC2c, LC3 and LC5c) were designed to amplify ketosynthase domain fragments from fungal PKS belonging to one of the two subclasses of NR- and PR-PKS, WA-type and MSAS-type, respectively (Bingle et al., 1999). In a subsequent study, additional sets of degenerate primers were developed to target conserved regions (about seven amino acids) of particular fungal PKS domains, such as ␤-ketoacylsynthase, ketoreductase and C-methyltransferase domains, allowing the selective and rapid cloning of specific fungal PKS genes (Nicholson et al., 2001). It has been demonstrated that these primers allow for distinguishing between NRPKS, PR-PKS, and HR-PKS. Primers derived from the consensus sequences of the KS domain (DTACS) and the AT domain (HSSGEIA), as well as the abovementioned primers designed by Bingle et al. were successfully employed for cloning the PKS gene encoding the citrinin synthase. Citrinin is a nephrotoxic agent produced by various Aspergillus, Penicillium and Monascus sp. that may occur in contaminated food (Shimizu et al., 2005) (Fig. 4). Primers derived from intrinsic C-methyltransferase motifs proved to be a valuable tool for locating genes encoding HR-PKS, e.g. the PKS involved in squalestatin biosynthesis (Cox et al., 2004). Squalestatin S1, also known as zaragozic acid, is a potent and selective inhibitor of squalene synthase and a potential drug for the treatment of high serum cholesterol concentrations (Dawson et al., 1992; Bergstrom et al., 1993). Degenerated C-MT primers were used to amplify a sequence from the cDNA template of squalestatin producing Phoma sp. C2932 with high similarity to diverse fungal C-MT sequences. Probing

Fig. 4. Structures of the mycotoxins citrinin and ochratoxin.

696

J. Sch¨umann, C. Hertweck / Journal of Biotechnology 124 (2006) 690–703

Fig. 5. Structure of squalestatin S1. The tetraketide moiety is boxed.

a cDNA library from Phoma sp. C2932 with this fragment helped identifying a HR-PKS (Phoma PKS1, Squalestatin Tetraketide Synthase SQTKS) involved in squalestatin biosynthesis (Cox et al., 2004) (Fig. 5). Other sets of KS primers were developed by Lee et al. in order to screen a group of insect- and nematodeassociated fungi (Lee et al., 2001). Sauer et al. designed two degenerated primers for KS domains of PKS to examine non-sporulating endophytic fungi associated with wild-type Vaccinium macrocarpon. The cloned and amplified domains were sequenced and could be segregated into three groups, one clustering with PKS involved in pigment (melanin) formation, one with aflatoxin encoding fungal PKS, while another one did not cluster with any of the known fungal PKS groups (Sauer et al., 2002). The same set of primers was used to screen the genome of the darkly pigmented fungus Glarea lozoyensis and to amplify three PKS genes. One of these genes, pks1, was shown to code for a PKS involved in the 1,8-dihydroxynaphtalene melanin (DHN) biosynthesis, responsible for the deep coloration of many fungi (Zhang et al., 2003). Degenerated KS and AT primers were used to screen the Penicillium citrinum genome for its biosynthetic potential. One of four detected PKS genes is correlated to the compactin biosynthesis. Like lovastatin, which is produced by A. terreus, compactin inhibits the HMGCoA reductase and differs from lovastatin only in a missing methyl group on the nonaketide chain derived from S-adenosyl-l-methionine (SAM) (Moore et al., 1985; Wagschal et al., 1996). Another viable approach to target fungal PKS genes is differential display. Such an approach, based on nitrogen repression, was used to isolate a PKS gene involved in the biosynthesis of the red pigment bikaverin of G. fujikuroi. RNA isolated from mycelia grown under repressed and derepressed conditions was

subjected to reverse transcription and the resulting cDNAs were used as templates in differential display PCR (Linnemannstons et al., 2002). The PKS gene required for ochratoxin A biosynthesis in Aspergillus ochraceus was detected by a suppression subtractive hybridization PCR-based approach (SSH-PCR) (O’Callaghan et al., 2003) (Fig. 4). Finally, when searching for particular PKS genes in fungi, one should be aware that probably not all metabolites isolated from fungi are truly fungal metabolites. A recent study revealed that Rhizopus microsporus, which does not seem to bear any PKS genes, harbours bacterial endosymbionts for the production of the polyketide rhizoxin, the causative agent of rice seedling blight (Partida-Martinez and Hertweck, 2005).

3. Functional analyses of fungal PKS genes (transformation and inactivation) Proving the identity of a particular fungal PKS gene can be a challenging task. As a prerequisite for gene inactivation or heterologous expression of fungal PKS and related genes DNA has to be introduced into a fungal strain. In 1979 the first genetic manipulation of Neurospora crassa was reported by Case et al. (1979) followed by transformation of Aspergillus nidulans by Turner and co-workers in 1983 (Ballance et al., 1983), Tilburn et al. in 1983 (Tilburn et al., 1983), John and Peberdy in 1984 (John and Peberdy, 1984) and Yelton et al. in 1984 (Yelton et al., 1984). Typical fungal transformation methods require protoplasts, cell wall degradation with lytic enzymes and permeabilization of cell membranes with polyethylene glycol (PEG) and calcium chloride (Brakhage and Langfelder, 2002). Protocols for electroporation of germinating conidia have been reported for filamentous fungi like Aspergillus oryzae (Chakraborty and Kapoor, 1990; Chakraborty et al., 1991), Aspergillus niger (Ozeki et al., 1994), A. nidulans (Sanchez et al., 1998) and Aspergillus fumigatus (Weidner et al., 1998). A viable alternative is the use of Agrobacterium tumefaciens, which is well known for its ability to efficiently transfer DNA into both plants and fungi (de Groot et al., 1998). It should be noted that also particle bombardment is a feasible method for the introduction of heterologous DNA into fungi. The most commonly

J. Sch¨umann, C. Hertweck / Journal of Biotechnology 124 (2006) 690–703

used selection markers for transformants are either nutritional markers, which complement an auxotrophic requirement (pyrG, amdS, argB, trpC, niaA) or dominant antibiotic resistance markers against hygromycin B, phleomycin, bialaphos (BASTA), sulfonylurea, and benomyl (Talbot, 2001). Despite the various methods available, a major problem for functional gene analyses is that the integration of transformed DNA can occur in a homologous or nonhomologous manner. The frequency of homologous integration varies greatly between different organisms and depends on the length of homologous DNA. With two separate homologous sites within one DNA fragment a double homologous integration can be achieved, resulting in gene replacement. Non-homologous integration occurs when the inserted DNA integrates at a random site in the fungal genome. This can also happen at high frequency if the DNA contains homologous regions. REMI transformation can be used to facilitate integration of transforming DNA (Brown et al., 1998), as inclusion of restriction enzymes with DNA causes nicks in chromosomal DNA and facilitates recognition of the insertion sites (Talbot, 2001; Brakhage and Langfelder, 2002). In this context it should be highlighted that a PKS gene (pks12) from Fusarium graminearum has recently been used as visible marker to develop a highly efficient gene targeting system (Maier et al., 2005). Complementation of randomly generated mutants is another viable method for analyzing the functions of targeted genes with recognizable phenotypes. Today several techniques are known to generate fungal mutants, such as REMI (Brown et al., 1998), UVmutagenesis (Jahn et al., 1997) or transposon mutagenesis (Kempken et al., 1998; Kempken and Kuck, 2000; Brakhage and Langfelder, 2002). Most of the above-mentioned inactivation techniques have been applied to the functional analyses of fungal polyketide biosynthesis genes that are, e.g. involved in the formation of pigments and/or pathogenicity factors. An important example is 1,8dihydroxynaphtalene (DHN), a widely distributed type of melanin among ascomycetous fungi (Bell and Wheeler, 1986). In plant pathogens, DHN-melanin is well documented as a virulence factor required for appressorium formation (Takano et al., 1997). DHN is derived from 1,3,6,8-tetrahydroxynaphthalene (THN), which is biosynthesized via the polyketide

697

route (Takano et al., 1995; Fulton et al., 1999; Fujii et al., 2000). The functional analysis of pks1, a PKS gene involved in melanin formation, was achieved by a gene replacement via Agrobacterium-mediated transformation. Knockout mutants with a disruption of the pks1 via double cross-over displayed an albino phenotype, indicating that pks1 is required for the synthesis of DHN-melanin in G. lozoyensis (Zhang et al., 2003). Aflatoxins are infamous mycotoxins produced by certain strains of Aspergillus parasiticus, Aspergillus flavus and Aspergillus nominus. They are known to be immunosuppressive, mutagenetic, teratogenetic and hepatocarcinogenic in animals and humans (Eaton and Groopman, 1994). Numerous ascomycetes and deuteromycetes including A. nidulans produce the mycotoxin sterigmatocystin (ST), an intermediate in aflatoxin biosynthesis. The well-understood biosynthetic pathway has been thoroughly reviewed (Trail et al., 1995; Bennett et al., 1997; Minto and Townsend, 1997; Bhatnagar et al., 2003). Aflatoxin biosynthesis genes are located in a 70 kb gene cluster containing genes for DNA-binding proteins, regulatory proteins, cytochrome P450-type monooxygenases, dehydrogenases, methyltransferases and polyketide and fatty acid synthases. A hexanoyl starter unit derived from fatty acid metabolism is used as PKS primer unit to synthesize norsolorinic acid (Brown et al., 1996). This polyphenolic pigment then undergoes several intriguing enzymatic conversions until aflatoxins are formed (Fig. 6). PksA was found to be the polyketide synthase responsible for the conversion of hexanoyl-CoA to norsolorinic acid as targeted gene knockout experiments in 1995 revealed. The pksA gene is an orthologue of the A. nidulans wA gene and codes for a PKS with KS, AT, ACP, and TE domains (Chang et al., 1995; Feng and Leonard, 1995). The lovastatin biosynthesis gene cluster contains two type I PKS genes. Elegant experiments by Hutchinson, Vederas and co-workers revealed that the iterative lovastatin nonaketide synthase (LNKS, LovB) interacts with protein LovC, which is necessary for the correct processing of the growing polyketide chain and production of dihydromonacolin L. The noniterative lovastatin diketide synthase (LDKS, LovF) is responsible for 2-methylbutyrate biosynthesis that is assembled with monacolin J through catalysis of an additional transesterase LovD to form lovastatin. Gene

698

J. Sch¨umann, C. Hertweck / Journal of Biotechnology 124 (2006) 690–703

Fig. 6. Simplified model of sterigmacystin and aflatoxin biosynthesis via norsolorinic acid.

disruption of lovC led to the production of less reduced shunt products of the nonaketide, while coexpression with LNKS restored the correct enoyl reduction and chain extension. Gene disruption of lovF led to accumulation of monacolin J, revealing that this gene plays a role in the biosynthesis of the (2R)-2-methylbutyryl side chain of lovastatin. Gene disruption of the putative transesterase gene lovD also resulted in an accumulation of monacolin J (Kennedy et al., 1999) (Fig. 7). The compactin biosynthesis pathway is assumed to involve very similar biosynthetic steps. Screening a cosmid library identified a gene cluster with two genes, mlcA and mlcB, putatively encoding PKS with high similarity to LNKS (LovB) and LDKS (LovF), except for a non-functional MT domain. Disruption of mlcA resulted in a mutant that could not produce compactin or any nonaketide-derived intermediates (Abe et al., 2002). In addition to gene disruption or deletion, RNAmediated gene silencing represents another genetic tool that could be helpful for exploring gene functions in fungi. This method represents a posttranscriptional gene-silencing phenomenon, in which double stranded RNA triggers degradation of sequence homologous mRNA. In 2005 Nakayashiki et al. successfully silenced specific genes in Magnaporthe oryzae and

Colletotrichum lagenarium (Nakayashiki et al., 2005). While this is – in principle – an attractive method to shut down gene expression relatively easily and quickly, RNA silencing may be partial and not necessarily lead to a null phenotype like the gene knockout. On the other hand partial silencing enables the analysis of lethal knockouts. Furthermore, this method provides flexibility in gene inactivation since it suppresses sequence specific, and not locus specific. Homologous genes have been shown to be silenced simultaneously (Thierry and Vaucheret, 1996; Baulcombe, 1999; Allen et al., 2004; Nakayashiki et al., 2005).

4. Heterologous expression of fungal PKS genes Production of fungal polyketide metabolites by large-scale fermentation may be hampered by the requirement of sophisticated growth conditions, inefficient production, and a large biosynthetic background, which obstructs work-up and isolation of metabolites. For this reason, a universal fungal PKS gene expression system would be convenient. It might not only facilitate the production of native polyketides, but also serve for engineering fungal polyketide biosynthesis. However, to date the heterologous production of fungal polyketide synthases is still at its infancy.

J. Sch¨umann, C. Hertweck / Journal of Biotechnology 124 (2006) 690–703

699

Fig. 7. Context-dependent lovastatin biosynthesis.

In light of the impressive efforts in expressing bacterial PKS genes one may ask what complicates the heterologous expression of fungal PKS genes. First of all, the presence of introns and strain-specific splicing mechanisms may require the use of hard to obtain full length cDNA or cumbersome removal of introns. Second, the ACP domains require posttranslational 4-phosphopantetheinylation catalyzed by 4phosphopantetheinyl transferases (PPTase) (Fig. 8). This enzyme can be quite specific, which makes it necessary to coexpress a species specific PPTase with the PKS in order to obtain a functioning holo-ACP. Third, in various cases, the large size of the gene locus encoding the biosynthetic pathway may render expression constructs instable. In general, a variety of heterologous expression hosts could be suitable for the production of polyketides, as outlined in an extensive review by Pfeifer and Khosla (2001). Apart from filamen-

tous fungi, such as A. nidulans, yeast (Saccharomyces cerevisiae), bacteria (in particular Actinomycetes), and plants have been used for sustainable polyketide production. The first example of a heterologous expression of a fungal PKS gene in bacteria was reported by Schweizer and co-workers in 1995. The multifunctional 6-methylsalicylic acid synthase gene (6-MSAS) from P. patulum was successfully expressed in Streptomyces coelicolor CH999, an engineered host strain lacking a genomic region encoding actinorhodin biosynthesis, which has previously been used for expression of bacterial PKS genes (Bedford et al., 1995). Prior to expression the 69 bp intron of the 6-MSAS gene was removed, a Shine–Dalgarno sequence complementary to the 3 region of S. coelicolor 16S rRNA was included and four of the first seven codons were exchanged to codons more

700

J. Sch¨umann, C. Hertweck / Journal of Biotechnology 124 (2006) 690–703

Fig. 8. Phosphopantheteinylated 6-MSAS and biosynthesis of 6-methyl salicylic acid (6-MSA).

frequently used in Streptomyces sp. (Wright and Bibb, 1992). In the expression vector the 6-MSAS gene was cloned downstream of the actinorhodin actI promoter, which is activated at the onset of the stationary phase. Transformants proved to produce 6-methylsalicylic acid in significant amounts (20 mg from 12 agar plates, 300 ml medium). This experiment implicated that the enzyme was folded into the correct tertiary structure and that the acyl carrier protein domain was successfully pantotheinylated (Bedford et al., 1995). Almost simultanously, in 1996, the successful heterologous expression of a PKS gene in a fungal host has been reported. The 6-MSAS encoding gene atX was identified in the A. terreus genome via Southern blot analysis with the 6-MSAS gene of P. patulum. Sequencing of atX revealed a 5,5 kb open reading frame containing a 70 bp intron near the N-terminus. atX was cloned downstream of the amyB promoter (promoter of the Taka-amylase A gene of A. oryzae) of the fungal expression vector pTAex3, which was then introduced into the heterologous host A. nidulans. Indeed the transformant produced significant amounts of 6MSA, establishing the function of AtX as a 6-MSAS (Fujii et al., 1996). As already mentioned above, in 1999 Kennedy et al. succeeded in the heterologous expression of the lovastatin PKS LNKS gene (lovB) in A. nidulans. The expression was achieved using the alcA promoter (alcohol dehydrogenase promoter of A. nidulans). The production of LNKS was confirmed by antibody staining. However, it should be underlined that the

analysis of the produced metabolites indicated that – although the PKS retained all activities – it was malfunctioning and unable to coordinate these activities properly. The produced polyketides featured a shorter carbon chain and a lower degree of reduction than the original intermediate dihydromonacolin L, which might indicate the inactivity of the ER domain. Only the coexpression of the LNKS and LovC genes (see above) resulted in dihydromonacolin L production, showing that these two enzymes work closely together in lovastatin biosynthesis (Kennedy et al., 1999). Thus, care must be taken when expressing individual components of a particular fungal PKS gene cluster. More recently, Cox et al. succeeded in the expression of a HR-PKS (Phoma PKS1 or SQTKS) involved in the biosynthesis of the tetraketide side chain of squalestatin S1, using A. oryzae as heterologous host. The transformant was capable of producing the tetraketide side chain of squalestatin in detectable concentrations, indicating that all domains of SQTKS are active under these conditions (Cox et al., 2004). The expression of a fungal PKS gene in E. coli and S. cerevisiae was reported in 1998 by Kealy et al. In the bacterial and in the yeast host coexpression of the 6-MSAS and a heterologous PPTase (Sfp from Bacillus subtilis) was required to convert the apo-PKS to its holo form. Both the sfp and the 6-MSAS genes were expressed under the control of the alcohol dehydrogenase 2 (ADH2) and T5 RNA polymerase promoters in S. cerevisiae and E. coli, respectively. A twofold higher 6-MSA level (1.7 g of 6-MSA/l culture) compared to the native host could be reached by expression in yeast

J. Sch¨umann, C. Hertweck / Journal of Biotechnology 124 (2006) 690–703

when cultivated in 50 ml YPD medium culture for 142 h at 30 ◦ C. Recombinant E. coli strains produced 6-MSA (75 mg/l culture) in the presence of 10% glycerol in ATCC medium 765 after cultivation for 24 h at 30 ◦ C (Kealey et al., 1998). The yet only example of fungal PKS gene expression in plants was reported by Yalpani and co-workers, who succeeded in the expression of the 6-MSAS gene from P. patulum in tobacco. The aim of this study was to investigate if 6-MSA can mimic salicylic acid (SA), the native activator of disease resistance (Fig. 8). To target heterologously produced 6-MSAS to tobacco plastids, the intron-free 6-MSAS gene was linked to a fragment coding for a signal sequence from the petunia ribulose bisphosphate carboxylase small subunit. Using a binary expression vector and Agrobacterium-mediated transformation, the construct was successfully introduced into tobacco. High level constitutive expression was achieved by using the SCP1 and UCP3 promoters. Leaves of transgenic tobacco expressing the 6-MSAS gene accumulated significant amounts of 6-MSA, up to 20 ␮g/g fresh weight. Interestingly, the production of 6-MSA induces enhanced defence protein levels and virus resistance similar to the native signal compound, SA. Furthermore, this example not only demonstrates that a complex fungal biosynthetic machinery may be functional in plants, but also that plant PPTases appear to be sufficiently flexible to phosphopantetheinylate ACP domains of fungal PKS. Possibly, plants could also be engineered to functionally express other fungal PKS genes to produce pharmacologically relevant polyketides.

5. Conclusion This short review demonstrates that locating fungal PKS genes, their functional analyses, and their expression in a heterologous host can be cumbersome, ambitious, but also highly rewarding. Although it will still take a while until fungal PKS research can catch up with the impressive body of results obtained with bacterial systems, it is obvious that this is a rapidly emerging field. The molecular tools available allow for fascinating ecological studies, as well as for detecting food-borne mycotoxin producers. Furthermore, recent pioneering work reveals that it seems to be possible to engineer fungal polyketide biosynthesis pathways for

701

sustainable metabolite production and drug development. An enormous biosynthetic potential remains to be tapped.

Acknowledgement Financial support by the European Community in the FP5 (“EUKETIDES”) for the authors’ original work is gratefully acknowledged.

References Abe, Y., Suzuki, T., et al., 2002. Molecular cloning and characterization of an ML-236B (compactin) biosynthetic gene cluster in Penicillium citrinum. Mol. Genet. Genom. 267 (5), 636– 646. Allen, R.S., Millgate, A.G., et al., 2004. RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy. Nat. Biotechnol. 22 (12), 1559–1566. Ballance, D.J., Buxton, F.P., et al., 1983. Transformation of Aspergillus nidulans by the orotidine-5 -phosphate decarboxylase gene of Neurospora crassa. Biochem. Biophys. Res. Commun. 112, 284–289. Baulcombe, D.C., 1999. Gene silencing: RNA makes RNA makes no protein. Curr. Biol. 22, 1559–1566. Beck, J., Ripka, S., et al., 1990. The multifunctional 6-methylsalicylic acid synthase gene of Penicillium patulum. Its gene structure relative to that of other polyketide synthases. Eur. J. Biochem. 192 (2), 487–498. Bedford, D.J., Schweizer, E., et al., 1995. Expression of a functional fungal polyketide synthase in the bacterium Streptomyces coelicolor A3(2). J. Bacteriol. 177, 4544–4548. Bell, A., Wheeler, M., 1986. Biosynthesis and functions of fungal melanins. Ann. Rev. Phytopathol. 24, 411–451. Bennett, J.W., Chang, P.K., et al., 1997. One gene to whole pathway: the role of norsolorinic acid in aflatoxin research. Adv. Appl. Microbiol. 45, 1–15. Bergstrom, J.D., Kurtz, M.M., et al., 1993. Zaragozic acids: a family of fungal metabolites that are picomolar competitive inhibitors of squalene synthase. Proc. Natl. Acad. Sci. USA 90 (1), 80– 84. Bhatnagar, D., Ehrlich, K.C., et al., 2003. Molecular genetic analysis and regulation of aflatoxin biosynthesis. Appl. Microbiol. Biotechnol. 61 (2), 83–93. Bingle, L.E.H., Simpson, T.J., et al., 1999. Ketosynthase domain probes identify two subclasses of fungal polyketide synthase genes. Fungal Genet. Biol. 26, 209–223. Brakhage, A.A., Langfelder, K., 2002. Menacing mold: the molecular biology of Aspergillus fumigatus. Ann. Rev. Microbiol. 56, 433–455. Brown, D.W., Adams, T.H., et al., 1996. Aspergillus has distinct fatty acid synthases for primary and secondary metabolism. Proc. Natl. Acad. Sci. USA 93, 14873–14877.

702

J. Sch¨umann, C. Hertweck / Journal of Biotechnology 124 (2006) 690–703

Brown, J.S., Aufauvre-Brown, A., et al., 1998. Insertional mutagenesis of Aspergillus fumigatus. Mol. Gen. Genet. 259 (3), 327– 335. Burkart, M.D., 2003. Metabolic engineering—a genetic toolbox for small molecule organic synthesis. Org. Biomol. Chem. 1, 1–4. Cane, D.E., Walsh, C.T., et al., 1998. Harnessing the biosynthetic code: combinations, permutations, and mutations. Science 282, 63–68. Case, M.E., Schweizer, M., et al., 1979. Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA. Proc. Natl. Acad. Sci. USA 76, 2563–5259. Chakraborty, B.N., Kapoor, M., 1990. Transformation of filamentous fungi by electroporation. Nucleic Acids Res. 18 (22), 6737. Chakraborty, B.N., Patterson, N.A., et al., 1991. An electroporationbased system for high-efficiency transformation of germinated conidia of filamentous fungi. Can. J. Microbiol. 37 (11), 858–863. Chang, P.K., Cary, J.W., et al., 1995. The Aspergillus parasiticus polyketide synthase gene pksA, a homolog of Aspergillus nidulans wA, is required for aflatoxin B1 biosynthesis. Mol. Gen. Genet. 248 (3), 270–277. Chu, F.S., 1991. Mycotoxins: food contamination, mechanism, carcinogenic potential and preventive measures. Mutat. Res. 259 (3–4), 291–306. Cox, R.J., Glod, F., et al., 2004. Rapid cloning and expression of a fungal polyketide synthase gene involved in squalestatin biosynthesis. Chem. Commun. 20, 2260–2261. Dawson, M.J., Farthing, J.E., et al., 1992. The squalestatins, novel inhibitors of squalene synthase produced by a species of Phoma. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activity. J. Antibiot. 45 (5), 639–647. de Groot, M.J., Bundock, P., et al., 1998. Agrobacterium tumefaciensmediated transformation of filamentous fungi. Nat. Biotechnol. 16 (9), 839–842. Eaton, D.L., Groopman, J.D., 1994. The Toxicology of Aflatoxins: Human Health, Veterinary, and Agricultural Significance. Academic Press, San Diego. Feng, G.H., Leonard, T.J., 1995. Characterization of the polyketide synthase gene (pksL1) required for aflatoxin biosynthesis in Aspergillus parasiticus. J. Bacteriol. 177 (21), 6246–6254. Feng, G.H., Leonard, T.J., 1998. Culture conditions control expression of the genes for aflatoxin and sterigmatocystin biosynthesis in Aspergillus parasiticus and A. nidulans. Appl. Environ. Microbiol. 64 (6), 2275–2277. Fujii, I., Mori, Y., et al., 2000. Enzymatic synthesis of 1,3,6,8tetrahydroxynaphthalene solely from malonyl coenzyme A by a fungal iterative type I polyketide synthase PKS1. Biochemistry 39 (30), 8853–8858. Fujii, I., Ono, Y., et al., 1996. Cloning of the polyketide synthase gene atX from Aspergillus terreus and its identification as the 6methylsalicylic acid synthase gene by heterologous expression. Mol. Gen. Genet. 253 (1–2), 1–10. Fujii, I., Watanabe, A., et al., 1998. Structures and functional analyses of fungal polyketide synthase genes. Actinomycetologica 12, 1–14. Fujii, I., Watanabe, A., et al., 2001. Identification of a Claisen cyclase domain in fungal polyketide synthase WA, a naphthopyrone synthase of Aspergillus nidulans. Chem. Biol. 8, 189–197.

Fulton, T.R., Ibrahim, N., et al., 1999. A melanin polyketide synthase (PKS) gene from Nodulisporium sp. that shows homology to the pks1 gene of Colletotrichum lagenarium. Mol. Gen. Genet. 262 (4–5), 714–720. Galagan, J.E., Calvo, S.E., et al., 2003. The genome sequence of the filamentous fungus Neurospora crassa. Nature 422 (6934), 859–868. Graziani, S., Vasnier, C., et al., 2004. Novel polyketide synthase from Nectria haemotococca. Appl. Environ. Microbiol. 70, 2984–2988. Grube, M., Blaha, J., 2003. On the phylogeny of some polyketide synthase genes in the lichenized genus Lecanora. Mycol. Res. 107, 1419–1426. Hendrickson, L., Davis, C.R., et al., 1999. Lovastatin biosynthesis in Aspergillus terreus: characterization of blocked mutants, enzyme activities and a multifunctional polyketide synthase gene. Chem. Biol. 6 (7), 429–439. Hutchinson, C.R., Kennedy, J., et al., 2000. Aspects of the biosynthesis of non-aromatic fungal polyketides by iterative polyketide synthases. Antonie van Leeuwenhoek 78, 287–295. Jahn, B., Koch, A., et al., 1997. Isolation and characterization of a pigmentless-conidium mutant of Aspergillus fumigatus with altered conidial surface and reduced virulence. Infect. Immun. 65 (12), 5110–5117. John, M.A., Peberdy, J.F., 1984. Transformatin of Aspergillus nidulans using the argB gene. Enzyme Microb. Technol. 6, 386–389. Kealey, J.T., Liu, L., et al., 1998. Production of a poyketide natural product in nonpolyketide-producing procaryotic and eukaryotic hosts. Proc. Natl. Acad. Sci. USA 95, 505–509. Keller, N.P., Brown, D., et al., 1995. A conserved polyketide mycotoxin gene cluster in Aspergillus nidulans. In: Eklund, M., Richard, R.L., Mise, K. (Eds.), Molecular Approaches to Food Safety Issues Involving Toxic Microorganisms. Alaken Inc., Fort Collins, pp. 263–277. Kempken, F., Jacobsen, S., et al., 1998. Distribution of the fungal transposon Restless: full-length and truncated copies in closely related strains. Fungal Genet. Biol. 25 (2), 110–118. Kempken, F., Kuck, U., 2000. Tagging of a nitrogen pathway-specific regulator gene in Tolypocladium inflatum by the transposon Restless. Mol. Gen. Genet. 263 (2), 302–308. Kennedy, J., Auclair, K., et al., 1999. Modulation of polyketide synthase activity by accessory proteins during lovastatin biosynthesis. Science 284, 1368–1372. Kroken, S., Glass, N.L., et al., 2003. Phylogenetic analysis of type I polyketide synthase genes in pathogenic and saprobic ascomycetes. Proc. Natl. Acad. Sci. USA 100, 15670–15675. Lee, T., Yun, S.H., et al., 2001. Polyketide synthase genes in insectand nematode-associated fungi. Appl. Microbiol. Biotechnol. 56 (1–2), 181–187. Leistner, L., 1984. Toxigenic penicillia occurring in feeds and foods: a review. Food Technol. Aust. 36 (9), 404–406. Linnemannstons, P., Schulte, J., et al., 2002. The polyketide synthase gene pks4 from Gibberella fujikuroi encodes a key enzyme in the biosynthesis of the red pigment bikaverin. Fungal Genet. Biol. 37 (2), 134–148. Maier, F.J., Malz, S., et al., 2005. Development of a highly efficient gene targeting system for Fusarium graminearum using the dis-

J. Sch¨umann, C. Hertweck / Journal of Biotechnology 124 (2006) 690–703 ruption of a polyketide synthase gene as a visible marker. FEMS Yeast Res. 5, 653–662. Minto, R.E., Townsend, C.A., 1997. Enzymology and molecular biology of aflatoxin biosynthesis. Chem. Rev. 97 (7), 2537– 2556. Moore, R.N., Gigam, G., et al., 1985. Biosynthesis of the hypercholesterolemic agent mevinolin by Aspergillus terreus: determination of the origin of carbon, hydrogen and oxygen atoms by carbon-13 NMR and mass spectrometory. J. Am. Chem. Soc. 107, 3694–3701. M¨uller, R., 2004. Don’t classify polyketide synthases. Chem. Biol. 11 (1), 4–6. Nakayashiki, H., Hanada, S., et al., 2005. RNA silencing as a tool for exploring gene function in ascomycete fungi. Fungal Genet. Biol. 42 (4), 275–283. Nicholson, T.P., Rudd, P.A.M., et al., 2001. Design and utlity of oligonucleotide gene probes for fungal polyketide synthases. Chem. Biol. 8, 157–178. Nierman, W.C., May, G., et al., 2005. What the Aspergillus genomes have told us. Med. Mycol. 43 (1), S3–S5. O’Callaghan, J., Caddick, M.X., et al., 2003. A polyketide synthase gene required for ochratoxin A biosynthesis in Aspergillus ochraceus. Microbiology 149, 3485–3491. O’Hagan, D., 1991. The Polyketide Metabolites. Ellis Horwood, Chichester. Ozeki, K., Kyoya, F., et al., 1994. Transformation of intact Aspergillus niger by electroporation. Biosci. Biotechnol. Biochem. 58 (12), 2224–2227. Partida-Martinez, L.P., Hertweck, C., 2005. Pathogenic fungus harbours endosymbiotic bacteria for toxin production. Nature 437, 884–888. Pfeifer, B.A., Khosla, C., 2001. Biosynthesis of polyketides in heterologous hosts. Microbiol. Mol. Biol. Rev. 65, 106– 118. Proctor, R.H., Desjardins, A.E., et al., 1999. A polyketide synthase gene required for biosynthesis of fumonisin mycotoxins in Gibberella fujikuroi mating population A. Fungal Genet. Biol. 27 (1), 100–112. Rawlings, B.J., 1999. Biosynthesis of polyketides (other than actinomycete macrolides). Nat. Prod. Rep. 16 (4), 425–484. Reeves, C.D., 2003. The enzymology of combinatorial biosynthesis. Crit. Rev. Biotechnol. 23, 95–147. Sanchez, O., Navarro, R.E., et al., 1998. Increased transformation frequency and tagging of developmental genes in Aspergillus nidulans by restriction enzyme-mediated integration (REMI). Mol. Gen. Genet. 258 (1–2), 89–94. Sauer, M., Lu, P., et al., 2002. Estimating polyketide metabolic potential among nonsporulating fungal endophytes of Vaccinium macrocarpon. Mycol. Res. 106, 460–470. Schmitt, I., Martin, M.P., et al., 2005. Diversity of non-reducing polyketide synthase genes in the Pertusariales (lichenized Ascomycota): a phylogenetic perspective. Phytochemistry 66 (11), 1241–1253. Shimizu, T., Kinoshita, H., et al., 2005. Polyketide synthase gene responsible for citrinin biosynthesis in Monascus purpureus. Appl. Environ. Microbiol. 71, 3453–3457.

703

Spencer, J.B., Jordan, P.M., 1992. Purification and properties of 6-methylsalicylic acid synthase from Penicillium patulum. Biochem. J. 288, 839–846. Staunton, J., Weissman, K.J., 2001. Polyketide biosynthesis: a millenium review. Nat. Prod. Rep. 18, 380–416. Sweeney, M.J., Dobson, A.D., 1999. Molecular biology of mycotoxin biosynthesis. FEMS Microbiol. Lett. 175 (2), 149–163. Takano, Y., Kubo, Y., et al., 1995. Structural analysis of PKS1, a polyketide synthase gene involved in melanin biosynthesis in Colletotrichum lagenarium. Mol. Gen. Genet. 249 (2), 162–167. Takano, Y., Kubo, Y., et al., 1997. The alternaria alternata melanin biosynthesis gene restores appressorial melanization and penetration of cellulose membranes in the melanin-deficient Albino mutant of Colletotrichum lagenarium. Fungal Genet. Biol. 21 (1), 131–140. Talbot, N., 2001. Molecular and Cellular Biology of Filamentous Fungi. Oxford University Press. Thierry, D., Vaucheret, H., 1996. Sequence homology requirements for transcriptional silencing of 35S transgenes and posttranscriptional silencing of nitrite reductase (trans)genes by the tobacco 271 locus. Plant Mol. Biol. 32 (6), 1075–1083. Tilburn, J., Scazzocchio, C., et al., 1983. Transformation by integration in Aspergillus nidulans. Gene 26, 205–221. Trail, F., Mahanti, N., et al., 1995. Molecular biology of aflatoxin biosynthesis. Microbiology 141, 755–765. Wagschal, K., Yoshizawa, Y., et al., 1996. Biosynthesis of ML-236C and the hypercholesterolemic agents compactin by Penicillium aurantiogriseum and lovastatin by Aspergillus terreus: determination of the origin of carbon, hydrogen and oxygen atoms by 13 C NMR spectrometry and observation of unusual labeling of acetate-derived oxygens by 18 O2 . J. Chem. Soc. Perkin Trans. 1 (1), 2357–2363. Walsh, C.T., 2002. Combinatorial biosynthesis of antibiotics: challenges and opportunities. ChemBioChem 3, 124–134. Weber, T., Welzel, K., et al., 2003. Exploiting the genetic potential of polyketide producing Streptomycetes. J. Biotechnol. 106, 221–232. Weidner, G., d’Enfert, C., et al., 1998. Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine 5 -monophosphate decarboxylase. Curr. Genet. 33 (5), 378–385. Wright, F., Bibb, M.J., 1992. Codon usage in the G + C-rich Streptomyces genome. Gene 113 (1), 55–65. Yang, G., Rose, M.S., et al., 1996. A polyketide synthase is required for fungal virulence and production of the polyketide T-toxins. Plant Cell 8, 2139–2150. Yelton, M.M., Hamer, J.E., et al., 1984. Transformation of Aspergillus nidulans by using a trpC plasmid. Proc. Natl. Acad. Sci. USA 81, 1470–1474. Yu, J., Chang, P.-K., et al., 1995. Comparative mapping of aflatoxin pathway gene clusters in Aspergillus parasiticus and Aspergillus flavus. Appl. Environ. Microbiol. 61 (6), 2365–2371. Zhang, A., Lu, P., et al., 2003. Efficient disruption of a polyketide synthase gene (pks1) required for melanin synthesis through Agrobacterium-mediated transformation of Glarea lozoyensis. Mol. Genet. Genom. 268 (5), 645–655.