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Penicillium Genomics
Applied Mycology& Biotechnology An International Series. Volume4. Fungal Genomics 9 2004 ElsevierB.V. All rights reserved
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Penicillium Genomics John C. Royer, Kevin T. Madden, Thea C. Norman, Katherine F. LoBuglio 1
Microbia, Inc., 320 Bent St., Cambridge MA 02142 U.S.A. [email protected]); IHarvard University Herbaria, 22 Divinity Ave., Cambridge, MA 02138 U.S.A.
Penicillium species exhibit wide variability in metabolite production, morphology, pathogenicity and lifestyle. Despite their economic importance, relatively little publicly available genomic analysis has been performed on Penicillium species. In this review, we summarize karyotype analysis of Penicillium species, and compare penicillin and statin gene clusters of Penicillium and closely related Aspergillus species. In addition, we present a preliminary genomic comparison between wild type and commercial penicillin producing strains of P. chrysogenum generated using genomic fragment microarrays. Finally, we summarize initial results of an EST sequencing project for the human pathogen P. marneffei. 1. INTRODUCTION The genus Penicillium comprises a ubiquitous group of fungi commonly found in soil, and as decomposers of various types of organic matter (Pitt 1985). The genus is characterized by the production of asexual spores (conidia) from verticels of phialides supported on a conidiophore of varying complexity termed the penicillus (Raper and Thom 1949; Pitt 1979). Pitt and Samson (1993) list 162 Penicillium species for which only asexual spores are produced, and 61 species that have a sexual cycle (either a Talaromyces or Eupenicillium teleomorph) as well as the asexual Penicillium state. Penicillium species are phylogenetically associated with the order of sexual ascomycete fungi known as the Eurotiales (Geiser and LoBuglio 2001). Phylogenetic analyses of DNA sequence data has demonstrated that anamorphic (asexual) genera such as Penicillium and its close relatives in the genus Aspergillus have not evolved independently as asexual lineages but rather have evolved multiple times from meiotic genera (Geiser et al. 1996; LoBuglio et al. 1993; Peterson et al. 1993). It has been estimated that Aspergillus and Penicillium diverged at least 60 million years ago based on 18S ribosomal DNA analysis under the constraint of a molecular clock (Berbee and Taylor 1993). Penicillium strains are important producers of secondary metabolites; many of which have been developed into antibiotics and other pharmaceuticals. Penicillium chrysogenum has been utilized for commercial production of penicillin for over 50 years. Intensive random mutagenesis and selection, coupled with improvements in fermentation processes have resulted in an increase of approximately 3 orders of magnitude in the titre of penicillin produced by P. chrysogenum (Backus and Stauffer 1955; Lein 1986; Demain and Elander 1999). Penicillium citrinum is
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currently utilized for production of the statin compactin, which is a precursor for the cholesterol lowering drug pravastatin (Endo et al. 1976; Brown et al. 1976). In addition, Penicillium strains are important in the food industry, both in food production and food contamination (Pitt 1985). One member of the genus, Penicillium marneffei, has become a well-recognized human pathogen in Southeast Asia (Wong et al. 1999). It typically infects immunocompromised individuals afflicted with diseases such as Hodgkin's disease, tuberculosis, or AIDS as well as those undergoing treatment with cortical steroids (Deng and Connor 1985, Hilmardottir et al. 1993, Viviani et al. 1993). Penicillium marneffei appears to be unique among Penicillium species in its capability to undergo a temperature-dependent dimorphic growth switch (Andrianopoulis 2002; Garrison and Boyd 1973). A symbiotic interaction occurs with another member of the genus, Penicillium nodositatum. This species has been shown to induce root nodule formation on roots of alder trees (Valla et al. 1989) and to exist as a neutral microsymbiont within the host tissue (Sequerra et al. 1995). Interestingly, RFLP analysis of the nuclear ribosomal DNA region containing the two internal transcribed spacers and 5.8S rRNA gene suggest that P. nodositatum is phylogenetically related to the Penicillium subgenus Biverticillium (Pitt 1979), which includes P. marneffei (LoBuglio and Taylor 1995; Sequerra et al. 1997). Thus, considerable variability with regard to metabolite production, pathogenicity, morphology, and lifestyle exists within the genus Penicillium. Genomic tools offer powerful methods for elucidating the genetic basis for these variations. Despite its importance, genomic analysis of Penicillium appears to have lagged behind that of other model, and economically important fungi. In this review, we summarize results on karyotype analysis of various Penicillium strains and compare structure of penicillin and statin gene clusters in Penicillium and Aspergillus strains. We also present preliminary genomic profiling analysis of penicillin production strains of P. chrysogenum, and briefly summarize initial results of an EST (expressed sequence tag) sequencing project underway for P. marneffei. 2. ELECTROPHORETIC KARYOTYPE AND GENOME SIZE Electrophoretic karyotype analysis has been performed on a number of Penicillium species (Table 1). Most strains examined, including P. chrysogenum possess 4 or 5 resolvable bands (Farber and Geisen 2000; Fierro et al. 1993; Chavez et al. 2001). Penicillium paxilli contains at least 6 chromosomes (Itoh et al. 1994) while the P. janthinellum genome has been resolved into 8-10 bands (Kayser and Shulz 1991). Genome sizes vary from 17.8-26.2 MB (P. marneffei) and 22.1 MB (P. purpurogenum) to 39-46 MB for P. janthinellum. Table 1. Electrophoretickaryotypesof various Penicillium species. Penicillium
Species P. chrysogenum P. notatum P. janthinellum P. paxilli P. nalgiovense P. purpurogenum P. marneffei
Chromosome Number 4 4 6-8 8 4 5 3-6
Chromosome Size Range (Mbp) 6.8-10.4 5.4-10.8 2.0-8.0 2.5-6.0 4.1-9.1 2.3-7.1 2.2-5.0
GenomeSize (Mbp) 34.1 32.1 39.0-49.0 23.4 26.5 21.2 17.8-26.2
Reference Fierro et al. 1993 Fierro et al. 1993 Kayser and Shulz 1991 Young et al. 1998 Farber and Geisen 2000 Chavez et al. 2001 Wong and Yuen, pers. comm., 2003
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3. SECONDARY METABOLITE GENE CLUSTERS Genes involved in the synthesis of complex secondary metabolites are often grouped together as clusters in filamentous fungi (Keller and Hohn 1997). This clustering has been proposed to be a remnant of horizontal gene transfer from prokaryotes, where genes involved in a particular function are often physically linked and transcribed together as operons (Landan et al. 1990; Penalva et al. 1990; Weigel et al. 1988). Clustering of secondary metabolite genes has also been proposed to facilitate coordinate regulation, and to allow for horizontal gene transfer between fungi, which may be particularly important in species that lack a sexual cycle (Keller and Hohn 1997). Walton (2000) proposed a "selfish cluster" hypothesis to explain the evolution and maintenance of secondary metabolite gene clusters in fungi. 3.1 Penicillin Clusters in Penicillium and Aspergillus Penicillins and cephalosporins are B-lactam containing antibiotics produced by filamentous
fungi from a number of genera. The penicillin gene cluster is composed of 3 genes; ACV synthase (pcbAB), IPN synthase (pcbC) and acyltransferase synthase (penDE) (Diez et al. 1990; Barredo et al. 1989a; Barredo et al. 1989b; Samson et al. 1985; Cart et al. 1986). These genes are clustered together in an approximately 20 kb region in both P. chrysogenum and A. nidulans (Fig.1.; Diez et al. 1990; MacCabe et al. 1990). Gene order and direction of transcription are maintained in these fungi, and protein identity is quite high; approximately 80% for the IPN synthetases. The degree of protein identity between the fungal genes and bacterial IPN synthetases is approximately 60%, and the fungal genes lack introns. These results have been cited as evidence for a horizontal gene transfer of this cluster from bacteria to fungi (Penalva et al. 1990; Weigel et al. 1988). Recently, Laich et al. (2002) demonstrated that the presence of the penicillin gene cluster is variable among Penicillium strains used in food production. Penicillium griseofulvum contains a complete gene cluster, P. verrucosom contains a truncated cluster, while many strains (including P. roquefortii and P. camembertii) lack any of the cluster genes. Interestingly, the cephalosporin production strain Acremonium chrysogenum contains two of the penicillin biosynthetic genes as a cluster. This partial cluster along with a second, separate cluster of genes is involved in synthesis of cephalosporin C.
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Industrial penicillin production strains generated by random mutagenesis have been shown to contain amplifications of a region of either 106.5 or 57.6 kb units, which contain the 3 penicillin biosynthetic genes, and are flanked by conserved hexanucleotide repeats (Fierro et al. 1995). Thus, the increased penicillin production capability of these strains is linked with increased copy number of the biosynthetic genes. The variation in presence and copy number of the penicillin
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cluster in wild type and production strains suggests great plasticity of this cluster (Gutierrez et al. 1999; Laich et al. 2002). 3.2 Statin Gene Clusters in Penicillium and Aspergillus
Members of the statin family of secondary metabolites are potent inhibitors of HMGCoA reductase, the key enzyme in cholesterol biosynthesis in humans. These polyketides have found utility for reduction of cholesterol, and natural and modified statins represent multibillion-dollar drugs. Penicillium citrinum produces the statin compactin, which is used as a substrate for microbial conversion to the commercial product pravastatin sodium. Aspergillus terreus naturally produces the statin lovastatin, which is identical to compactin except that a methyl group is present at the C-6 position (Moore et al. 1985). As with many fungal secondary metabolites, the genes for these polyketides are clustered together; and the gene clusters for both compactin and lovastatin have been cloned (Kennedy et al. 1999; Abe et al. 2002). As expected, many homologous gene products (with 57-75% identity) are shared between the two clusters. Identity between MlcR and LovE, transcription factors that regulate compactin and lovastatin production, respectively, is significantly lower at 34%. The presence of introns, which are lacking in the genes of the penicillin cluster, argues against a "recent" horizontal gene transfer from a prokaryotic source. The low level of homology between the proteins of the two clusters (similar to the 60% identity between the homologous nitrogen regulatory genes Nre of P. chrysogenum and AreA of A. nidulans, (Haas et al. 1995)) suggests that the cluster may have been present in the shared ancestor of these two fungi and has evolved considerably since the divergence of the genera Penicllium and Aspergillus. P450 Lovastatin cluster < (Kennedy et aL 1999)
Compactin cluster (Abe et a/. 2002)
NKS
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Fig. 2. Comparison of the compactin cluster of P. citrinum with the lovastatin cluster of A. terreus. Region A is flipped between the two clusters, while the relationship between region B is more complex. P450, P450 monooxygenase; NKS, nonaketide synthase; OX, oxidoreductase; DH, dehydrogenase; TE, transesterase; HMG, HMG-CoA reductase; TF, transcription factor; EP, effiux pump; DKS, diketide synthase.
While gene functions are conserved and the genes are maintained as clusters in these two fungi of differing genera, gene order and direction of transcription have been altered (Fig 2). Interestingly, the 5' portion of the two clusters (comprising 5 genes) maintains synteny if the orientation is flipped. The 3' end of the cluster can be restored to synteny by a more complex flipping and insertion. These results contrast with comparisons of penicillin clusters, where the degree of protein identity, as well as synteny within the cluster is maintained to a higher degree between Penicillium and Aspergillus species.
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4. RANDOM FRAGMENT GENOMIC ARRAYS TO CHARACTERIZE G E N O M E S OF PENICILLIN P R O D U C T I O N STRAINS
The lineage of stratus with varying penicillin production potentials generated during the penicillin strain improvement process represents a unique source of genetic material to examine directed evolution, and to identify genes whose modifications affect metabolite production. DNA microarray technology is a particularly well-suited tool for uncovering variations between closely related strains. Microarrays are often generated using DNA representing known open reading frames (where genomic sequence is available) or ESTs. Since neither sequence information nor ESTs were available for P. chrysogenum, we utilized a random fragment genomic microarray approach to assess the changes that have occurred within the Penicillium production strains (Fig. 3, see Askenazi et al. 2003 for experimental details). DNA from the progenitor strain (ATCC 9480, NRRL 1951) was isolated, digested to produce fragments of approximately 2 KB, and cloned. Approximately 13,000 PCR products were generated from the resulting fragment library using a common primer pair. In addition, PCR fragments were generated for all known P. chrysogenum genes. Resulting PCR products were purified and transferred to 384-well plates to generate the microarray.
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Microarrays are typically utilized to examine mRNA, ie the pattern of expression of genes. However, microarrays can also be utilized to directly compare the genomes of closely related strains. In the current study, the microarray of strain ATCC 9480 was utilized to compare the genomes of the starting, wild type strain and the improved P-2 strain (ATCC 48271, Lein 1986) of the P. chrysogenum production lineage. Genomic DNA was prepared from each strain, partially digested to a length of approximately 1 KB, and differentially labeled with fluorescent nucleotides (Cy3-dCTP or Cy5-dCTP) using a random priming reaction. Competitive microarray hybridizations were performed using DNA from the starting strain and differentially labeled DNA from the P2 strain. Wild type DNA was also competitively hybridized to itself to determine the experimental reproducibility. Genomic fragments that displayed signal intensities with absolute value log2 ratios >0.8 were considered significantly amplified or deleted, relative to the wild type strain, and were selected for sequence analysis (See Askenazi et al. 2003 for experimental details). A number of genomic fragments were identified which were associated with stronger
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hybridization signals in the improved P-2 strain. Not surprisingly, sequence analysis revealed that many of these genomic fragments contained genes of the penicillin biosynthetic cluster. These results were consistent with results of previous studies (Fierro et al. 1995) in which Southern blot hybridizations and sequencing techniques were used to show that the penicillin biosynthetic cluster has been amplified from 1 copy in the progenitor strain to 5 to 7 copies in the P-2 strain. The genomic fragment microarray approach has, in addition identified at least 2 more genes that are either contained within or closely linked to the 55kb amplified region and could be associated with penicillin production. These include a putative isoamyl alcohol oxidase, which could regulate formation of valine, a precursor of penicillin; and a zinc binuclear cluster transcription factor that could regulate transcription of genes involved in penicillin production. The microarray approach also identified a number of amplified genes that do not reside within the penicillin amplification unit, and might be important for penicillin production. Specific ABC transporter effiux pumps have been either amplified or deleted. This seemingly contradictory finding is plausible, since certain pumps could positively impact penicillin production by increasing penicillin effiux into the medium, while other pumps could negatively impact penicillin production by transporting precursors into the medium. Multiple amplified fragments were identified that encode a glucose transporter/sensor which may regulate the flux of carbon into central metabolism or the transition from primary to secondary metabolism. A fragment that contains a putative HMG CoA synthase; which can regulate flux into branch chain amino acid biosynthesis was also identified. Finally, two clones were found to contain sequence that suggests amplification of a transposon. It is possible that this transposon could be involved in the amplification of the penicillin biosynthetic cluster. Genomic profiling is well suited for detecting significant size deletions and insertions in wild type, and particularly in mutagenized production strains. Clearly, such gross changes can be detected in enhanced penicillin strains, and similarly, genomic rearrangements are expected to be associated with the increased production of other secondary metabolites. Furthermore, genomic profiling using genomic fragment microarrays can be employed in combination with gene expression studies, more sensitive genomic profiling methods, and sequencing efforts in order to gain a detailed understanding of the genetic control of secondary metabolite production. For example, in strains such as P. chrysogenum and P. citrinum, for which the secondary metabolite gene clusters have been cloned, but whole genomic sequence is lacking, genomic profiling is particularly well suited for identifying important genes that are not physically linked to the cluster. In uncharacterized systems, transcriptional profiling using random fragment microarrays can be used to rapidly identify biosynthetic genes that have not been previously identified (Askenazi et al. 2003). 5. P E N I C I L L I U M M A R N E F F E I EST SEQUENCING PROJECT An EST sequencing project has recently been initiated for P. marneffei (S. S. Y.Wong and K.
Y. Yuen, personal communication, 2003). Initial analysis of 2303 sequence tags has revealed an overall G+C content of 48.8%. More than 3.4% of the genes encode secondary metabolism genes for non-ribosomal peptide synthesis and polyketide synthesis; among these is a homologue of the lovastatin nonaketide synthase gene of A. terreus (Kennedy et al. 1999). This finding is particularly interesting, given the observation that plant pathogens also appear to contain more genes dedicated to secondary metabolism than do saprophytes (Yoder and Turgeon 2001). Significantly, the sequencing project has revealed the presence of homologues to mating type and pheromone genes in this asexual species. In addition, nuclear small (18S) and large (28S)
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ribosomal gene sequences were highly similar to ribosomal genes of Talaromyces. These results concur with a previous phylogeny study and suggest that if a cryptic sexual stage exists for P. marneffei it would most likely be a species of Talaromyces (LoBuglio and Taylor 1995). 6. C O N C L U S I O N S Examination of the penicillin gene cluster in P. chrysogenum and A. nidulans reveals a conservation of synteny, high degree of sequence similarity and lack of introns. A comparison of the two statin clusters; compactin in P. citrinum, and lovastatin in A. terreus, reveals a reduced level of protein identity, lack of synteny, and the presence of introns. These results are consistent with a relatively recent introduction of the penicillin cluster from a bacterial source, and a more ancient evolution of the statin clusters. Preliminary genomic profiling of P. chrysogenum in the current study has identified a number of genes that are amplified or deleted in the mutated, high producing strains. These include, but are not limited to the biosynthetic genes directly involved in penicillin production. Despites it's importance, relatively little publicly available genomic analysis has been performed with Penicillium species. Surprisingly, there is no publicly available sequence, or sequencing project underway for the penicillin production strain P. chrysogenum. Detailed results from the P. marneffei sequencing project are forthcoming, and will highlight potential benefits of genomic analyses of other Penicillium species. REFERENCES Abe Y, Suzuki T, Ono C, Iwamoto K, Hosobuchi M, Yoshikawa H (2002). Molecular cloning and characterization of an ML-236B (compactin) biosynthetic gene cluster in Penicillium citrinum. Mol. Genet. Genomics 267: 636646. Andrianopoulos A (2002). Control of morphogenesis in the human fungal pathogen Penicillium marneffei. Int J Med Microbio1292:331-347. Askenazi M, Driggers EM, Holtzman DA, Norman TC, Iverson S, Zimmer DP, Boers M, Blomquist PR, Martinez EJ, Monreal AW, Feibelman TP, Mayorga ME, Maxon ME, Sykes K, Tobin JV, Cordero E, Salama SR, Trueheart J, Royer JC, and Madden K (2003) Integratingtranscriptional and metabolite profiles to direct the engineering of lovastatin-producing strains. Nature Biotechnol. 21:1-7. Backus MP, Stauffer JF (1955). The production and selection of a family of strains in Penicillium chrysogenum. Mycologia 47: 429-463. Barredo, JL, Cantoral JM, Alvarez E, Diez B, and Martin JF (1989a). Cloning, sequence analysis and transcriptional study of the isopenicillin N synthase of Penicillium chrysogenum AS-P-78. Mol Gen Genet 216:91-98 Barredo JL, van Solingen P, Diez B, Alvarez E, Cantoral JM, KatevilderA, Smaal EB, Groenen MAM, Veenstra AE and Martin JF (1989b) Cloning and characterization of acyI-CoA:6-APA acyltransferasegene of Penicillium chrysogenum. Gene 83:291-300. Brown AG, Smale TC, King TJ, Hasenkamp R, Thompson RH (1976). Crystal and molecular structure of compactin, a new antifungal metabolite from Penicillium brevicompactin. J Chem Soc Perkin Trans 1:11651170. Berbee ML, and Taylor JW (1993). Datingthe evolutionaryradiations of the true fungi. Can. J Bot 71(8):11141127. Carr LG, Skatrud PL, Scheetz ME II, Queener SW and Ingolia TD. 1986. Cloning and expression of the isopenicillin N synthetase gene from Penicillium chrysogenum. Gene 48:257-266. Chavez R, Fierro F, Gordillo F, Martin JF, and Eyzaguirre J (2001). Electrophoretickaryotype of the filamentous fungus Penicilliumpurpurogenum and chromosomal location of several xylanolytic genes. FEMS Microbiol. Letters 205:379-383. Demain AL, and Elander RP (1999). The B-lactam antibiotics: past, present, and future. Antonie van Leeuwenhoek 75:5-19.
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Deng Z, and Connor DH (1985). Progressive disseminated penicilliosis caused by Penicillium marneffei. Am. J. Clin. Pathol. 84:323-327. Diez B, Gutierrez S, Barredo J, van Solingen P, van der Vort LHM, and Martin JF (1990). The cluster of penicillin biosynthetic genes. Identification and characterization of the pcb AB gene encoding the alpha-aminoadipylcysteinyl-valine synthetase and linkage to the pcbC and penDE genes. J Biol Chem 265:16358-16365 Endo A, Kuroda M, Tsujita M (1976). ML-236A, ML236B, and ML236C, new inhibitors of cholesterogenesis produced by Penicillium citrinum. J Antibiot 29: 1346-1348. Farber P, and Geisen R (2000). Karyotype of Penicillium nalgiovense and assignment of the penicillin biosynthetic genes to chromosome IV. lnt J of Food Microbiol. 58:59-63. Fierro F, Gutierrez S, Diez B, and Martin JF (1993). Resolution of four large chromosomes in penicillin-producing filamentous fungi: the penicillin gene cluster is located on chromosome II (9.6 Mb) in Penicillium notatum and chromosome I (10.4 Mb) in Penicillium chrysogenum. Mol Gen Genet 241:573-578. Fierro F, Barredo JL, Diez B, Gutierrez S, Fernandez F, and Martin JF (1995). The penicillin gene cluster is amplified in tandem repeats linked by conserved hexanucleotide sequences. Proc Natl Acad Sci 92: 62006204. Garrison RG, and Boyd KS (1973). Dimorphism of Penicillium marneffei as observed by electron microscopy. Can J Microbiol 19:1305-1309. Geiser DM, Timberlake WE, and Arnold ML (1996). Loss of Meiosis in Aspergillus. Mol. Biol. Evol. 13:809-817. Geiser DM, and LoBuglio KF (2001). The monophyletic Plectomycetes: Ascosphaerales, Onygenales, Eurotiales. In: McLaughlin, McLaughlin, Lemke, eds. The Mycota VII Part A, Systematics and Evolution. Berlin Heidelberg: Springer-Verlag, pp.201-219. Gutierrez S, Fierro F, Casqueiro J, and JF Martin (1999). Gene organization and plasticity of the B-lactam genes in different filamentous fungi. Antonie van Leeuwenhoek 75:81-94. Haas H, Bauer B, Redl B, Stoffler G, and MarzlufGA (1995). Molecular cloning and analysis ofnre, the major nitrogen regulatory gene of Penicillium chrysogenum. Curr Genet 27: 150-158. Hilmarsdottit I, Meynard JL, Rogeaux O, Guermonprez G, Datry A, Katlama C, Brucker G, Coutellier A, Danis M, and Gentilini M (1993). Disseminated Penicillium marneffei infection associated with human immunodeficiency virus: a report of two cases and a review of 35 published cases. J Acquired Immune Defic Syndr 6:466-471. Itoh Y, Johnson R, and Scott B (1994). Integrative transformation of the mycotoxin-producing fungus, Penicillium paxilli. Curr Genet 25:508-513. Kayser T, and Schulz G (1991). Electrophoretic karyotype of cellulolytic Penicillium janthinellum strains. Curr Genet 20:289-291. Keller NP, and Hohn TM (1997). Metabolic pathway gene clusters in filamentous fungi. Fungal Genetics and Biol 21:17-29. Kennedy J, Auclair K, Kendrew SG, Park C, Vederas JC, and Hutchinson CR (1999). Modulation of polyketide synthase activity by accessory proteins during lovastatin biosynthesis. Science 284:1368-1372. Laich F, Fierro F, and Martin JF (2002). Production of Penicillin by fungi growing on food products: Identification of a complete Penicillin gene cluster in Penicillium griseofulvum and a truncated cluster in Penicillium verrucosum. Applied and Environ Microbiol 68:1211-1219. Landan G, Cohen F, Aharonowitz Y, Shuali Y, Graur D, and Shiffman D (1990). Evolution of isopenicillin N synthase genes may have involved horizontal gene transfer. Mol Biol Evol 7: 399-406. Lein, J. 1986. The Panlabs penicillin strain improvement program, p. 105-139. In Z. Vanek and A. Hostakek (ed.), Overproduction of microbial metabolites. Butterworths, Boston, Mass. LoBuglio KF, Pitt JI, and Taylor JW (1993). Phylogentic analysis of two ribosomal DNA regions indicates multiple independent losses of a sexual Talaromyces state among asexual Penicillium species in subgenus Biverticillium. Mycologia 85:592-604. LoBuglio KF, and Taylor JW (1995). Phylogeny and PCR identification of the human pathogenic fungus Penicillium marneffei. J Clinical Microbio133:85-89. MacCabe AP, Riach MBR, Unkles SE and Kinghorn JR (1990) The Aspergillus nidulans npeA locus consists of three contiguous genes required for penicillin biosynthesis. EMBO J 9:279-287 Moore RN, Gigam G, Chan JK, Hogg AM, Nakashima TT, and Vederas JC (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 spectrometry. J Am Chem Soc 107:3694-3701. Penalva MA, Moya A, Dopazo J, and Ramon D (1990). Sequences of isopenicillin N synthetase genes suggest horizontal gene transfer from prokaryotes to eukaryotes. Proc R Soc London 241:164-169.
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Peterson SW (1993). Molecular genetic assessment of relatedness of PeniciUium subgenus Penicillium. In: DR Reynolds, and JW Taylor eds. The Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics. Wallingford: CAB International, pp 121-128. Pitt Jl (1979). The genus Penicillium and its Teleomorphic States Eupenicillium and Talaromyces. London: Academic Press. Pitt JI (1985). A Laboratory Guide to Common Penicillium Species. North Ryde: CSIRO Division of Food Processing. Pitt JI and Samson RA (1993). Trichocomaceae. In: W Greuter ed. Names in Current Use in the Families Trichocomaceae, Cladonaceae, Pinaceae, and Lemnaceae. Konigstein: Koeltz Scientififc Books, pp. 13-57. Raper KB, and Thom C (1949). A manual of Penicillia. Baltimore:Williams and Wilkins. Samson SM, Belagaje R, Blankenship DT, Chapman JL, Perry D, Skatrud PL, Frank RM, Abraham EP, Baldwin JE, Queener SE and lngolia TD (1985). Isolation, sequence determination and expression in E. coli of the isopenicillin N synthetase gene from Cephalosporium acremonium. Nature 318:191-194. Sequerra J, Capellano A, Gianinazzi, Pearson V, and Moiroud A (1995). Ultrastructure of cortical root cells of Alnus incana infected by Penicillium nodositatum. New Phytol 130:545-555. Sequerra J, Marmeisse IL Valla G, Normand P, Capellano A, and Moiroud A (1997). Taxonomic position and intraspecific variability of the nodule forming Penicillium nodositatum inferred from RFLP analysis of the ribosomal intergenic spacer and Random Amplified Polymorphic DNA. Mycol Res 101:465-472. Valla G, Capellano A, Hugueny R, and Moiroud A (1989). Penicillium nodositatum Valla, a new species inducing myconodules on Alnus roots. Plant Soil 114:142-146. Viviani MA, Hill JO, and Dixon DM (1993). Penicillium marneffei: dimorphisim and treatment. HV Bossche, FC Odds, and D Kerridge, eds. Dimorphic fungi in biology and medicine. New York: Plenum Press. Walton JD (2000). Horizontal gene transfer and the evolution of secondary metabolite gene clusters in fungi: An hypothesis. Fungal Genetics and Biol. 30:167-171. Wong SSY, Slau H, and Yuen KY (1999). Penicilliosis marneffei - West meets East J Med Microbiol 48: 973975. Weigel BJ, Burger SG, Chen VJ, Skatrud PL, Frolik CA, Queener SW, and Ingolia TD (1988). Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans. J Bacteriol 170:3817-3826. Yoder OC, and Turgeon BG (2001). Fungal genomics and pathogenicity. Current Opinions in Plant Biol 4:315-321.
11
Penicillium Genomics John C. Royer, Kevin T. Madden, Thea C. Norman, Katherine F. LoBuglio 1
Microbia, Inc., 320 Bent St., Cambridge MA 02142 U.S.A. [email protected]); IHarvard University Herbaria, 22 Divinity Ave., Cambridge, MA 02138 U.S.A.
Penicillium species exhibit wide variability in metabolite production, morphology, pathogenicity and lifestyle. Despite their economic importance, relatively little publicly available genomic analysis has been performed on Penicillium species. In this review, we summarize karyotype analysis of Penicillium species, and compare penicillin and statin gene clusters of Penicillium and closely related Aspergillus species. In addition, we present a preliminary genomic comparison between wild type and commercial penicillin producing strains of P. chrysogenum generated using genomic fragment microarrays. Finally, we summarize initial results of an EST sequencing project for the human pathogen P. marneffei. 1. INTRODUCTION The genus Penicillium comprises a ubiquitous group of fungi commonly found in soil, and as decomposers of various types of organic matter (Pitt 1985). The genus is characterized by the production of asexual spores (conidia) from verticels of phialides supported on a conidiophore of varying complexity termed the penicillus (Raper and Thom 1949; Pitt 1979). Pitt and Samson (1993) list 162 Penicillium species for which only asexual spores are produced, and 61 species that have a sexual cycle (either a Talaromyces or Eupenicillium teleomorph) as well as the asexual Penicillium state. Penicillium species are phylogenetically associated with the order of sexual ascomycete fungi known as the Eurotiales (Geiser and LoBuglio 2001). Phylogenetic analyses of DNA sequence data has demonstrated that anamorphic (asexual) genera such as Penicillium and its close relatives in the genus Aspergillus have not evolved independently as asexual lineages but rather have evolved multiple times from meiotic genera (Geiser et al. 1996; LoBuglio et al. 1993; Peterson et al. 1993). It has been estimated that Aspergillus and Penicillium diverged at least 60 million years ago based on 18S ribosomal DNA analysis under the constraint of a molecular clock (Berbee and Taylor 1993). Penicillium strains are important producers of secondary metabolites; many of which have been developed into antibiotics and other pharmaceuticals. Penicillium chrysogenum has been utilized for commercial production of penicillin for over 50 years. Intensive random mutagenesis and selection, coupled with improvements in fermentation processes have resulted in an increase of approximately 3 orders of magnitude in the titre of penicillin produced by P. chrysogenum (Backus and Stauffer 1955; Lein 1986; Demain and Elander 1999). Penicillium citrinum is
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currently utilized for production of the statin compactin, which is a precursor for the cholesterol lowering drug pravastatin (Endo et al. 1976; Brown et al. 1976). In addition, Penicillium strains are important in the food industry, both in food production and food contamination (Pitt 1985). One member of the genus, Penicillium marneffei, has become a well-recognized human pathogen in Southeast Asia (Wong et al. 1999). It typically infects immunocompromised individuals afflicted with diseases such as Hodgkin's disease, tuberculosis, or AIDS as well as those undergoing treatment with cortical steroids (Deng and Connor 1985, Hilmardottir et al. 1993, Viviani et al. 1993). Penicillium marneffei appears to be unique among Penicillium species in its capability to undergo a temperature-dependent dimorphic growth switch (Andrianopoulis 2002; Garrison and Boyd 1973). A symbiotic interaction occurs with another member of the genus, Penicillium nodositatum. This species has been shown to induce root nodule formation on roots of alder trees (Valla et al. 1989) and to exist as a neutral microsymbiont within the host tissue (Sequerra et al. 1995). Interestingly, RFLP analysis of the nuclear ribosomal DNA region containing the two internal transcribed spacers and 5.8S rRNA gene suggest that P. nodositatum is phylogenetically related to the Penicillium subgenus Biverticillium (Pitt 1979), which includes P. marneffei (LoBuglio and Taylor 1995; Sequerra et al. 1997). Thus, considerable variability with regard to metabolite production, pathogenicity, morphology, and lifestyle exists within the genus Penicillium. Genomic tools offer powerful methods for elucidating the genetic basis for these variations. Despite its importance, genomic analysis of Penicillium appears to have lagged behind that of other model, and economically important fungi. In this review, we summarize results on karyotype analysis of various Penicillium strains and compare structure of penicillin and statin gene clusters in Penicillium and Aspergillus strains. We also present preliminary genomic profiling analysis of penicillin production strains of P. chrysogenum, and briefly summarize initial results of an EST (expressed sequence tag) sequencing project underway for P. marneffei. 2. ELECTROPHORETIC KARYOTYPE AND GENOME SIZE Electrophoretic karyotype analysis has been performed on a number of Penicillium species (Table 1). Most strains examined, including P. chrysogenum possess 4 or 5 resolvable bands (Farber and Geisen 2000; Fierro et al. 1993; Chavez et al. 2001). Penicillium paxilli contains at least 6 chromosomes (Itoh et al. 1994) while the P. janthinellum genome has been resolved into 8-10 bands (Kayser and Shulz 1991). Genome sizes vary from 17.8-26.2 MB (P. marneffei) and 22.1 MB (P. purpurogenum) to 39-46 MB for P. janthinellum. Table 1. Electrophoretickaryotypesof various Penicillium species. Penicillium
Species P. chrysogenum P. notatum P. janthinellum P. paxilli P. nalgiovense P. purpurogenum P. marneffei
Chromosome Number 4 4 6-8 8 4 5 3-6
Chromosome Size Range (Mbp) 6.8-10.4 5.4-10.8 2.0-8.0 2.5-6.0 4.1-9.1 2.3-7.1 2.2-5.0
GenomeSize (Mbp) 34.1 32.1 39.0-49.0 23.4 26.5 21.2 17.8-26.2
Reference Fierro et al. 1993 Fierro et al. 1993 Kayser and Shulz 1991 Young et al. 1998 Farber and Geisen 2000 Chavez et al. 2001 Wong and Yuen, pers. comm., 2003
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3. SECONDARY METABOLITE GENE CLUSTERS Genes involved in the synthesis of complex secondary metabolites are often grouped together as clusters in filamentous fungi (Keller and Hohn 1997). This clustering has been proposed to be a remnant of horizontal gene transfer from prokaryotes, where genes involved in a particular function are often physically linked and transcribed together as operons (Landan et al. 1990; Penalva et al. 1990; Weigel et al. 1988). Clustering of secondary metabolite genes has also been proposed to facilitate coordinate regulation, and to allow for horizontal gene transfer between fungi, which may be particularly important in species that lack a sexual cycle (Keller and Hohn 1997). Walton (2000) proposed a "selfish cluster" hypothesis to explain the evolution and maintenance of secondary metabolite gene clusters in fungi. 3.1 Penicillin Clusters in Penicillium and Aspergillus Penicillins and cephalosporins are B-lactam containing antibiotics produced by filamentous
fungi from a number of genera. The penicillin gene cluster is composed of 3 genes; ACV synthase (pcbAB), IPN synthase (pcbC) and acyltransferase synthase (penDE) (Diez et al. 1990; Barredo et al. 1989a; Barredo et al. 1989b; Samson et al. 1985; Cart et al. 1986). These genes are clustered together in an approximately 20 kb region in both P. chrysogenum and A. nidulans (Fig.1.; Diez et al. 1990; MacCabe et al. 1990). Gene order and direction of transcription are maintained in these fungi, and protein identity is quite high; approximately 80% for the IPN synthetases. The degree of protein identity between the fungal genes and bacterial IPN synthetases is approximately 60%, and the fungal genes lack introns. These results have been cited as evidence for a horizontal gene transfer of this cluster from bacteria to fungi (Penalva et al. 1990; Weigel et al. 1988). Recently, Laich et al. (2002) demonstrated that the presence of the penicillin gene cluster is variable among Penicillium strains used in food production. Penicillium griseofulvum contains a complete gene cluster, P. verrucosom contains a truncated cluster, while many strains (including P. roquefortii and P. camembertii) lack any of the cluster genes. Interestingly, the cephalosporin production strain Acremonium chrysogenum contains two of the penicillin biosynthetic genes as a cluster. This partial cluster along with a second, separate cluster of genes is involved in synthesis of cephalosporin C.
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Industrial penicillin production strains generated by random mutagenesis have been shown to contain amplifications of a region of either 106.5 or 57.6 kb units, which contain the 3 penicillin biosynthetic genes, and are flanked by conserved hexanucleotide repeats (Fierro et al. 1995). Thus, the increased penicillin production capability of these strains is linked with increased copy number of the biosynthetic genes. The variation in presence and copy number of the penicillin
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cluster in wild type and production strains suggests great plasticity of this cluster (Gutierrez et al. 1999; Laich et al. 2002). 3.2 Statin Gene Clusters in Penicillium and Aspergillus
Members of the statin family of secondary metabolites are potent inhibitors of HMGCoA reductase, the key enzyme in cholesterol biosynthesis in humans. These polyketides have found utility for reduction of cholesterol, and natural and modified statins represent multibillion-dollar drugs. Penicillium citrinum produces the statin compactin, which is used as a substrate for microbial conversion to the commercial product pravastatin sodium. Aspergillus terreus naturally produces the statin lovastatin, which is identical to compactin except that a methyl group is present at the C-6 position (Moore et al. 1985). As with many fungal secondary metabolites, the genes for these polyketides are clustered together; and the gene clusters for both compactin and lovastatin have been cloned (Kennedy et al. 1999; Abe et al. 2002). As expected, many homologous gene products (with 57-75% identity) are shared between the two clusters. Identity between MlcR and LovE, transcription factors that regulate compactin and lovastatin production, respectively, is significantly lower at 34%. The presence of introns, which are lacking in the genes of the penicillin cluster, argues against a "recent" horizontal gene transfer from a prokaryotic source. The low level of homology between the proteins of the two clusters (similar to the 60% identity between the homologous nitrogen regulatory genes Nre of P. chrysogenum and AreA of A. nidulans, (Haas et al. 1995)) suggests that the cluster may have been present in the shared ancestor of these two fungi and has evolved considerably since the divergence of the genera Penicllium and Aspergillus. P450 Lovastatin cluster < (Kennedy et aL 1999)
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Fig. 2. Comparison of the compactin cluster of P. citrinum with the lovastatin cluster of A. terreus. Region A is flipped between the two clusters, while the relationship between region B is more complex. P450, P450 monooxygenase; NKS, nonaketide synthase; OX, oxidoreductase; DH, dehydrogenase; TE, transesterase; HMG, HMG-CoA reductase; TF, transcription factor; EP, effiux pump; DKS, diketide synthase.
While gene functions are conserved and the genes are maintained as clusters in these two fungi of differing genera, gene order and direction of transcription have been altered (Fig 2). Interestingly, the 5' portion of the two clusters (comprising 5 genes) maintains synteny if the orientation is flipped. The 3' end of the cluster can be restored to synteny by a more complex flipping and insertion. These results contrast with comparisons of penicillin clusters, where the degree of protein identity, as well as synteny within the cluster is maintained to a higher degree between Penicillium and Aspergillus species.
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4. RANDOM FRAGMENT GENOMIC ARRAYS TO CHARACTERIZE G E N O M E S OF PENICILLIN P R O D U C T I O N STRAINS
The lineage of stratus with varying penicillin production potentials generated during the penicillin strain improvement process represents a unique source of genetic material to examine directed evolution, and to identify genes whose modifications affect metabolite production. DNA microarray technology is a particularly well-suited tool for uncovering variations between closely related strains. Microarrays are often generated using DNA representing known open reading frames (where genomic sequence is available) or ESTs. Since neither sequence information nor ESTs were available for P. chrysogenum, we utilized a random fragment genomic microarray approach to assess the changes that have occurred within the Penicillium production strains (Fig. 3, see Askenazi et al. 2003 for experimental details). DNA from the progenitor strain (ATCC 9480, NRRL 1951) was isolated, digested to produce fragments of approximately 2 KB, and cloned. Approximately 13,000 PCR products were generated from the resulting fragment library using a common primer pair. In addition, PCR fragments were generated for all known P. chrysogenum genes. Resulting PCR products were purified and transferred to 384-well plates to generate the microarray.
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Microarrays are typically utilized to examine mRNA, ie the pattern of expression of genes. However, microarrays can also be utilized to directly compare the genomes of closely related strains. In the current study, the microarray of strain ATCC 9480 was utilized to compare the genomes of the starting, wild type strain and the improved P-2 strain (ATCC 48271, Lein 1986) of the P. chrysogenum production lineage. Genomic DNA was prepared from each strain, partially digested to a length of approximately 1 KB, and differentially labeled with fluorescent nucleotides (Cy3-dCTP or Cy5-dCTP) using a random priming reaction. Competitive microarray hybridizations were performed using DNA from the starting strain and differentially labeled DNA from the P2 strain. Wild type DNA was also competitively hybridized to itself to determine the experimental reproducibility. Genomic fragments that displayed signal intensities with absolute value log2 ratios >0.8 were considered significantly amplified or deleted, relative to the wild type strain, and were selected for sequence analysis (See Askenazi et al. 2003 for experimental details). A number of genomic fragments were identified which were associated with stronger
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hybridization signals in the improved P-2 strain. Not surprisingly, sequence analysis revealed that many of these genomic fragments contained genes of the penicillin biosynthetic cluster. These results were consistent with results of previous studies (Fierro et al. 1995) in which Southern blot hybridizations and sequencing techniques were used to show that the penicillin biosynthetic cluster has been amplified from 1 copy in the progenitor strain to 5 to 7 copies in the P-2 strain. The genomic fragment microarray approach has, in addition identified at least 2 more genes that are either contained within or closely linked to the 55kb amplified region and could be associated with penicillin production. These include a putative isoamyl alcohol oxidase, which could regulate formation of valine, a precursor of penicillin; and a zinc binuclear cluster transcription factor that could regulate transcription of genes involved in penicillin production. The microarray approach also identified a number of amplified genes that do not reside within the penicillin amplification unit, and might be important for penicillin production. Specific ABC transporter effiux pumps have been either amplified or deleted. This seemingly contradictory finding is plausible, since certain pumps could positively impact penicillin production by increasing penicillin effiux into the medium, while other pumps could negatively impact penicillin production by transporting precursors into the medium. Multiple amplified fragments were identified that encode a glucose transporter/sensor which may regulate the flux of carbon into central metabolism or the transition from primary to secondary metabolism. A fragment that contains a putative HMG CoA synthase; which can regulate flux into branch chain amino acid biosynthesis was also identified. Finally, two clones were found to contain sequence that suggests amplification of a transposon. It is possible that this transposon could be involved in the amplification of the penicillin biosynthetic cluster. Genomic profiling is well suited for detecting significant size deletions and insertions in wild type, and particularly in mutagenized production strains. Clearly, such gross changes can be detected in enhanced penicillin strains, and similarly, genomic rearrangements are expected to be associated with the increased production of other secondary metabolites. Furthermore, genomic profiling using genomic fragment microarrays can be employed in combination with gene expression studies, more sensitive genomic profiling methods, and sequencing efforts in order to gain a detailed understanding of the genetic control of secondary metabolite production. For example, in strains such as P. chrysogenum and P. citrinum, for which the secondary metabolite gene clusters have been cloned, but whole genomic sequence is lacking, genomic profiling is particularly well suited for identifying important genes that are not physically linked to the cluster. In uncharacterized systems, transcriptional profiling using random fragment microarrays can be used to rapidly identify biosynthetic genes that have not been previously identified (Askenazi et al. 2003). 5. P E N I C I L L I U M M A R N E F F E I EST SEQUENCING PROJECT An EST sequencing project has recently been initiated for P. marneffei (S. S. Y.Wong and K.
Y. Yuen, personal communication, 2003). Initial analysis of 2303 sequence tags has revealed an overall G+C content of 48.8%. More than 3.4% of the genes encode secondary metabolism genes for non-ribosomal peptide synthesis and polyketide synthesis; among these is a homologue of the lovastatin nonaketide synthase gene of A. terreus (Kennedy et al. 1999). This finding is particularly interesting, given the observation that plant pathogens also appear to contain more genes dedicated to secondary metabolism than do saprophytes (Yoder and Turgeon 2001). Significantly, the sequencing project has revealed the presence of homologues to mating type and pheromone genes in this asexual species. In addition, nuclear small (18S) and large (28S)
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ribosomal gene sequences were highly similar to ribosomal genes of Talaromyces. These results concur with a previous phylogeny study and suggest that if a cryptic sexual stage exists for P. marneffei it would most likely be a species of Talaromyces (LoBuglio and Taylor 1995). 6. C O N C L U S I O N S Examination of the penicillin gene cluster in P. chrysogenum and A. nidulans reveals a conservation of synteny, high degree of sequence similarity and lack of introns. A comparison of the two statin clusters; compactin in P. citrinum, and lovastatin in A. terreus, reveals a reduced level of protein identity, lack of synteny, and the presence of introns. These results are consistent with a relatively recent introduction of the penicillin cluster from a bacterial source, and a more ancient evolution of the statin clusters. Preliminary genomic profiling of P. chrysogenum in the current study has identified a number of genes that are amplified or deleted in the mutated, high producing strains. These include, but are not limited to the biosynthetic genes directly involved in penicillin production. Despites it's importance, relatively little publicly available genomic analysis has been performed with Penicillium species. Surprisingly, there is no publicly available sequence, or sequencing project underway for the penicillin production strain P. chrysogenum. Detailed results from the P. marneffei sequencing project are forthcoming, and will highlight potential benefits of genomic analyses of other Penicillium species. REFERENCES Abe Y, Suzuki T, Ono C, Iwamoto K, Hosobuchi M, Yoshikawa H (2002). Molecular cloning and characterization of an ML-236B (compactin) biosynthetic gene cluster in Penicillium citrinum. Mol. Genet. Genomics 267: 636646. Andrianopoulos A (2002). Control of morphogenesis in the human fungal pathogen Penicillium marneffei. Int J Med Microbio1292:331-347. Askenazi M, Driggers EM, Holtzman DA, Norman TC, Iverson S, Zimmer DP, Boers M, Blomquist PR, Martinez EJ, Monreal AW, Feibelman TP, Mayorga ME, Maxon ME, Sykes K, Tobin JV, Cordero E, Salama SR, Trueheart J, Royer JC, and Madden K (2003) Integratingtranscriptional and metabolite profiles to direct the engineering of lovastatin-producing strains. Nature Biotechnol. 21:1-7. Backus MP, Stauffer JF (1955). The production and selection of a family of strains in Penicillium chrysogenum. Mycologia 47: 429-463. Barredo, JL, Cantoral JM, Alvarez E, Diez B, and Martin JF (1989a). Cloning, sequence analysis and transcriptional study of the isopenicillin N synthase of Penicillium chrysogenum AS-P-78. Mol Gen Genet 216:91-98 Barredo JL, van Solingen P, Diez B, Alvarez E, Cantoral JM, KatevilderA, Smaal EB, Groenen MAM, Veenstra AE and Martin JF (1989b) Cloning and characterization of acyI-CoA:6-APA acyltransferasegene of Penicillium chrysogenum. Gene 83:291-300. Brown AG, Smale TC, King TJ, Hasenkamp R, Thompson RH (1976). Crystal and molecular structure of compactin, a new antifungal metabolite from Penicillium brevicompactin. J Chem Soc Perkin Trans 1:11651170. Berbee ML, and Taylor JW (1993). Datingthe evolutionaryradiations of the true fungi. Can. J Bot 71(8):11141127. Carr LG, Skatrud PL, Scheetz ME II, Queener SW and Ingolia TD. 1986. Cloning and expression of the isopenicillin N synthetase gene from Penicillium chrysogenum. Gene 48:257-266. Chavez R, Fierro F, Gordillo F, Martin JF, and Eyzaguirre J (2001). Electrophoretickaryotype of the filamentous fungus Penicilliumpurpurogenum and chromosomal location of several xylanolytic genes. FEMS Microbiol. Letters 205:379-383. Demain AL, and Elander RP (1999). The B-lactam antibiotics: past, present, and future. Antonie van Leeuwenhoek 75:5-19.
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Deng Z, and Connor DH (1985). Progressive disseminated penicilliosis caused by Penicillium marneffei. Am. J. Clin. Pathol. 84:323-327. Diez B, Gutierrez S, Barredo J, van Solingen P, van der Vort LHM, and Martin JF (1990). The cluster of penicillin biosynthetic genes. Identification and characterization of the pcb AB gene encoding the alpha-aminoadipylcysteinyl-valine synthetase and linkage to the pcbC and penDE genes. J Biol Chem 265:16358-16365 Endo A, Kuroda M, Tsujita M (1976). ML-236A, ML236B, and ML236C, new inhibitors of cholesterogenesis produced by Penicillium citrinum. J Antibiot 29: 1346-1348. Farber P, and Geisen R (2000). Karyotype of Penicillium nalgiovense and assignment of the penicillin biosynthetic genes to chromosome IV. lnt J of Food Microbiol. 58:59-63. Fierro F, Gutierrez S, Diez B, and Martin JF (1993). Resolution of four large chromosomes in penicillin-producing filamentous fungi: the penicillin gene cluster is located on chromosome II (9.6 Mb) in Penicillium notatum and chromosome I (10.4 Mb) in Penicillium chrysogenum. Mol Gen Genet 241:573-578. Fierro F, Barredo JL, Diez B, Gutierrez S, Fernandez F, and Martin JF (1995). The penicillin gene cluster is amplified in tandem repeats linked by conserved hexanucleotide sequences. Proc Natl Acad Sci 92: 62006204. Garrison RG, and Boyd KS (1973). Dimorphism of Penicillium marneffei as observed by electron microscopy. Can J Microbiol 19:1305-1309. Geiser DM, Timberlake WE, and Arnold ML (1996). Loss of Meiosis in Aspergillus. Mol. Biol. Evol. 13:809-817. Geiser DM, and LoBuglio KF (2001). The monophyletic Plectomycetes: Ascosphaerales, Onygenales, Eurotiales. In: McLaughlin, McLaughlin, Lemke, eds. The Mycota VII Part A, Systematics and Evolution. Berlin Heidelberg: Springer-Verlag, pp.201-219. Gutierrez S, Fierro F, Casqueiro J, and JF Martin (1999). Gene organization and plasticity of the B-lactam genes in different filamentous fungi. Antonie van Leeuwenhoek 75:81-94. Haas H, Bauer B, Redl B, Stoffler G, and MarzlufGA (1995). Molecular cloning and analysis ofnre, the major nitrogen regulatory gene of Penicillium chrysogenum. Curr Genet 27: 150-158. Hilmarsdottit I, Meynard JL, Rogeaux O, Guermonprez G, Datry A, Katlama C, Brucker G, Coutellier A, Danis M, and Gentilini M (1993). Disseminated Penicillium marneffei infection associated with human immunodeficiency virus: a report of two cases and a review of 35 published cases. J Acquired Immune Defic Syndr 6:466-471. Itoh Y, Johnson R, and Scott B (1994). Integrative transformation of the mycotoxin-producing fungus, Penicillium paxilli. Curr Genet 25:508-513. Kayser T, and Schulz G (1991). Electrophoretic karyotype of cellulolytic Penicillium janthinellum strains. Curr Genet 20:289-291. Keller NP, and Hohn TM (1997). Metabolic pathway gene clusters in filamentous fungi. Fungal Genetics and Biol 21:17-29. Kennedy J, Auclair K, Kendrew SG, Park C, Vederas JC, and Hutchinson CR (1999). Modulation of polyketide synthase activity by accessory proteins during lovastatin biosynthesis. Science 284:1368-1372. Laich F, Fierro F, and Martin JF (2002). Production of Penicillin by fungi growing on food products: Identification of a complete Penicillin gene cluster in Penicillium griseofulvum and a truncated cluster in Penicillium verrucosum. Applied and Environ Microbiol 68:1211-1219. Landan G, Cohen F, Aharonowitz Y, Shuali Y, Graur D, and Shiffman D (1990). Evolution of isopenicillin N synthase genes may have involved horizontal gene transfer. Mol Biol Evol 7: 399-406. Lein, J. 1986. The Panlabs penicillin strain improvement program, p. 105-139. In Z. Vanek and A. Hostakek (ed.), Overproduction of microbial metabolites. Butterworths, Boston, Mass. LoBuglio KF, Pitt JI, and Taylor JW (1993). Phylogentic analysis of two ribosomal DNA regions indicates multiple independent losses of a sexual Talaromyces state among asexual Penicillium species in subgenus Biverticillium. Mycologia 85:592-604. LoBuglio KF, and Taylor JW (1995). Phylogeny and PCR identification of the human pathogenic fungus Penicillium marneffei. J Clinical Microbio133:85-89. MacCabe AP, Riach MBR, Unkles SE and Kinghorn JR (1990) The Aspergillus nidulans npeA locus consists of three contiguous genes required for penicillin biosynthesis. EMBO J 9:279-287 Moore RN, Gigam G, Chan JK, Hogg AM, Nakashima TT, and Vederas JC (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 spectrometry. J Am Chem Soc 107:3694-3701. Penalva MA, Moya A, Dopazo J, and Ramon D (1990). Sequences of isopenicillin N synthetase genes suggest horizontal gene transfer from prokaryotes to eukaryotes. Proc R Soc London 241:164-169.
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Peterson SW (1993). Molecular genetic assessment of relatedness of PeniciUium subgenus Penicillium. In: DR Reynolds, and JW Taylor eds. The Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics. Wallingford: CAB International, pp 121-128. Pitt Jl (1979). The genus Penicillium and its Teleomorphic States Eupenicillium and Talaromyces. London: Academic Press. Pitt JI (1985). A Laboratory Guide to Common Penicillium Species. North Ryde: CSIRO Division of Food Processing. Pitt JI and Samson RA (1993). Trichocomaceae. In: W Greuter ed. Names in Current Use in the Families Trichocomaceae, Cladonaceae, Pinaceae, and Lemnaceae. Konigstein: Koeltz Scientififc Books, pp. 13-57. Raper KB, and Thom C (1949). A manual of Penicillia. Baltimore:Williams and Wilkins. Samson SM, Belagaje R, Blankenship DT, Chapman JL, Perry D, Skatrud PL, Frank RM, Abraham EP, Baldwin JE, Queener SE and lngolia TD (1985). Isolation, sequence determination and expression in E. coli of the isopenicillin N synthetase gene from Cephalosporium acremonium. Nature 318:191-194. Sequerra J, Capellano A, Gianinazzi, Pearson V, and Moiroud A (1995). Ultrastructure of cortical root cells of Alnus incana infected by Penicillium nodositatum. New Phytol 130:545-555. Sequerra J, Marmeisse IL Valla G, Normand P, Capellano A, and Moiroud A (1997). Taxonomic position and intraspecific variability of the nodule forming Penicillium nodositatum inferred from RFLP analysis of the ribosomal intergenic spacer and Random Amplified Polymorphic DNA. Mycol Res 101:465-472. Valla G, Capellano A, Hugueny R, and Moiroud A (1989). Penicillium nodositatum Valla, a new species inducing myconodules on Alnus roots. Plant Soil 114:142-146. Viviani MA, Hill JO, and Dixon DM (1993). Penicillium marneffei: dimorphisim and treatment. HV Bossche, FC Odds, and D Kerridge, eds. Dimorphic fungi in biology and medicine. New York: Plenum Press. Walton JD (2000). Horizontal gene transfer and the evolution of secondary metabolite gene clusters in fungi: An hypothesis. Fungal Genetics and Biol. 30:167-171. Wong SSY, Slau H, and Yuen KY (1999). Penicilliosis marneffei - West meets East J Med Microbiol 48: 973975. Weigel BJ, Burger SG, Chen VJ, Skatrud PL, Frolik CA, Queener SW, and Ingolia TD (1988). Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans. J Bacteriol 170:3817-3826. Yoder OC, and Turgeon BG (2001). Fungal genomics and pathogenicity. Current Opinions in Plant Biol 4:315-321.