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Efficient disruption of a polyketide synthase gene (pks1) required for melanin synthesis through Agrobacterium-mediated transformation of Glarea lozoyensis

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Abstract

Glarea lozoyensis produces pneumocandin B0, a potent inhibitor of fungal glucan synthesis. This industrially important filamentous fungus is slow-growing, is very darkly pigmented, and has not been easy to manipulate genetically. Using a PCR strategy to survey the G. lozoyensis genome for polyketide synthase (PKS) genes, we have identified pks1, a gene that consists of five exons interrupted by four introns of 56, 400, 50 and 341 bp. It encodes a 2124-amino acid protein with five catalytic modules: ketosynthase, acyltransferase, two acyl carrier sites, and thioesterase/Claisen cyclase. The transcriptional initiation and termination sites were found 43 bp upstream of the translational start codon and 295 bp downstream of the translational stop codon, respectively. Cluster analysis of 37 fungal ketosynthase modules grouped the Pks1p with PKSs involved in the biosynthesis of 1,8-dihydroxynaphthalene melanin. Disruption of pks1 yielded knockout mutants that displayed an albino phenotype, suggesting that pks1 encodes a tetrahydroxynaphthalene synthase. Gene replacement was achieved by Agrobacterium-mediated transformation, which proved to be simple and efficient. Loss of pigmentation occurred in more than half the transformants, and examination of six non-pigmented transformants showed that the functional genomic copy of the pks1 gene had been replaced by the disruption cassette in each case. A putative 1215-bp ORF (dsg) devoid of introns was present downstream from pks1. BLAST analysis of the 405-amino acid sequence of its predicted product showed a high degree of similarity with Zn(II)2Cys6 binuclear cluster DNA-binding proteins, a class of fungal transcription factors involved in the regulation of polyketide production and other pathways.

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References

  • Adefarati AA, Giacobbe RA, Hensens OD, Tkacz JS (1991) Biosynthesis of L-671,329, an echinocandin-type antibiotic produced by Zalerion arboricola: origins of some of the unusual amino acids and the dimethylmyristic acid side chain. J Am Chem Soc 113:3542–3545

    CAS  Google Scholar 

  • An Z, Farman ML, Budde A, Taura S, Leong SA (1996) New cosmid vectors for library construction, chromosome walking and restriction mapping in filamentous fungi. Gene 176:93–96

    Article  CAS  PubMed  Google Scholar 

  • Aparicio JF, Molnar I, Schwecke T, Konig A, Haydock SF, Khaw LE, Staunton J, Leadlay PF (1996) Organization of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of the enzymatic domains in the modular polyketide synthase. Gene 169:9–16

    Article  CAS  PubMed  Google Scholar 

  • Ballance DJ (1991) Transformation systems for filamentous fungi and an overview of fungal gene structure. In: Leong SA, Berka RM (eds) Molecular industrial mycology: systems and applications for filamentous fungi. Marcel Dekker, New York, pp 1–29

    Google Scholar 

  • Bélanger C, Loubens I, Nester EW, Dion P (1997) Variable efficiency of a Ti plasmid-encoded VirA protein in different agrobacterial hosts. J Bacteriol 179:2305–2313

    PubMed  Google Scholar 

  • Bell AA, Wheeler MH (1986) Biosynthesis and functions of fungal melanins. Annu Rev Phytopathol 24:411–451

    Article  CAS  Google Scholar 

  • Bevan M (1984) Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12:8711–8721

    PubMed  Google Scholar 

  • Bills GF, Platas G, Pelaez F, Masurekar P (1999) Reclassification of a pneumocandin-producing anamorph, Glarea lozoyensis gen. et sp. nov., previously identified as Zalerion arboricola. Mycol Res 103:179–192

    Article  CAS  Google Scholar 

  • Bingle LE, Simpson TJ, Lazarus CM (1999) Ketosynthase domain probes identify two subclasses of fungal polyketide synthase genes. Fungal Genet Biol 26:209–223

    Article  CAS  PubMed  Google Scholar 

  • Bird D, Bradshaw R (1997) Gene targeting is locus dependent in the filamentous fungus Aspergillus nidulans. Mol Gen Genet 255:219–225

    Article  CAS  PubMed  Google Scholar 

  • Brown DW, Yu J-H, Kelkar HS, Fernandes M, Nesbitt TC, Keller NP, Adams TH, Leonard TJ (1996) Twenty-five coregulated transcripts define a sterigmatocystin gene cluster in Aspergillus nidulans. Proc Natl Acad Sci USA 93:1418–1422

    Article  CAS  PubMed  Google Scholar 

  • Bruns TD, Fogel R, Taylor JW (1990) Amplification and sequencing of DNA from fungal herbarium specimens. Mycologia 82:175–184

    CAS  Google Scholar 

  • Bundock P, den Dulk-Ras A, Beijersbergen A, Hooykaas PJJ (1995) Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J 14:3206–3214

    CAS  PubMed  Google Scholar 

  • Bundock P, Mróczek K, Winkler AA, Steensma HY, Hooykaas PJJ (1999) T-DNA from Agrobacterium tumefaciens as an efficient tool for gene targeting in Kluyveromyces lactis. Mol Gen Genet 261:115–121

    CAS  PubMed  Google Scholar 

  • Byrd AD, Schardl CL, Songlin PJ, Mogen KL, Siegel MR (1990) The β-tubulin gene of Epichloe typhina from perennial ryegrass (Lolium perenne). Curr Genet 18:347–354

    CAS  PubMed  Google Scholar 

  • Cangelosi GA, Best EA, Martinetti G, Nester EW (1991) Genetic analysis of Agrobacterium. Methods Enzymol 204:384–397

    CAS  PubMed  Google Scholar 

  • Chang PK, Ehrlich KC, Yu J, Bhatnagar D, Cleveland TE (1995) Increased expression of Aspergillus parasiticus aflR, encoding a sequence-specific DNA-binding protein, relieves nitrate inhibition of aflatoxin biosynthesis. Appl Environ Microbiol 61:2372–2377

    CAS  PubMed  Google Scholar 

  • Chang PK, Yu J, Bhatnagar D, Cleveland TE (1999) The carboxy-terminal portion of the aflatoxin pathway regulatory protein AFLR of Aspergillus parasiticus activates GAL1::lacZ gene expression in Saccharomyces cerevisiae. Appl Environ Microbiol 65:2508–2512

    CAS  PubMed  Google Scholar 

  • Chen X, Stone M, Schlagnhaufer C, Romaine CP (2000) A fruiting body tissue method for efficient Agrobacterium-mediated transformation of Agaricus bisporus. Appl Environ Microbiol 66:4510–4513

    CAS  PubMed  Google Scholar 

  • De Groot MJA, Bundock P, Hooykaas PJJ, Beijersbergen AGM (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nature Biotechnol 16:839–842

    Google Scholar 

  • Ehrlich KC, Montalbano BG, Bhatnagar D, Cleveland TE (1998) Alteration of different domains in AFLR affects aflatoxin pathway metabolism in Aspergillus parasiticus transformants. Fungal Genet Biol 23:279–287

    CAS  PubMed  Google Scholar 

  • Frisch DA, Harris-Haller LW, Yokubaitia NT, Thomas TL, Hardin SH, Hall TC (1995) Complete sequence of the binary vector Bin 19. Plant Mol Biol 27:405–409

    CAS  PubMed  Google Scholar 

  • Fujii I, Mori Y, Watanabe A, Kubo Y, Tsuji G, Ebizuka Y (2000) Enzymatic synthesis of 1,3,6,8-tetrahydroxynaphthalene solely from malonyl coenzyme A by a fungal iterative type I polyketide synthase PKS1. Biochemistry 39:8853–8858

    Article  CAS  PubMed  Google Scholar 

  • Fujii I, Watanabe A, Sankawa U, Ebizuka Y (2001) Identification of Claisen cyclase domain in fungal polyketide synthase WA, a naphthopyrone synthase of Aspergillus nidulans. Chem Biol 8:189–197

    Article  CAS  PubMed  Google Scholar 

  • Fulton TR, Ibrahim N, Losada MC, Grzegorski D, Tkacz JS (1999) A melanin polyketide synthase (PKS) gene from Nodulisporium sp. that shows homology to the pks1 gene of Colletotrichum lagenarium. Mol Gen Genet 262:714–720

    Article  CAS  PubMed  Google Scholar 

  • Gouka RJ, Gerk C, Hooykaas PJJ, Bundock P, Musters W, Verrips CT, de Groot MJ (1999) Transformation of Aspergillus awamori by Agrobacterium tumefaciens-mediated homologous recombination. Nature Biotechnol 17:598–601

    Article  CAS  Google Scholar 

  • Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary plant vector strategy based on separation of the vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180

    CAS  Google Scholar 

  • Hooykaas PJJ, Beijersbergen AGM (1994) The virulence system of Agrobacterium tumefaciens. Annu Rev Phytopathol 32:157–179

    CAS  Google Scholar 

  • Ikeda H, Nonomiya T, Usami M, Ohta T, Omura S (1999) Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin in Streptomyces avermitilis. Proc Natl Acad Sci USA 96:9509–9514

    Article  CAS  PubMed  Google Scholar 

  • Jahn B, Koch A, Schmidt A, Wanner G, Gehringer H, Bhakdi S, Brakhage AA (1997) Isolation and characterization of a pigmentless-conidium mutant of Aspergillus fumigatus with altered conidial surface and reduced virulence. Infect Immun 65:5110–5117

    Google Scholar 

  • Jeanmougin F, Thompson JD, Gouy M, Higgins DG, Gibson TJ (1998) Multiple sequence alignment with Clustal X. Trends Biochem Sci 23:403–405

    CAS  PubMed  Google Scholar 

  • Jin S, Song Y-N, Pan SQ, Nester EW (1993) Characterization of a virG mutation that confers constitutive virulence gene expression in Agrobacterium. Mol Microbiol 7:555–562

    CAS  PubMed  Google Scholar 

  • Keller NP, Brown D, Butchko RAE, Fernandes M, Kelkar H, Nesbitt C, Segner S, Bhatnagar D, Cleveland TE, Adams T (1995) A conserved polyketide mycotoxin gene cluster in Aspergillus nidulans. In: Eklund M, Richard RL, Mise K (eds) Molecular approaches to food safety issues involving toxic microorganisms. Alaken Inc, Fort Collins, Colo., pp 263–277

  • Kelly R, Register E, Sosa M (1994) Heterologous transformation of Zalerion arboricola. Curr Genet 26:217–224

    Article  CAS  PubMed  Google Scholar 

  • Kennedy J, Auclair K, Kendrew SG, Park C, Vederas JC, Hutchinson CR (1999) Modulation of polyketide synthase activity by accessory proteins during lovastatin biosynthesis. Science 284:1368–1372

    Article  CAS  PubMed  Google Scholar 

  • Langfelder, K, Jahn, B., Gehringer, H, Schmidt A, Wanner G, Brakhage AA (1998) Identification of a polyketide synthase gene (pksP) of Aspergillus fumigatus involved in conidial pigment biosynthesis and virulence. Med Microbiol Immunol 187:79–89

    CAS  PubMed  Google Scholar 

  • Langfelder K, Philippe B, Jahn B, Latge J-P, Brakhage AA (2001) Differential expression of the Aspergillus fumigatus pksP gene detected in vitro and in vivo with green fluorescent protein. Infect Immun 69:6411–6418

    Article  CAS  PubMed  Google Scholar 

  • Lee T, Yun S-H, Hodge KT, Humber RA, Krasnoff SB, Turgeon GB, Yoder OC, Gibson DM (2001) Polyketide synthase genes in insect- and nematode-associated fungi. Appl Microbiol Biotechnol 56:181–187

    Article  CAS  PubMed  Google Scholar 

  • Mayorga ME, Timberlake WE (1992) The developmentally regulated Aspergillus nidulans wA gene encodes a polypeptide homologous to polyketide and fatty acid synthases. Mol Gen Genet 235:205–212

    CAS  PubMed  Google Scholar 

  • Miao V, Coeffet-LeGal MF, Brown D, Sinnemann S, Donaldson G, Davies J (2001) Genetic approaches to harvesting lichen products. Trends Biotechnol 19:349–55

    Article  CAS  PubMed  Google Scholar 

  • Morris SA, Schwartz RE, Sesin DF, Masurekar P, Hallada TC, Schmatz DM, Bartizal K, Hensens OD, Zink DL (1994) Pneumocandin D0, a new antifungal agent and potent inhibitor of Pneumocystis carini. J Antibiot 47:755–764

    CAS  PubMed  Google Scholar 

  • Nicholson, TP, Rudd BA, Dawson M, Lazarus CM, Simpson TJ, Cox RJ (2001) Design and utility of oligonucleotide gene probes for fungal polyketide synthases. Chem Biol 8:157–178

    Article  CAS  PubMed  Google Scholar 

  • Noble HM, Langley D, Sidebottom PJ, Lane SJ, Fisher PJ (1991) An echinocandin from an endophytic Cryptosporiopsis sp. and Pezicula sp. in Pinus sylvestris and Fagus sylvatica. Mycol Res 95:1439–1440

    CAS  Google Scholar 

  • Pazour GJ, Ta CN, Das A (1992) Constitutive mutations of Agrobacterium tumefaciens transcriptional activator virG. J Bacteriol 174:4169–4174

    CAS  PubMed  Google Scholar 

  • Peralta EG, Hellmiss R, Ream W (1986) Overdrive, a T-DNA transmission enhancer on the A. tumefaciens tumour-inducing plasmid. EMBO J 5:1137–1142

    CAS  Google Scholar 

  • Radford A, Parish JH (1997) The genome and genes of Neurospora crassa. Fungal Genet Biol 21:258–266

    Article  CAS  PubMed  Google Scholar 

  • Ream W (1989) Agrobacterium tumefaciens and interkingdom genetic exchange. Annu Rev Phytopathol 27:583–618

    Article  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

    Google Scholar 

  • Sauer M, Lu P, Sangari R, Kennedy S, Polishook J, Bills G, An Z (2002) Estimating polyketide metabolic potential among non-sporulating fungal endophytes of Vaccinium macrocarpon. Mycol Res 106:460–470

  • Scheeren-Groot EP, Rodenburg KW, den Dulk-Ras A, Turk SCHJ, Hooykaas PJJ (1994) Mutational analysis of the transcriptional activator virG of Agrobacterium tumefaciens. J Bacteriol 176:6418–6426

    CAS  PubMed  Google Scholar 

  • Schwartz RE, Giacobbe RA, Bland JA, Monaghan RL (1989) L-671,329, a new antifungal agent. I. Fermentation and isolation. J Antibiot 42:163–167

    CAS  PubMed  Google Scholar 

  • Schwartz RE, Sesin DF, Joshua H, Wilson KE, Kempf AJ, Gocklen KA, Kuehner D, Gailliot P, Gleason C, White R, Inamine E, Bills G, Salmon P, Zitano L (1992) Pneumocandins from Zalerion arboricola. I. Discovery and isolation. J Antibiot 45:1853–1866

    CAS  PubMed  Google Scholar 

  • Shiotani H, Tsuge T (1995) Efficient gene targeting in the filamentous fungus Alternaria alternata. Mol Gen Genet 248:142–50

    Google Scholar 

  • Swofford DL (2000) PAUP: phylogenetic analysis using parsimony version 4.0b4a. Sinauer Associates, Sunderland, Mass.

  • Takano Y, Kubo Y, Shimizu K, Mise K, Okuno T, Furusawa I (1995) Structural analysis of PKS1, a polyketide synthase gene involved in melanin biosynthesis in Colletotrichum lagenarium. Mol Gen Genet 249:162–167

    CAS  PubMed  Google Scholar 

  • Takano Y, Kubo Y, Kawamura C, Tsuge T, Furusawa I (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 21:131–140

    Article  CAS  PubMed  Google Scholar 

  • Timberlake WE (1991) Cloning and analysis of fungal genes. In: Bennett JW, Lasure LL (eds) More gene manipulation in fungi. Academic Press, San Diego, pp 51–85

  • Tkacz JS (2000) Polyketide and peptide products of endophytic fungi: variations on two biosynthetic themes of secondary metabolism. In: Bacon CW, White JF (eds) Microbial endophytes. Marcel Dekker, New York, pp 263–294

  • Tkacz JS, DiDomenico B (2001) Antifungals: what's in the pipeline. Curr Opin Microbiol 4:540–545

    CAS  PubMed  Google Scholar 

  • Tkacz JS, Giacobbe RA, Monaghan RL (1993) Improvement in the titer of echinocandin-type antibiotics: a magnesium-limiting medium supporting the biphasic production of pneumocandins A0 and B0. J Ind Microbiol 11:95–103

    CAS  PubMed  Google Scholar 

  • Todd RB, Andrianopoulos A (1997) Evolution of a fungal regulatory gene family: the Zn(II)2Cys6 binuclear cluster DNA binding motif. Fungal Genet Biol 21:388–405

    Article  CAS  PubMed  Google Scholar 

  • Tsai H-F, Chang YC, Washburn RG, Wheeler MH, Kwon-Chung KJ (1998) The developmentally regulated alb1 gene of Aspergillus fumigatus: its role in modulation of conidial morphology and virulence. J Bacteriol 180:3031–3038

    CAS  PubMed  Google Scholar 

  • Tsai H-F, Fujii I, Watanabe A, Wheeler MH, Chang YC, Yasuoka Y, Ebizuka Y, Kwon-Chung KJ (2001) Pentaketide melanin biosynthesis in Aspergillus fumigatus requires chain-length shortening of a heptaketide precursor. J Biol Chem 276:29292–29298

    Article  CAS  PubMed  Google Scholar 

  • Watanabe A, Fujii I, Sankawa U, Mayorga ME, Timberlake WE, Ebizuka Y (1999) Re-identification of Aspergillus nidulans wA gene to code for a polyketide synthase of naphthopyrone. Tetrahedron Lett 40:91–94

    Article  CAS  Google Scholar 

  • Winans SC (1992) Two-way chemical signaling in Agrobacterium-plant interactions. Microbiol Rev 56:12–31

    CAS  PubMed  Google Scholar 

  • Xiang C, Han P, Lutziger I, Wang K, Oliver DJ (1999) A mini binary vector series for plant transformations. Plant Mol Biol 40:711–717

    Article  CAS  PubMed  Google Scholar 

  • Yu J-H, Butchko RAE, Fernandes M, Keller NP, Leonard TJ, Adams TH (1995) Conservation of structure and function of the aflatoxin regulatory gene aflR from Aspergillus nidulans and A. flavus. Curr Genet 29:549–555

    Article  Google Scholar 

  • Zhu J, Oger PM, Schrammeijer B, Hooykaas PJJ, Farrand SK, Winans SC (2000) The bases of crown gall tumorigenesis. J Bacteriol 182:3885–3895

    CAS  PubMed  Google Scholar 

  • Zupan JR, Zambryski P (1995) Transfer of T-DNA from Agrobacterium to the plant cell. Plant Physiol 107:1041–1047

    Article  CAS  PubMed  Google Scholar 

  • Zwiers LH, De Waard MA (2001) Efficient Agrobacterium tumefaciens-mediated gene disruption in the phytopathogen Mycosphaerella graminicola. Curr Genet 39:388–393

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank C. DeSanti, W. Strohl, and the Department of Microbiology at Ohio State University for making pANT846 available.

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  1. Merck Research Laboratories, P.O. Box 2000/RY80Y-325, Rahway, NJ 07065, USA

    A. Zhang, P. Lu, A. M. Dahl-Roshak, P. S. Paress, S. Kennedy, J. S. Tkacz & Z. An

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  1. A. Zhang
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  2. P. Lu
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  3. A. M. Dahl-Roshak
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Correspondence to Z. An.

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Communicated by C.A.M.J.J. van den Hondel

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Zhang, A., Lu, P., Dahl-Roshak, A.M. et al. Efficient disruption of a polyketide synthase gene (pks1) required for melanin synthesis through Agrobacterium-mediated transformation of Glarea lozoyensis . Mol Gen Genomics 268, 645–655 (2003). https://doi.org/10.1007/s00438-002-0780-4

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