Abstract
Acremonium chrysogenum, a producer of cephalosporin C, was subjected to DNA-mediated transformaations using a vector without bacterial DNA sequences. Recombinant fungal strains were generated with a gel-purified DNA fragment, carrying only the mutated β-tubulin gene from A. chrysogenum. The lack of any bacterial DNA was verified by Southern hybridization analysis and polymerase chain reaction amplifications to detect even residual DNA sequences. This procedure can be referred to as a self-cloning experiment for which less restricted working regulations are needed. Finally, the transfer of a synthetic hirudin gene by contransformation demonstrated that any DNA molecule can be introduced into the A. chrysogenum genome without bacterial marker genes. This seems to be highly relevant for biotechnical processes in which safe recombinant producer strains are required to satisfy governmental restrictions.
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References
Austin B, Hall RM, Tyler BM (1990) Optimized vectors and selection for transformation of Neurospora crassa and Aspergillus nidulans to bleomycin and phleomycin resistance. Gene 93:157–162
Benson F, Zagurski R (1984) An alternative method for extraction of DNA from agarose. In: Silhavy TJ, Bermon ML, Enquist LW (eds). Experiments with gene fusions. Cold Spring Harbor Laboratory Cold Spring Harbor, NY
Fincham JRS (1989) Transformation in fungi. Microbiol Rev 53:148–170
Finkelstein DB (1992) Transformation. In: Finkelstein DB, Ball C (eds) Biotechnology of filamentous fungi. Butterworth-Heinemann, Boston London Singapore, pp 113–156
Goldsbrough AP, Lastrella CN, Yoder JI (1993) Transposition mediated repositioning and subsequent elimination of marker genes from transgenic tomato. Biotechnology 11:1286–1292
Gutiérrez S, Diez B, Alvarez E, Barredo JL, Martin JF (1991) Expression of the penDE gene of Penicillium chrysogenum encoding isopenicillin N acyltransferase in Cephalosporium acremonium: production of benzylpenicillin by the transformants. MGG 225:56–64
Isogai T, Fukagawa M, Aramori I, Iwami M, Kojo H, Ono T, Ueda Y, Kohsaka M, Imanaka H (1991) Construction of a 7-aminocephalosporanic acid (7ACA) biosynthetic operon and direct production of 7ACA in Acremonium chrysogenum. Biotechnology 9:188–191
Kinghorn JR, Turner G (eds.) (1992) Applied molecular genetics of filamentous fungi. Blackie, London Glasgow New York
Kück U, Walz M, Mohr G, Mracek M (1989) The 5′ sequence of the isopenicillin N-synthetase gene (pcbC) from Cephalosporium acremonium directs the expression of the prokaryotic hygromycin B phosphotransferase gene (hph) in Aspergillus niger. Appl Microbiol Biotechnol 31:358–365
Lecellier G, Silar P (1994) Rapid methods for nucleic acids extraction from Petri dish-grown mycelia. Curr Genet 25:122–123
Lemke PA, Peng M (1995) Genetic manipulation of fungi by DNA-mediated transformation. In: U. Kück (ed) The mycota II. Springer, Berlin Heidelberg New York pp 109–139 (in press)
Mathison L, Soliday C, Stepan T, Aldrich T, Rambosek J (1993) Cloning, characterization, and use in strain improvement of the Cephalosporium acremonium gene cefG encoding acetyl transferase. Curr Genet 23:33–41
Menne S, Walz M, Kück U (1994) Expression studies with the bidirectional pcbAB-pcbC promoter region from Acremonium chrysogenum using reporter gene fusions. Appl Microbiol Biotechnol 42:27–35
Nowak C, Kück U (1994) Development of an homologous transformation system for Acremonium chrysogenum based on the β-tubulin gene. Curr Genet 25:34–40
Skatrud PL, Queener SW, Carr LG, Fisher DL (1987) Efficient integrative transformation of Cephalosporium acremonium. Curr Genet 12:337–348
Skatrud PL, Tietz AJ, Ingolia TD, Cantwell CA, Fisher DL, Chapman JL, Queener SW (1989) Use of recombinant DNA to improve production of cephalosporin C by Cephalosporium acremonium. Biotechnology 7:477–485
Stahl U, Tudzynski P (1992) (eds.) Molecular biology of filamentous fungi. VCH Weinheim New York Basel
Walz M, Kück U (1993) Targeted integration into the Acremonium chrysogenum genome: disruption of the pcbC gene. Curr Genet 24:421–427
Wernars K, Goosen T, Wennekes LMJ, Sewart K, Hondel CAMJJ van den, Broek HWJ van den (1987) Contransformation of Aspergillus nidulans: a tool for replacing fungal genes. Mol Gen Genet 209:71–77
Yoder JI, Goldsbrough AP (1994) Transformation systems for generating marker-free transgenic plants. Biotechnology 12:263–267
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Nowak, C., Radzio, R. & Kück, U. DNA-mediated transformation of a fungus employing a vector devoid of bacterial DNA sequences. Appl Microbiol Biotechnol 43, 1077–1081 (1995). https://doi.org/10.1007/BF00166928
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DOI: https://doi.org/10.1007/BF00166928