Abstract
Aspergillus niger var. awamori was previously transformed with a vector designed to express a fused glucoamylase-prochymosin gene and bearing the Neurospora crassa pyr4 gene as a selectable marker. Mutant strains that overproduced the glucoamylase-prochymosin fusion protein were derived from one of the transformants. Despite the fact that the expression vector was integrated into the genome of these strains it was possible to obtain strains from which the vector sequences had been removed. This was performed by selection against the pyr4 gene present on the expression vector using 5-fluoroorotic acid. The cured strains were retransformed in order to investigate production of heterologous proteins using other expression vectors. In addition to the glucoamylase-prochymosin fusion protein, the mutant Aspergillus strains also over-produced Rhizomucor miehei aspartic proteinase but not preprochymosin produced as a non-fusion protein. The ability to select for loss of integrated plasmid from Aspergillus transformants may prove to be important for a variety of applications.
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References
Ballance DJ, Buxton FP, Turner G (1983) Transformation of Aspergillus nidulans by the orotidine-5'-phosphate decarboxylase gene of Neurospora crassa. Biochem Biophys Res Commun 112:284–289
Berka RM, Barnett CC (1989) The development of gene expression systems for filamentous fungi. Biotechnol Adv 7:127–154
Berka RM, Ward M, Wilson LJ, Hayenga KJ, Kodama KH, Carlomagno LP, Thompson SA (1990) Molecular cloning and delection of the gene encoding aspergillopepsin A from Aspergillus awamori. Gene 86:153–162
Bussink HJD, Hombergh JPTW van den, Ijssel PRLA van den, Visser J (1992) Characterization of polygalacturonase-overproducing Aspergillus niger transformants. Appl Microbiol Biotechnol 37:324–329
Campbell EI, Unkles SE, Kinghorn JR (1989) Improved transformation efficiency of Aspergillus niger using the homologous niaD gene for nitrate reductase. Curr Genet 16:53–56
Cullen D, Gray GL, Wilson LJ, Hayenga KJ, Lamsa MH, Rey MW, Norton S, Berka RM (1987) Controlled expression and secretion of bovine chymosin in Aspergillus nidulans. Bio/Technology 5:369–376
Dunn-Coleman NS, Bloebaum P, Berka RM, Bodie E, Robinson N, Armstrong G, Ward M, Przetak M, Carter G, LaCost R, Wilson LJ, Kodama KH, Baliu EF, Bower B, Lasma M, Heinsohn H (1991) Commercially viable levels of chymosin production by Aspergillus. Bio/Technology 9:976–981
Gray GL, Hayenga K, Cullen D, Wilson LJ, Norton S (1986) Primary structure of Mucor meihei aspartyl protease: evidence for a zymogen intermediate. Gene 48:41–53
Hartingsveldte W van, Mattern IE, Zeijl CMJ van, Pouwels PH, Hondel CAMJJ van den (1986) Development of a homologous transformation system for Aspergillus niger based on the pyrG gene. Mol Gen Genet 206:71–75
Hynes MJ, Paterman JAJ (1970) The genetic analysis of amidase synthesis in Aspergillus nidulans. II. Mutants resistant to fluoroacetamide. Mol Gen Genet 108:107–116
Hynes MJ, Corrick CM, King JA (1983) Isolation of genomic clones containing the amdS gene of Aspergillus nidulans and their use in the analysis of structural and regulatory mutations. Mol Cell Biol 3:1430–1439
Kelly JM, Hynes MJ (1985) Transformation of Aspergillus niger by the amdS gene of Aspergillus nidulans. EMBO J 4:475–479
Lamsa M, Bloebaum P (1990) Mutation and screening to increase chymosin yield in a genetically-engineered strain of Aspergillus awamori. J Ind Microbiol 5:229–238
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd edn.Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Sleep D, Belfield GP, Ballance DJ, Steven J, Jones S, Evans LR, Moir PD, Goodey AR (1991) Saccharomyces cerevisiae strains that overexpress heterologous proteins. Bio/Technology 9:183–187
Smith RA, Duncan MJ, Moir DT (1985) Heterologous protein secretion from yeast. Science 229:1219–1224
Ward M (1991) Chymosin production in Aspergillus. In: Leong SA, Berka RM (eds) Molecular industrial mycology. Systems and applications for filamentous fungi. Dekker, New York, pp 83–105
Ward M, Kodama KH (1992) Introduction to fungal proteinases and expression in fungal systems. In: Dunn BM (ed) Structure and function of the aspartic proteinases: genetics, structure, mechanisms. Plenum, New York, pp 149–160
Ward M, Wilson LJ, Kodama KH, Rey MW, Berka RM (1990) Improved production of chymosin in Aspergillus by expression as a glucoamylase-chymosin fusion. Bio/Technology 8:435–440
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Ward, M., Wilson, L.J. & Kodama, K.H. Use of Aspergillus overproducing mutants, cured for intergrated plasmid, to overproduce heterologous proteins. Appl Microbiol Biotechnol 39, 738–743 (1993). https://doi.org/10.1007/BF00164459
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DOI: https://doi.org/10.1007/BF00164459