Summary
Aspergillus oryzae was transformed with a synthetic gene consisting of a chicken lysozyme signal sequence and a mature human lysozyme (HLY) sequence. The transformants secreted active HLY (about 1.2 mg/l) when the HLY gene was expressed under the control of the Taka-amylase A gene (amyB) promoter. Western blot analysis suggested that the secreted protein was immunoreactive with anti-human lysozyme antibody and the signal peptide was correctly cleavaged off in the A. oryzae transformants. The transcriptional level of the HLY gene was investigated by Northern blot analysis using a probe that was equivalently specific to both the HLY gene and the amyB gene. The HLY gene was expressed of a higher level compared with the amyB gene because of its multi-copy intergration. The efficient transcription of the HLY gene suggested that A. oryzae is a promising host for production of heterologous proteins from higher eukaryotes.
Similar content being viewed by others
References
Archer DB, Jeenes DJ, MacKenzie DA, Brightwell G, Lambert N, Lowe G, Radford SE, Dobson CM (1990a) Hen egg white lysozyme expressed in, and secreted from, Aspergillus niger is correctly processed and folded. Bio/Technology 8:741–745
Archer DB, Roberts IN, MacKenzie DA (1990b) Heterologous protein secretion from Aspergillus niger in phosphate-buffered batch culture. Appl Microbiol Biotechnol 34:313–315
Barbesgaard P, Heldt-Hansen HP, Diederichsen B (1992) On the safety of Aspergillus oryzae: a review. Appl Microbiol Biotechnol 36:569–572
Castanon MJ, Spevak W, Adorf GR, Chlebowicz-Sledziewska E, Sledziewski A (1988) Cloning of human lysozyme gene and expression in yeast Saccharomyces cerevisiae. Gene 66:223–234
Cathala G, Savouret JF, Mendez B, West BL, Karin M, Martial JA, Baxter JD (1983) A method for isolation of intact, translationally active ribonucleic acid. DNA 2:329–335
Christensen T, Woeldike H, Boel E, Mortensen SB, Hjortshoej K, Thim L, Hansen MT (1988) High level expression of recombinant genes in Aspergillus oryzae. Bio/Technology 6:1419–1422
Cohen BL (1977) The proteases of Aspergilli. In: Smith JE, Pateman JA (eds) Genetics and physiology of Aspergillus. Academic Press, New York, pp 281–292
Feinberg AP, Vogelstein B (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13
Gomi K, Iimura Y, Hara S (1987) Integrative transformation of Aspergillus oryzae with a plasmid containing the Aspergillus nidulans amyB gene. Agric Biol Chem 51:2549–2555
Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580
Hung-Jensen B, Andeasen F, Christensen T, Christensen M, Thim L, Boel E (1989) Rhizomucor miehei triglyceride lipase is processed and secreted from transformed Aspergillus oryzae. Lipids 24:781–785
Jigami Y, Muraki M, Harada N, Tanaka H (1986) Expression of synthetic human-lysozyme gene in Saccharomyces cerevisiae: use of a synthetic chicken-lysozyme signal sequence for secretion and processing. Gene 43:273–279
Laemmli UK (1970) Cleavaged structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685
Morsky P (1983) Turbidimetric determination of lysozyme with Micrococcus lysodeikticus cells: reexamination of reaction conditions. Anal Biochem 128:77–85
Muraki M, Jigami Y, Tanaka H, Harada N, Kishimoto F, Agui H, Ogino S, Nakasato S (1986) Expression of synthetic human lysozyme gene in Escherichia coli. Agric Biol Chem 50:713–723
Osserman EF, Canfield RE, Beychok S (1974) Lysozyme. Academic Press, New York
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.
Saunders G, Picknett TM, Tuite MF, Ward M (1989) Heterologous gene expression in filamentous fungi. TIBTECH 7:283–287
Tada S, Iimura Y, Gomi K, Takahashi K, Hara S, Yoshizawa K (1989) Cloning and nucleotide sequence of the genomic Taka-amylase A gene of Aspergillus oryzae. Agric Biol Chem 53:593–599
Tada S, Gomi K, Kitamoto K, Takahashi K, Tamura G, Hara S (1991a) Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli β-glucuronidase gene and analysis of its expression in A. oryzae. Mol Gen Genet 229:301–306
Tada S, Gomi K, Kitamoto K, Kumagai C, Tamura G, Hara S (1991b) Identification of the promoter region of the Taka-amylase A gene required for starch induction. Agric Biol Chem 55:1939–1941
Ward M (1991) Chymosin production in Aspergillus. In: Leong SA, Berka RM (eds) Molecular industrial mycology. Dekker, New York, pp 83–105
Yoshimura T, Toibara A, Kikuchi K, Kobayashi M, Hayakawa T, Nakahara K, Kikuchi M, Ikehara M (1987) Differences between Saccharomyces cerevisiae and Bacillus subtilis in secretion of human lysozyme. Biochem Biophys Res Commun 145:712–718
Yoshimura T, Toibara A, Nakahama K (1988) Human lysozyme: sequencing of cDNA and expression and secretion by Saccharomyces cerevisiae. Biochem Biophys Res Commun 150:794–801
Author information
Authors and Affiliations
Additional information
Correspondence to: K. Kitamoto
Rights and permissions
About this article
Cite this article
Tsuchiya, K., Tada, S., Gomi, K. et al. High level expression of the synthetic human lysozyme gene in Aspergillus oryzae . Appl Microbiol Biotechnol 38, 109–114 (1992). https://doi.org/10.1007/BF00169428
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00169428