Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-05-07
2001-08-21
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S254600, C435S320100, C536S024100
Reexamination Certificate
active
06277596
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a high-yield production system for a protein or peptide, and more specifically it relates to a production system using a regulator sequence derived from
Trichoderma viride
, and to a production technique for a protein or peptide which employs the production system.
2. Description of the Related Art
Filamentous fungi are known to secrete significant amounts of extracellular proteins, particular enzymes. For example, the genus Aspergillus secretes amylases, proteases, lipases, cellulases and other enzymes, which are therefore utilized in various different fields. As to their yields, it has been reported, for example, that
Aspergillus niger
produces over 20 g of glucoamylase per liter of liquid culture, while
Aspergillus oryzae
produces about 50 g per kilogram of solid culture (Katuya Gomi: Kagaku to Seibutsu (1994), 32, 269).
Recent years have brought an accumulation of knowledge regarding production techniques for target proteins utilizing the protein-producing abilities of these filamentous fungi. Examples of filamentous fungi-derived foreign proteins which have been produced include Mucor miehei-derived rennin in
Aspergillus nidulans
hosts (G. L. Gray, et al.; Gene (1986), 48, 41),
Aspergillus ficuum
-derived phytase in
Aspergillus niger
hosts (R. F. M. van Gorcom, et al.: European Patent Application (1991), 0420358A1),
Mucor miehei
-derived rennin (T. Christensen, et al.: Bio/Technology (1988), 6, 1419) and lipase (B. Huge-Jensen, et al.: Lipids (1989), 24, 781) in
Aspergillus oryzae
hosts,
Phlebia radiata
-derived laccase in
Trichoderma reesei
hosts (M. Saloheimo, et al.: Bio/Technology (1991), 9, 987),
Aspergillus oryzae
-derived &agr;-amylase in
Trichoderma viride
hosts (C. Cheng, et al.: Agric. Biol. Chem. (1991), 55, 1817) and Fusarium genus-derived alkali proteases in
Acremonium chrysogenum
hosts (Shigeru Morita, et al.: Summary of Meeting Lectures of the Nihon Nogei Kagakukai (1993), p.140). Production of human, bovine and other animal proteins as well as plant proteins has also been confirmed in
Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae
and
Trichoderma reesei
hosts.
Thus, filamentous fungi are clearly excellent as production hosts for proteins and polypeptides. The productivity of the target protein also becomes an important issue in terms of industrial applications. The factors considered to be important in determining productivity include (1) regulation for effective and high-yield transcription and translation of the target protein by a regulator region (for example, promoter and terminator) which is capable of expression in the host, (2) a translation product with the desired higher-order structure (activity type) and (3) stable extracellular secretion of the same. To this end there have been developed many effective promoters, such as the Aspergillus amylase gene promoter and the Trichoderma cellulase gene promoter. It is currently known that the use of an &agr;-amylase gene promoter in an
Aspergillus oryzae
host can produce 3.3 g of
Mucor miehei
rennin per liter of liquid culture.
No matter how powerful the promoter, however, it is usually not possible to obtain the target protein in an amount exceeding the protein yield of the host. In fact, only 30% protein productivity is achieved in the case of
Aspergillus oryzae
mentioned above. This is thought to be due to the fact that the gene which is used remains in the host, that the codon use frequency in the translated region differs among species, that the secretion mechanism differs among species, and other reasons as well, but as yet no technique has been discovered to solve these problems.
It has therefore been considered that target protein yields could be enhanced by improving the protein production of the hosts themselves.
Filamentous fungi belonging to the genus Trichoderma are known to be excellent cellulase producing cells. In particular,
Trichoderma reesei
has been widely studied in terms of its foreign protein production, and much research is being conducted on foreign protein expression using a promoter for the cellobiohydrolase 1 (cbh1) gene, said to constitute about 70% of the secreted protein of that species (Uusitalo J M., et al.: J. Biotechnol. (1991), 17, 35. Joutsjoki V V., et al.: Curr. Genet. (1993), 24, 223. Barnett C C., et al.: Biotechnology (1991), 9, 562. Berges T., et al.: Curr. Genet. (1993), 24, 53. Saloheimo M., et al.: Gene (1989), 85, 343. Saarelainen R., et al.: Mol. Gen. Genet. (1993), 241, 497). However, only C. Cheng et al. have reported a foreign protein expression system using
Trichoderma viride
as the host. According to C. Cheng et al., the &agr;-amylase gene was introduced into a protease-deficient strain of
Trichoderma viride
as the host using the cbh
1
promoter and signal sequence, to produce &agr;-amylase at 1 g per liter of liquid culture. While this demonstrates that foreign protein production is possible with
Trichoderma viride
, that production level is not satisfactory in terms of cost on a practical production scale.
SUMMARY OF THE INVENTION
The present inventors have now found that a cellulase gene regulator sequence derived from
Trichoderma viride
gives high expression of target proteins and have subsequently succeeded in using this regulator sequence to produce
Humicola insolens
-derived endoglucanase at 15 g/L. Such productivity has not been achieved in any foreign protein production system of filamentous fungi disclosed to date. The present invention has been accomplished on the basis of this finding.
It is therefore an object of the present invention to provide a regulator sequence which gives high expression of target proteins and a high yield production system for proteins which employs the sequence.
It is a further object of the invention to provide a method for high-yield production of proteins or peptides using this production system.
The regulator sequence of the invention which gives high expression of target proteins is a regulator sequence of the cellulase cbh1 gene derived from
Trichoderma viride.
The high-yield production system for proteins according to the invention utilizes this
Trichoderma viride
-derived cellulase cbh1 gene regulator sequence.
REFERENCES:
patent: 5-509223 (1993-12-01), None
patent: WO 91/17243 (1991-11-01), None
patent: WO 95/16782 (1995-06-01), None
Shoemaker et al. Molecular cloning of . . . T.reesie strain L27. Bio/Technology vol. 1:691-95. 1983.*
Harkki et al. A novel fungal expression system. Bio/Technology vol. 7:596-603. 1989.*
Uusitalo et al. enzyme production by recombinant T.reseei strains. J Biotechnology. vol. 17:35-50. 1991.*
Saarrelainen et al. Cloning, sequencing and enhanced expression. Mol. Gen. Genet. vol. 241: 497-503. 1993.*
Udaka et al. GenBank Accession No. X53931, dated Apr. 10, 1993.*
C. Cheng et al., “Nucleotide sequence of the cellobiohydrolase gene fromTrichoderma viride”, Nucleic Acids Research, vol. 18, No. 18, p. 5559.
W. Sung et al., “Expression ofTrichoderma reeseiandTrichoderma viridexylanases inEscherichia coli”, Biochemistry and Cell Biology, vol. 73, Nos. 5 and 6, pp. 253-259, 1995.
C. Cheng et al., “Efficient Production of Taka-amylase A byTrichoderma viride”, Agricultural and Biological Chemistry, vol. 55, No. 7, pp. 1817-1822, 1991.
D. Kim et al., “Purification and Characterization of Endoglucanase and Exoglucanase Components fromTrichoderma viride”, Journal of Fermentation and Bioengineering, vol. 77, No. 4, pp. 363-369, 1994.
Aoyagi Kaoru
Moriya Tatsuki
Murakami Takeshi
Sumida Naomi
Watanabe Manabu
Meiji Seika Kaisha Ltd.
Prouty Rebecca E.
Rao Manjunath N.
Wenderoth , Lind & Ponack, L.L.P.
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