Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1989-09-07
1991-07-02
Schwartz, Richard A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 697, 435 698, 435 711, 435 91, 435161, 435171, 4351721, 4351723, 435254, 4353201, 435940, 536 27, 935 6, 935 8, 935 9, 935 10, 935 22, 935 28, 935 33, 935 37, 935 47, 935 59, 935 66, C12N 116, C12N 1510, C12N 1552, C12N 1579
Patent
active
050285335
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to the field of recombinant DNA biotechnology.
BACKGROUND OF THE INVENTION
It has been suggested that starch (or dextrin)-degrading yeasts could be usefully exploited in fermentation processes, such as the production of ethanol or beer, which utilise starch-containing raw materials (Tubb, 1983, 1984: full reference to be found at the end of this specification), and in the production of amylolytic enzymes such as amyloglucosidases (Eveleigh, 1981; Fogarty, 1983). Dex.sup.+ strains of Saccharomyces cerevisiae (formerly called S. diastaticus; van der Walt, 1970; Yarrow, 1984) have at least one DEX or STA gene (Erratt and Stewart, 1978; Tamaki, 1978) and are able to ferment soluble starch or dextrins as a consequence of producing an extracellular amylo-.alpha.-1,4-glucosidase (AMG) during vegetative growth (Hopkins, 1955; Searle and Tubb, 1981). However, common brewing strains of Saccharomyces cerevisiae do not have the ability to produce extracellular AMG during vegetative growth. Dex.sup.+ strains of Saccharomyces cerevisiae have been hybridised with brewing strains, and progeny have been derived which have the ability to produce AMG during vegetative growth. However these progeny strains produce unacceptable low carbohydrate beers (Tubb et al., 1981), unless steps are taken to eliminate a gene (POFl) responsible for a `herbal phenolic` off-flavor (Goodey and Tubb, 1982).
Recombinant DNA techniques offer a more specific approach to conferring amylolytic character on strains of yeast already possessing many other desirable commercial characteristics. Recently, .alpha.-amylase genes from mice (Thomsen, 1983) and wheat (Rothstein et al., 1984) have been expressed in S. cerevisiae, and a gene for AMG production has been cloned from a STA1 strain of yeast (Yamashita and Fukui, 1983).
A 3.6kb DNA fragment has been cloned from a Saccharomyces diastaticus genome (strain BRG536): DEX1) and shown to confer production of extracellular amylo-.alpha.-1,4-glucosidase (AMG), and thereby, the ability to hydrolyse starch and dextrins, on Dex.sup.- strains of S. cerevisiae (Tubb, R. S., Brewers Guardian, Sept. 1984, 34-37). (A preliminary report of this work was given at the ALKO Symposium on gene expression in yeast at Helsinki in June, 1983).
The use of a eukaryotic signal sequence to promote product transport from a eukaryotic host cell harbouring a recombinant vector is known. Published European patent application EP-A1-0127304 describes fusion polypeptides comprising a signal (or "pre") sequence and a desired polypeptide produced by expression of a gene in a host cell. The fusion polypeptides are transported through the host cell membrane and cleaved to produce extracellular, mature, polypeptides. The yeast invertase signal sequence is specifically mentioned and its use in the preparation of host cells capable of producing extracellular interferon is described. Published European patent application EP-A1-0116201 describes an essentially similar use of eukaryotic signal sequences and exemplifies the use of the yeast .alpha.-factor signal sequence in the production of extracellular human epidermal growth factor (hEGF). The existence and use of the signal sequence of a yeast amylolytic enzyme to promote product secretion of heterologous polypeptides from yeast has been the subject of speculation (Tubb, R. S., Brewers Guardian, Sept. 1984, 34-37).
The cloned 3.6kb DNA fragment referred to above has now been sequenced and the coding sequence for AMG has been identified and elucidated. In addition, it has been unequivocally shown that the AMG gene includes a leader sequence coding for a signal peptide capable of promoting product secretion.
According to the present invention, there is provided a precursor polypeptide having the amino acid sequence: -leu-gly-X
The precursor polypeptide, when produced in a eukaryotic host cell by the expression of a gene coding for the precursor polypeptide, is exported from the host cell and processed to produce the mature polypeptide, X.
The po
REFERENCES:
patent: 4588684 (1986-05-01), Brake
patent: 4663280 (1987-05-01), Sloma
patent: 4663294 (1987-05-01), Yamane et al.
patent: 4725535 (1988-02-01), Sonenshein et al.
Nature, vol. 308, No. 5960, Apr. 12, 1984, S. J. Rothstein et al., pp. 662-665.
Agricultural and Biological Chemistry, vol. 47, No. 11, Nov. 1983.
Brewers' Guardian, Sep. 1984, R. S. Tubb, "Genetic Development of Yeast Strains", pp. 34-37, see page 36.
P. Journal of Bacteriology, vol. 161, No. 2, Feb. 1985, I. Yamashita et al., pp. 567-573, See FIG. 1, Aminoacids 12-32.
Celltech Limited
Peet Richard C.
Schwartz Richard A.
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