Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1997-11-06
2000-03-28
Brusca, John S.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4353201, 4352542, 536 231, 536 241, 536 245, C12P 2100, C12N 1563, C12N 115, C12N 1531
Patent
active
060430516
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a transcriptionally controllable promoter, derived from yeast, useful in molecular bioengineering for the manufacture of useful proteins, metabolites, etc. employing a gene recombinant DNA technique and also to a DNA where said promoter is linked to a heterologous gene in an expressible manner as well as to a vector containing said DNA. The present invention further relates to a method for the manufacture of protein using a vector containing a DNA obtained by linking a nucleic acid coding for the protein downstream of said promoter in an expressible manner.
PRIOR ART
In order to produce a protein which is useful as pharmaceuticals, food, etc. in a genetic engineering manner, its production in yeast using the yeast as a host has been widely conducted. A technique of introducing a heterologous gene into a yeast of the genus Saccharomyces (especially, Saccharomyces cerevisiae; hereinafter simply referred to as S. cerevisiae) for the production of the corresponding heterologous protein has been known. When the heterologous gene (including genomic DNA and cDNA) is expressed in S. cerevisiae cells, it is necessary to give an promoter expressible in the yeast upstream thereof. Examples of the promoter for S. cerevisiae which has been known so far are a promoter of alcohol dehydrogenase 1 (ADH 1) gene or a promoter of 3-phosphoglycerate kinase (PGK) gene known to show a strong expression; GAL1 and GAL10 promoter where a transcription is induced in a galactose medium; and PHO5 promoter where a transcription is induced in a medium having a low concentration of inorganic phosphate. It is desirable that a promoter is capable of conducting a transcriptional control and resulting in a far higher expressi on.
For a large-scale production of a heterologous protein by of yeast, it is preferred that a promoter not only shows a strong expression but also is capable of conducting a transcriptional control. It has been known that, in a synthetic minimum medium, a doubling time of a haploid strain of yeast is about 140 minutes while, in a complete nutrient medium, the time is as quick as about 90 minutes [Methods in Enzymology, 194, 15 (1991)]. Thus, as a condition for culturing the yeast to produce a heterologous protein, a complete nutrient medium abundant in nutrition is preferred since it accelerates the proliferating rate of the cells and can keep a high cell density. However, promoters capable of inducing a transcription which have been known up to now employ a medium wherein galactose is used as a carbon source or a medium having a low concentration of phosphate. Therefore, they are not preferred in terms of a cell proliferation for an purpose of a large-scale production of a heterologous protein.
OBJECTS OF THE INVENTION
An object of the present invention is to offer a nucleotide sequence of a promoter derived from a yeast which is capable of conducting a transcriptional control by a PDR1 gene product, and which can be used in a complete nutrient medium so that the above-mentioned problems have been solved.
SUMMARY OF THE INVENTION
The present inventors have found that expression of a gene related to the sensitivity to aureobasidin obtained from an aureobasidin-sensitive cells (a wild-type strain) increase when the aureobasidin-sensitive cells are subjected to a treatment such as a mutagenization to make them aureobasidin-resistant and have considered in offering a promoter which participates in said gene expression.
As a result of an intensive investigation, the present inventors have cloned a promoter which participates in the above gene expression from a genomic DNA of a mutant which is made aureobasidin-resistant and have determined the nucleotide sequence. To our surprise, when further investigation has been conducted, the present inventors have found that the transcriptional activity of said promoter is controlled by a gene product of a PDR1 gene of a pleiotropic drug resistance gene whereupon the present invention has been achieved.
The p
REFERENCES:
F. Sherman, "Getting Started with Yeast", Method in Enzymology, vol. 194, pp. 1-21, 1991.
K. Takesako et al., "Aureobasidins, New Antifungal Antibiotics Taxonomy, Fermentation, Isolation, and Properties", Journal of Antibiotics, vol. 44, No. 9, pp. 919-924, 1991.
K. Ikai et al., "Structure of Aureobasidin A", The Journal of Antibiotics, vol. 44, No. 9, pp. 925-933, 1991.
K. Ikai et al. "Structures of Aureobasidins B to R", The Journal of Antibiotics, vol. 44, No. 11, pp. 1187-1198, 1991.
E. Balzi et al., "The Multidrug Resistance Gene PDR1 from Saccharomyces cerevisiae", The Journal of Biological Chemistry, vol. 262, No. 35, pp. 16871-16879, 1987.
Kato Ikunoshin
Okado Takashi
Takesako Kazutoh
Brusca John S.
Takara Shuzo Co. Ltd.
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