Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1997-05-09
1999-12-14
Railey, II, Johnny F.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435171, 4352523, 4352542, 4353201, 536 241, C12N 115, C12N 1531, C12N 1581, C12P 2100
Patent
active
06001590&
DESCRIPTION:
BRIEF SUMMARY
This application is a national stage application of PCT/JP96/02597 filed on Sep. 12, 1996, the entire contents of which are hereby incorporated by reference, which receives priority from JP 7-234133 filed Sep. 12, 1995 and from JP 8-42536, filed Feb. 29, 1996.
TECHNICAL FIELD
The present invention relates to a promoter and/or a terminator for a formate dehydrogenase gene from Candida boidinii, a gene expression cassette containing the promoter, a heterologous gene and the terminator, a vector comprising the expression cassette, transformant cells carrying the expression vector and a process for producing a useful gene product by use of the transformant cells.
BACKGROUND ART
Methanol-assimilating yeasts are those capable of growing on methanol as a sole carbon source. In the first reaction of the methanol metabolism in the methanol-assimilating yeasts, formaldehyde and hydrogen peroxide are produced from methanol and oxygen by alcohol oxidase.
The hydrogen peroxide produced is decomposed by catalase into water and oxygen. The formaldehyde, on the other hand, is oxidized into carbon dioxide by formaldehyde dehydrogenase, S-formylglutathione hydrolase and formate dehydrogenase, and NADH produced in these reactions is utilized as an energy source for cells. Simultaneously, the formaldehyde is condensed with xylulose-5-phosphate by dihydroxyacetone synthase to be converted into glyceraldehyde-3-phosphate and dihydroxyacetone which are then converted to cell constituents via the pentose phosphate pathway.
When the methanol-assimilating yeasts are cultured in the presence of methanol, the above-mentioned alcohol oxidase, dihydroxyacetone synthase and formate dehydrogenase are produced in significant amounts and their contents reach about 40% of the intracellular soluble proteins.
Because a large scale cultivation of the methanol-assimilating yeasts can be done with inexpensive methanol as described above, and because they possess methanol inducible promoters with a strong transcriptional activity not observed in other yeasts, the methanol-assimilating yeasts can be considered to be yeasts suitable for a heterologous gene expression system.
Candida boidinii is one of the methanol-assimilating yeasts, and this yeast is used for studying a method of expressing a heterologous gene by use of a regulatory region of an alcohol oxidase gene (AOD1) (Japanese Patent LOP Publication No. 344,895/1993).
Formate dehydrogenase, like alcohol oxidase, is an enzyme produced in a significant amount, but it is an enzyme located downstream in the methanol metabolism and considered to undergo expression regulation different from that of alcohol oxidase. For example, formate dehydrogenase can, as revealed in the present invention, be induced and expressed depending on culture conditions, even in the presence of glucose by which alcohol oxidase expression is completely inhibited. Therefore, it is expected that a method of expressing a large amount of a heterologous gene, which is different from a method using an alcohol oxidase promoter, can be established.
However, no knowledge of the expression and regulation of formate dehydrogenase from Candida boidinii has been gained up to now. There is a need for a promoter for formate dehydrogenase to elucidate the expression and regulation of said enzyme and to express a heterologous gene efficiently by its strong transcriptional activity.
DISCLOSURE OF THE INVENTION
The object of the present invention is to provide a promoter and/or a terminator with a strong transcriptional activity to express a heterologous gene, an expression vector containing the promoter and terminator, a transformant carrying the expression vector and a process for producing an expression product of a heterologous gene by use of the transformant.
The present inventors did extensive research to elucidate the expression system of a formate dehydrogenase gene from a methanol-assimilating yeast Candida boidinii in order to effectively express a heterologous gene, and as a result, they found a promoter and/or a termi
REFERENCES:
Allen et al., Gene 162:99-104 (1995).
Sakai et al., Journal of Bacteriology 179(14):4480-4485 (1997).
Y. Sakai et al., "Expression of Saccharomyces Adenylate Kinase Gene in Candida Biodinii Under the Regualation of its Alcohol Oxidase Promoter", Appl. Microbiol. Biotechnol., vol. 42, (1995), pp. 860-864.
Y. Sakai et al., "Cloning and Sequencing of the Alcohol Oxidase-Encoding Gene (AOD1) from the Formaldehyde-Producing Asporogeneous Methylotrophic Yeast, Candida Boidinii S2", Gene, vol. 114, (1992), pp. 67-73.
Iwamatsu Akihiro
Kato Nobuo
Komeda Toshihiro
Sakai Yasuyoshi
Suda Hisako
Kirin Beer Kabushiki Kaisha
Railey II Johnny F.
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