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
2000-08-18
2002-07-23
Nashed, Nashaat T. (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...
C435S320100, C435S354000, C536S024100
Reexamination Certificate
active
06423510
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a promoter for dihydroxyacetone synthase gene (also referred to as DAS) from
Candida boidinii
; a gene expression cassette containing said promoter, a heterologous gene and a terminator; an expression vector containing said gene expression cassette; a transformant transformed with said vector; and a method for producing a heterologous gene product using said transformant.
BACKGROUND OF THE INVENTION
Methanol-assimilating yeasts are those capable of growing on a medium containing methanol as a sole carbon source. The methanol metabolism in the methanol-assimilating yeasts is performed as follows. In the first reaction 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 eventually into carbon dioxide via actions of 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. This yeast is used for studying a method for expressing a heterologous gene by use of a regulatory region of an alcohol oxidase gene or a formate dehydrogenase gene (see JP-A-5-344895 and International Publication WO97/10345).
Dihydroxyacetone synthase, as well as alcohol oxidase or formate dehydrogenase, is an enzyme produced in a significant amount, but no knowledge about expression control of this enzyme has been obtained so far. There is a need for a promoter of this enzyme to elucidate the expression control of this enzyme and to express a heterologous gene efficiently using its strong transcriptional activity.
The object of this invention is to provide a promoter with a strong transcriptional activity to express a heterologous gene; an expression vector containing said promoter; a host cell transformed with said expression vector; and a method for producing an expression product of heterologous gene using said host cell.
The present inventors did intensive research to elucidate the expression control system of a dihydroxyacetone synthase gene from a methanol-assimilating yeast
Candida boidinii
in order to effectively express a heterologous gene. As a result, the inventors found a promoter sequence with a strong transcriptional activity, achieved high expression of the heterologous gene using this sequence, and thereby arrived at the completion of the present invention.
SUMMARY OF THE INVENTION
The present invention relates to a promoter for dihydroxyacetone synthase gene from
Candida boidinii
, which substantially comprises a nucleotide sequence shown in SEQ ID:1. More specifically, the promoter of this invention substantially comprises a continuous 498 bp nucleotide sequence of SEQ ID NO:1, 364 to 861. Examples of this promoter include, but are not limited to, those substantially comprising a continuous 861 bp nucleotide sequence of 1 to 861, a continuous 1281 bp nucleotide sequence of 1 to 1281, and a continuous 1843 bp nucleotide sequence of 1 to 1843, all of which contain the region of the continuous 498 bp nucleotide sequence from 364 to 861 of SEQ ID NO:1. In addition, a sequence that does not contain a sequence of 364 to 861 in SEQ ID NO: 1 but possesses a promoter activity falls into the scope of this invention (FIG.
5
).
As used herein, the term “substantially comprises a nucleotide sequence” means that the nucleotide sequence of SEQ ID NO:1 may have mutations, such as substitution, deletion, addition or insertion insofar as a desired promoter activity can be obtained to some degree. For example, the nucleotide sequence of SEQ ID NO:1 in which “A” at position 3 have been substituted by “T” also falls into the scope of this invention as long as the desired promoter activity can be obtained. That is, the present invention also includes a mutated or modified gene having a homology of 70% or higher, preferably 80% or higher, more preferably 90% or higher to the above defined nucleotide sequence of SEQ ID NO:1 and having a desired promoter activity.
The present invention also provides an expression cassette comprising the promoter and a heterologous gene. This expression cassette may further contain a terminator sequence or may contain a selectable marker sequence (e.g., a drug-resistant gene) or a ribosome binding site if necessary.
Further the present invention relates to an expression vector which enables the expression of a heterologous gene containing the above expression cassette.
The term “a heterologous gene” as used herein means any gene to be expressed. Such heterologous genes include, but are not limited to, an acid phosphatase gene, &agr;-amylase gene, various interferon genes, erythropoietin gene, and granulocyte colony stimulating factor gene. These genes may be obtained by any techniques.
The present invention further relates to a transformant transformed with said expression vector.
Further, the present invention relates to a method for producing an expression product of the heterologous gene, comprising culturing said transformant in an appropriate medium, and recovering the expression product of the heterologous gene from the cultured cell. Such a medium includes, but is not limited to, media containing, as a carbon source, methanol and methanol supplemented with glycerol.
This specification contains the whole or part of the content described in the specification and/or drawings of Japanese Patent Application No. 10-37263, which is the priority claimed in this application.
REFERENCES:
patent: 0 558 024 (1993-02-01), None
patent: 97/10345 (1997-03-01), None
Haywood G. et al, Microbial oxidation of amines. Distribution, purification and properties of two primary-amine oxidases from the yeastCandida boidinigrown on amines as sole nitrogen source, Biochem., J. 1981, 199, 187-201.*
Allen S. J. et al, Isolation, sequene and overexpression of the gene encoding NAD-dependent formate dehydrogenase from the methylotrophic yeastCandida methylica,Gene, 1995, 162, 99-104.*
Y. Sakai et al.,Journal of Bacteriology,vol. 180, No. 22, Regulation and Physiological Role of the DAS1 Gene, Encoding Dihydroxyacetone Synthase, in the Methylotrophic YeastCandida boidinii,pp. 5885-5890, (Nov. 1998).
Kato Nobuo
Komeda Toshihiro
Kondo Keiji
Sakai Yasuyoshi
Foley & Lardner
Kirin Beer Kabushiki Kaisha
Nashed Nashaat T.
Walicka Malgorzata A.
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