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
1999-04-28
2001-08-14
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S440000, C435S471000, C435S320100, C435S252300, C435S254110, C435S254220, C536S024100
Reexamination Certificate
active
06274340
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a promoter with a high transcriptional activity useful in high level expression of heterologous genes, to an expression vector carrying the promoter, to a transformant introduced with the expression vector, and to a method for producing a heterologous protein by culturing the transformant.
BACKGROUND ART
A methanol assimilating yeast, i.e. methylotrophic yeast, is able to grow on methanol as a sole carbon source. In the initial reaction of methanol metabolism by the methylotrophic yeast, formaldehyde and hydrogen peroxide are produced from methanol and oxygen by alcohol oxidase. The produced hydrogen peroxide is catabolized into water and oxygen by catalase. The formaldehyde is oxidized to carbon dioxide through the actions of formaldehyde dehydrogenase, S-formylglutathione hydrolase, and formate dehydrogenase. NADH formed upon these oxidations becomes an energy source of the yeast cell. Simultaneously, due to the action of dihydroxyacetone synthase, the formaldehyde condenses with xylulose-5-phosphate, and the condensate product is converted into glyceraldehyde-3-phosphate and dihydroxyacetone, which become components of the yeast cell via pentose phosphate cycle. When the methylotrophic yeast is cultured in the presence of methanol, the above-mentioned alcohol oxidase, dihydroxyacetone synthase and formate dehydrogenase are produced in an extremely large amount, reaching about 40% of the intracellular soluble proteins.
As described above, the methylotrophic yeast is considered to be a suitable host for expression system of heterologous genes in that it can be mass-cultured with inexpensive methanol and that it has a promoter for methanol-metabolizing enzyme with a strong transcriptional activity unfound in other yeasts.
Candida boidinii
is a kind of methylotrophic yeast. Utilizing this yeast, methods for expressing a heterologous gene by using regulatory regions of alcohol oxidase gene and formate dehydrogenase gene have been studied (JP-A-5-344895, WO97/10345, etc.). In most cases of such an expression system, a heterologous gene is produced in a larger amount within a transformant having the chromosome into which a higher copy number of an expression vector has been integrated (Appl. Microbiol. Biotechnol., 42, 860-864 (1995); Proceedings of Annual Meeting of Japan Society for Bioscience, Biotechnology, and Agrochemistry, 1997, p. 257; and Proceedings of Annual Meeting of Society for Bioscience and Bioengineering, Japan, 1997, p. 314). Considering the stability of an expression vector within a transformant, it seems to be more desirable to achieve a high amount of expression at a low copy number, and there has been a demand for developing promoters with stronger transcriptional activity.
DISCLOSURE OF INVENTION
The present invention aims at providing a promoter with strong transcriptional activity useful in expressing heterologous genes, an expression vector carrying the promoter, a transformant into which the expression vector has been introduced, and a method for preparing a heterologous gene expression product by utilizing the transformant.
For solution of the above problem, The present inventor has conducted intensive studies on development of promoters with high transcriptional activity. As a result, the present inventor has now found that a DNA fragment prepared by adding nucleotides with a specific nucleotide sequence to the promoter DNA fragment that functions in the methylotrophic yeast
Candida boidinii,
was used as a promoter to efficiently express a heterologous gene downstream of the promoter. By this finding, the present invention has been accomplished.
Accordingly, the present invention provides a DNA for enhancing promoter activity which comprises the nucleotide sequence shown in SEQ ID NO:1, or the nucleotide sequence having deletions, substitutions, additions or insertions of one or more nucleotides in the nucleotide sequence shown in SEQ ID NO:1.
The present invention also provides a mutant promoter wherein one or more fragments of the above-defined DNA are located at at least one position preceding, following or within any promoter in a forward or reverse direction.
The present invention further provides a recombinant expression vector comprising the mutant promoter together with a heterologous gene. The heterologous gene as used herein refers to any gene that is a target of expression unlimited to specific one. Examples of the heterologous gene include genes for acid phosphatase, &agr;-amylase, various interferons, erythropoietin, and granulocyte colony-stimulating factor. The heterologous genes useful in the present invention are not limited by the method for preparing them.
The present invention still further provides a transformant prepared by transforming a host cell with the vector.
The present invention still yet further provides a method for enhancing a promoter activity, characterized in that one or more fragments of the above-defined DNA are located at at least one position preceding, following or within any promoter in a forward or reverse direction.
Moreover, the present invention provides a process for preparing an expression product, comprising culturing the above defined transformant in a medium, and recovering the expression product of a heterologous gene from the obtained culture. Examples of the medium include a medium containing methanol as a carbon source, a medium containing methanol supplemented with glycerol, and a medium containing formic acid supplemented with any carbon and nitrogen sources.
Hereinafter, the present invention will be described in more detail.
In accomplishing the present invention, the present inventor has identified a region (hereinafter, referred to as “UAS sequence”) requisite for a transcriptional activity of a formate dehydrogenase gene promoter (hereinafter, often abbreviated as “FDH promoter”) of the methylotrophic yeast
Candida boidinii.
More specifically, the DNA of the present invention is an UAS sequence comprising the nucleotide sequence 841-880 of the formate dehydrogenase gene promoter from
Candida boidinii
having the nucleotide sequence shown in SEQ ID NO:2.
As used herein, “any promoter” refers to a DNA fragment exhibiting a transcriptional activity in a host cell, which is not limited to specific one. Therefore, any promoter (i.e., a promoter DNA fragment) useful in the present invention may be derived from any organisms as long as it has a transcriptional activity. Moreover, any promoter may partially include a mutation such as substitution, deletion, addition or insertion in a nucleotide sequence from the corresponding wild type promoter as long as it has a transcriptional activity. The location and the number of the substitution, deletion, addition or insertion are not limited to specific ones. As the promoter of the invention, a
Candida boidinii
formate dehydrogenase gene promoter (SEQ ID NO:2) and a
Candida boidinii
actin gene promoter (SEQ ID NO:3) are exemplified. For instance, even a DNA fragment having a deletion of the nucleotides 1-300 in the nucleotide sequence shown in SEQ ID NO:2 may also be included in any promoter of the present invention as long as it exhibits a transcriptional activity in
Candida boidinii.
The introduction of the mutation may be carried out by general genetic engineering techniques including restriction enzyme treatment, utilization of chemically synthesized DNA, PCR method, site-directed mutagenesis, and methods for producing deletion mutants using Exonuclease III.
The mutant promoter of the present invention can be prepared by locating the UAS sequence at any promoter DNA fragment as described above in either a forward direction or a reverse direction.
Locating the UAS sequence in a forward direction means locating (e.g., adding or inserting) a sequence homologous to the UAS sequence, i.e.,
5′-TTTACCACTATCCAATTAAAATCCATGGATCAGACGGTAG-3′ (SEQ ID NO:1),
at the promoter DNA fragment in the same 5′→3′ orientation as the orientation of the promoter DNA fragment. On the othe
Foley & Lardner
Kirin Beer Kabushiki Kaisha
Yucel Remy
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