JP2003210173A - New promoter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new promoter which can carry out the high efficient expression of a useful gene in a yeast belonging to the genus Candida. <P>SOLUTION: This modified GCN4 promoter enabling the high efficient expression of the gene is obtained by introducing a mutation into at least one translation-starting codon existing between a translation-starting point and a structural gene, on GCN4 gene originated from the yeast belonging to the genus Candida. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel promoter derived from Candida yeast. More specifically, the present invention relates to a promoter capable of expressing a useful gene constitutively and highly efficiently at the translation level in Candida yeasts without depending on induction conditions such as culture conditions and medium conditions.

[0002]

2. Description of the Related Art With the development of gene recombination technology, useful proteins and useful chemicals have been produced using microorganisms. First, development of a gene recombination system using prokaryotic Escherichia coli or Bacillus subtilis is actively progressed, and various useful substances are produced particularly using a host-vector system of Escherichia coli. However, proteins produced in E. coli sometimes form insoluble granules in the cells, and there is a problem that sugar chain addition characteristic of eukaryotes cannot be performed.

On the other hand, the development of a system using a eukaryotic yeast as a host has been advanced. Yeast has been used for brewing and bread making for a long time, and also has the experience of being produced for feed, which guarantees high safety. It is also possible to add sugar chains to the produced protein,
It is a characteristic that distinguishes it from prokaryotes. For example, in addition to Saccharomyces cerevisiae, which has abundant genetic knowledge, in the genera Shiwaniomyces, Kluyveromyces, Pichia, Hansenula, Yarrowia, and Candida,
Host-vector system is being developed (Nonconventional
Yeasts in Biotechno1ogy, Klaus Wolf, Springer).

Among these yeasts, there is one that can grow using a straight chain hydrocarbon (n-alkane) as the sole carbon source (referred to as "n-alkane assimilating yeast"). Candida ma1tos of the genus Candida
a), Candida tropicali
s) etc. and Hansenula po1ym
orpha), Yarrowia 1ipo1y
tica) etc. These yeasts have an oxidase system that oxidizes the ends of n-alkanes, and decompose the long-chain carboxylic acid generated by oxidation to acetyl-CoA, which is a substrate of the TCA cycle in the β-oxidation system of peroxisomes, and is the energy source. Can be used as

The n-alkane-assimilating yeast not only grows using a straight-chain hydrocarbon as a carbon source, but also is resistant to a hydrophobic substance that inhibits the growth of ordinary yeasts, and is a useful substance by conversion of a hydrophobic chemical substance. It is promising as a host that provides a place for production and reaction of. Therefore, by producing such a heterologous gene expression system in yeast, it is possible to construct a new useful chemical substance production system that does not generate harmful by-products and does not waste energy.

In Candida maltosa among n-alkane assimilating yeasts, a heterologous gene expression system has been actively constructed. M. Kawamura et al.
onability = hereinafter abbreviated as TRA) (M, Kawamur)
a, et a1., Gene, vol.24, 157, (1983)). It was found that this region contains a sequence involved in autonomous replication in Candida maltosa (Autonomously rep1icating sequence: abbreviated as ARS hereinafter) and a centromere sequence (hereinafter abbreviated as CEN). To date, a low copy vector having the entire TRA region and a high copy vector excluding the CEN region, which is expected to highly express the transgene, have been developed (M. Ohkuma, et al., Mo1. Gen. Genet., Vo.
l.249, 447, (1995)).

By the way, a promoter that functions in the yeast is required for gene expression in Candida maltosa, and a plurality of promoters are available. Candida maltosa highly produces enzymes of the n-alkane oxidation system in the presence of alkanes. In particular, the gene encoding cytochrome P450 (abbreviated as ALK), which performs the initial oxidation of alkanes, is strongly induced (M. Ohoku).
ma, et a1., DNA and Ce11 Biology, vol.14, 163,
(1995)), and transcription of genes encoding β-oxidation enzymes is also induced (Y. Masuda, et al., Gene, vol.16).
7, 157, (1995)). The promoter of the ALK1 gene in the ALK gene group is most strongly induced by n-alkane and can be used for gene expression. on the other hand,
In the presence of fatty acids, ALK2 and ALK5 gene promoters are suitable.

The promoter of phosphoglycerate kinase (hereinafter abbreviated as PGK), which is known as a glycolytic enzyme, is known to induce strong gene expression in the presence of glucose. C of Candida Maltosa
The GK promoter was cloned by Y. Masuda et al. (Y. Masuda, et a1., Curr. Genet., Vol.25,
412, (1994)). Furthermore, the GAL promoter, which has a strong gene expression-inducing activity in the presence of galactose, was also cloned (SM Park, et a1., Yeast, vo).
l.13, 21 (1997)). ALK promoter, PGK promoter and GA cloned in this way
The L promoter can be used for gene expression in Candida maltosa.

However, the ALK promoter hardly functions when glucose or the like is used as the carbon source, and the PGK promoter hardly functions when fatty acid or n-alkane is used as the carbon source. Therefore, in producing a useful substance in Candida maltosa, a carbon source suitable for producing the substance is not necessarily suitable for strong gene expression. Further, the GAL promoter is inducible only when galactose is used as a carbon source, and thus it is not suitable for industrial production because expensive galactose must be used.

Therefore, in order to produce a useful substance with high efficiency using Candida maltosa, a new promoter has been desired which is not restricted by the kind of carbon source and can express a gene more strongly than the above promoter. . Recently, the GCN4 gene of Candida maltosa (hereinafter abbreviated as C-GCN4) has been cloned (Takahisa et al.,
2000 Molecular Biology Society Annual Meeting). It is already known that the gene is closely related to the regulation of gene expression in the amino acid biosynthesis system in Saccharomyces cerevisiae (AG Hinnebusch, EW Jones et al., Ed., The M.
olecular and Ce11ular Bio1ogy of the Yeast Sacchar
omyces, vo1.2, 317, Co1d Spring Harbor Laboratory
Press).

Saccharomyces cerevisiae GCN4
The gene (hereinafter abbreviated as S-GCN4) has a long 5'untranslated region and has four upstream open reading frames (hereinafter abbreviated as uORF) in the same region. S-GCN4
Gene expression is suppressed in nutrient-rich culture conditions,
Induced under culture conditions such as amino acid starvation. Such gene expression induction is due to enhancement of translation activity.

Further, by introducing mutations into the translation initiation codons of the four uORFs in the 5'untranslated region of the S-GCN4 gene, the translation activity of the gene can be maintained even under culture conditions rich in nutrient sources. Known to rise (G. Thireos et a1., Proc. Natl. Acad. Sci. USA, v
ol.81, 5096 (1984), PP Muellereta1., Cell, vo
l.45, 201 (1986)). From this, it is considered that the gene expression using the S-GCN4 promoter having the above-mentioned mutation introduced is not affected by the difference in carbon source.

On the other hand, by adding 3-amino-1,2,4-triazole (hereinafter abbreviated as 3-AT) to the medium, S
-A gene expression system utilizing the GCN4 promoter has also been constructed (A. Mimran, et a1., Biotechniques,
vol.28, 552, (2000), W000 / 52181). This system can carry out gene expression to the same extent as the aforementioned mutagenized S-GCN4 promoter. Therefore, using Saccharomyces cerevisiae as a host, production of human serum albumin using this system was examined. However, the productivity of human serum albumin is very low at 0.5 mg / L, and in this production system, it is a histidine synthesis system inhibitor 3
-Because it is necessary to add AT, it was considered difficult to apply it to industrial production.

Therefore, S-GCN4 was not considered to be useful as a promoter for producing a useful substance in Saccharomyces cerevisiae, although the gene translation activity was increased by the introduction of mutation. Unlike Saccharomyces cerevisiae, the yeast of the genus Candida has no known sexual generation, and most have a diploid genome. It has also been reported that the reading of the genetic code is abnormal. Candida Cyrindrasse (Cand
ida cy1indraceae) (Y. Kawaguchi, et al., Nature, v
ol.341, 164 (1989)) and Candida Maltosa (H. Sugiyama, et a1., Yeast, vol11, 43 (1995))
Has been reported to read the CUG codon, which normally encodes a leucine codon, as serine. Thus, it can be said that the yeast of the genus Candida has properties greatly different from those of Saccharomyces cerevisiae.

The above-mentioned C-GCN4 cloned by Takahisa et al. Is different from S-GCN4 in that it has three types of u.
Contains the ORF. GCN found in Saccharomyces cerevisiae due to the difference in the structure of the GCN4 gene and the peculiarities of the Candida yeast described above.
It is unclear whether or not the increase in translation activity due to the introduction of mutations into the four genes is also observed in yeast of the genus Candida. Moreover, it was not considered that C-GCN4, like S-GCN4, has a strong promoter activity required for substance production.

[0016]

An object of the present invention is to provide a promoter capable of strong gene expression in yeast regardless of the type of carbon source.

[0017]

Means for Solving the Problems As a result of intensive investigations by the present inventors, the 5'of the Candida maltosa GCN4 gene was found.
The inventors have found that the promoter activity is increased by introducing mutations into the three types of translation initiation codons present in the untranslated region, and have completed the present invention. The mutated GCN
The promoter showed extremely high promoter activity as compared with the PGK promoter and GAL promoter. Moreover, it was considered that the promoter activity was not affected by the carbon source.

That is, the present invention provides the following (1) to (13). (1) Modified G obtained by introducing a mutation into at least one translation initiation codon existing between the transcription start point and the structural gene on the GCN4 gene derived from Candida yeast
CN4 promoter. (2) A modified GCN4 promoter obtained by introducing mutations into all translation initiation codons existing between the transcription start point and the structural gene on the GCN4 gene derived from Candida yeast.

(3) The promoter according to (1) or (2) above, wherein the GCN4 gene derived from Candida yeast is the GCN4 gene derived from Candida maltosa. (4) The GCN4 gene has the following DNA (a) or (b):
The promoter according to (3) above, which comprises: (a) DNA represented by SEQ ID NO: 1 (b) DNA hybridizing with a DNA having a nucleotide sequence complementary to the DNA represented by SEQ ID NO: 1 under stringent conditions and having GCN4 gene activity

(5) The promoter according to (3) above, wherein the GCN4 gene comprises the DNA represented by SEQ ID NO: 1. (6) Including the nucleotide sequence shown in SEQ ID NO: 2, above (1) to
The promoter according to any one of (5). (7) The promoter is wild type GCN due to mutation introduction.
50 more in protein expression than 4 promoters
The promoter according to any one of (1) to (6) above, which is improved by a factor of 2 or more.

(8) A promoter comprising the following DNA (a) or (b): (a) DNA represented by SEQ ID NO: 2 (b) Hybridized with a DNA consisting of a nucleotide sequence complementary to the DNA represented by SEQ ID NO: 2 under stringent conditions and consisting of the DNA represented by SEQ ID NO: 2 DNA having a promoter activity equivalent to that of the promoter (9) An expression vector containing the promoter according to any one of (1) to (8) above.

(10) A transformant containing the expression vector according to (9) above. (11) The host is yeast belonging to the genus Candida,
(10) The transformant described. (12) A method for producing a GCN4 promoter having improved promoter activity by introducing a mutation into at least one translation initiation codon existing between a transcription start point and a structural gene on a GCN4 gene derived from Candida yeast. (13) On the GCN4 gene derived from Candida yeast,
A method for improving the promoter activity of a gene by introducing a mutation into at least one translation initiation codon existing between the transcription start point and the structural gene.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. 1. Construction of Modified GCN4 Promoter (1) GCN4 Gene The “GCN4 gene” of the present invention is a transcription of a gene group that regulates an amino acid biosynthesis system (at least 12 different amino acid biosynthetic pathway networks). It is a gene encoding an active factor and is found in yeasts such as Saccharomyces and Candida.

In the present invention, the GCN4 gene is
The GCN gene may be derived from any species as long as it is derived from Candida yeast. Such yeasts of the genus Candida include, for example, Candida maltosa (Candida ma1tosa) and Candida tropicalis (C
andida tropicalis) and the like, but Candida maltosa is particularly preferable.

As a preferred example of the GCN4 gene according to the present invention, C-GCN derived from Candida maltosa
There are four genes. The outline of the structure of the gene is shown in FIG. The gene consists of, for example, DNA having the nucleotide sequence shown in SEQ ID NO: 1. However, a DNA that hybridizes under stringent conditions with a DNA having a base sequence complementary to the DNA represented by SEQ ID NO: 1
As long as it has GCN4 gene activity, it is included in the GCN4 gene. Here, the GCN4 activity means a function of activating transcription of a gene group involved in regulation of amino acid synthesis. In addition, stringent conditions include, for example, sodium concentration of 10 mM.
˜300 mM, temperature 25 ° C. to 70 ° C., preferably sodium concentration 50 mM to 100 mM, temperature 42 ° C. to 55 ° C.

As shown in FIG. 1, in the upstream region from the transcription initiation point to the translation initiation point of the C-GCN4 gene derived from Candida maltosa, G controlling the expression of GCN4 gene was expressed.
There are three upstream open reading frames (hereinafter abbreviated as uORF) that encode a CN4 promoter region and a peptide. Each position on the C-GCN4 gene has a promoter region of 62 in SEQ ID NO: 1.
6 to 1482, structural gene region is 1483 to 2427, uO
It is considered that RF1 is the base region of the 1154-1165th position, uORF2 is the 1289-1300th position, and uORF3 is the 1324-1350th base region. It should be noted that the promoter region is not defined academically, but exhibits promoter activity and is a region that can be used as a promoter in the present invention.

(2) Modified GCN4 promoter "Modified GCN4 promoter" according to the present invention
Can be obtained by introducing a mutation into at least one translation initiation codon ATG existing between the transcription start point and the structural gene on the GCN4 gene derived from Candida yeast. That is, the "modified GCN4 promoter" of the present invention means GCN whose promoter activity is improved by mutation introduction as compared with the wild type GCN4 promoter.
4 promoters. The improvement of the promoter activity is
The expression level of the protein is preferably 50 times or more that of the wild type GCN4 promoter, and particularly 8
It is preferably 0 times or more. Here, "Wild type GC
"N4 promoter" means a natural GCN without mutation
Means 4 promoters. Further, "mutation" means substitution, deletion or addition of bases. The number of bases to be substituted, deleted or added is not particularly limited as long as it can destroy the target uORF. The number of translation initiation codons to be mutated is not particularly limited, but it is preferably performed for all translation initiation codons existing on the GCN4 gene.

The reason why the promoter activity is improved by the introduction of the mutation is not clear, but it is considered that the translation activity is increased by introducing the mutation into the translation initiation codon ATG of uORF. The modified GCN4 promoter of the present invention means a gene in a region having promoter activity, and does not mean the entire region on the GCN4 gene including the disrupted uORF used in its production. .

(3) Mutation introduction into the GCN4 gene The above-mentioned mutation introduction into the GCN4 gene can be performed based on a known method, for example, by combining a mutation introduction primer and a PCR method. The mutation-introducing primer can specifically amplify a target gene fragment based on a known method.
It may be prepared by designing an oligonucleotide having a length of about 5 to 25 bases and introducing a desired mutation (base substitution, insertion or deletion) into the oligonucleotide. Introduction of multiple mutations is achieved by partially PCR-amplifying a target gene and cutting and ligating the obtained amplified fragment with an appropriate restriction enzyme.

As the amplification condition by PCR, any condition may be used as long as the target gene fragment can be amplified. In addition, the DNA polymerase used is of the amplified gene fragment.
It is preferable to use a type of polymerase that does not add A (adenine) to the 3'side. Examples of the DAN polymerase include KOD DNA polymerase (TOYOBO
Manufactured by the company) and the like.

As a preferred embodiment of the present invention, the three uORFs of the GCN4 gene of Candida maltosa (hereinafter, uORF1 and uORF from the gene 5'upstream, respectively)
2, referred to as uORF3)
A method for introducing a mutation into G will be described below.

First, the primer ORF31-FP (SEQ ID NO: 3)
And ORF31-RP (SEQ ID NO: 5), C-GCN4
PCR is performed using the gene (SEQ ID NO: 1) as a template, and OR
An amplified DNA fragment uORF31 having a mutation introduced into F3 is prepared. Similarly, using another primer pair ORF32-RP (SEQ ID NO: 4) and ORF32-FP (SEQ ID NO: 6), C-
Amplified DNA by PCR reaction using GCN4 gene as a template
Generate the fragment uORF32.

Next, the uORF31 and uORF32
Were digested with restriction enzymes SpeI, and the digested fragments were digested with DNA.
Connect with ligase. Using the ligated DNA fragment as a template, the ORF31-FP (SEQ ID NO: 3) and ORF3 described above were further prepared.
A PCR reaction is carried out using a 2-RP (SEQ ID NO: 4) primer to prepare an amplified DNA fragment m-uORF3.

Further, using the m-uORF3 as a template, OR
A PCR reaction was performed using F32-RP (SEQ ID NO: 4) and ORF23-FP (SEQ ID NO: 7) primers to obtain an amplified DNA fragment uO.
RF23 is produced. Also, with ORF31-FP (SEQ ID NO: 3)
Using the ORF21-RP (SEQ ID NO: 8) primer, C-GC
A PCR reaction is performed using the N4 gene as a template to prepare an amplified DNA fragment uORF21. Fabricated uORF21 and uO
RF23 is cleaved with restriction enzyme MunI, and the cleaved fragments are ligated with DNA ligase.

UORF21 and uOR connected in this way
ORF32-RP using the DNA fragment composed of F23 as a template
(SEQ ID NO: 4) and ORF13-FP (SEQ ID NO: 9) primers were used for PCR reaction to obtain amplified DNA fragment uORF13.
To make. In addition, ORF31-FP (SEQ ID NO: 3) and ORF1
C-GCN4 using the 1-RP (SEQ ID NO: 10) primer
PCR reaction is performed using the gene as a template, and amplified DNA fragment uO
RF11 is produced. Fabricated uORF11 and uORF
13 is cut with restriction enzymes Spel and XbaI, respectively, and the cut fragments are ligated with DNA ligase.

UORF11 and uOR thus connected
ORF31-FP using the DNA fragment composed of F13 as a template
(SEQ ID NO: 3) and ORF32-RP (SEQ ID NO: 4) primers were used for PCR reaction, and amplified DNA fragments m-ORF1 and m-ORF1,2 in which mutations were introduced in all translation initiation codons of three uORFs of C-GCN4 gene. , 3 can be manufactured.

M-ORFs 1, 2 and 3 contain 9 amino acids of the coding region of the C-GCN4 gene, but as long as they have a promoter function, they are 3'downstream of uORF3 having a mutation introduced in the translation initiation codon. Any region may be used as the promoter of the present invention. Any region may be used as the promoter of the present invention on the 5 ′ upstream side of the mutated uORF1 as long as it has a promoter function.

A preferred example of the promoter sequence of the present invention thus obtained is shown in SEQ ID NO: 2. A DNA capable of hybridizing with a DNA having a nucleotide sequence complementary to the DNA represented by SEQ ID NO: 2 under stringent conditions,
It is included in the promoter of the present invention as long as it has a promoter activity equivalent to that of the gene consisting of the DNA shown in SEQ ID NO: 2. Here, the stringent conditions include, for example, sodium concentration of 10 mM to 300 mM and temperature of 25 ° C.
˜70 ° C., preferably 50 mM to 100 mM in sodium concentration, and 42 ° C. to 55 ° C. in temperature. The promoter activity of the gene consisting of the DNA shown in SEQ ID NO: 2 is shown in Table 1 of Example 2 described later.

2. Analysis of promoter function (1) Analysis method The functional analysis of a novel promoter having a mutation introduced in the translation initiation codon of uORF is performed by quantifying mRNA transcribed by the promoter or a gene product translated from the mRNA. You can In particular, it is preferably carried out by connecting a so-called reporter gene such as β-galactosidase gene or acid phosphatase gene, which can measure enzyme activity, downstream of the promoter. As a preferable example of the analysis method, an analysis method using a plasmid pPL1 containing a β-galactosidase gene derived from yeast Kluyveromyces 1actis (M. Ohkuma, et a1., Biosci. Biote
ch. Biochem., vol.59, 1328, (1995)) (Fig. 2).

(2) Construction of vector First, pPL1 was cleaved with restriction enzymes SalI and SmaI to give m-uOR.
Cleavage F1,2,3 with the restriction enzyme Sa1I. These DN
The A fragment was ligated with DNA ligase to obtain pPL-CGCN4
^{Build u0RFl, 2,3} . pPL-CGCN4 ^{u0RFl, 2,3} is an expression vector containing the β-galactosidase gene downstream of the promoter of the present invention.

(3) Preparation of transformant Then, according to a known method, Candida maltosa is transformed with the above plasmid. For example, the calcium method (EM Lederberg, et a1., J. Bacterio
1., vol.119, 1072 (1974)) and electroporation method (Currnt Protoco1s in Molecular Biology, Volume 1,
1.8.4, 1994) etc. can be used. Also, Fa
st TrackTM-Yeast Transformation KitSM (GenoTechnol
It is also possible to use a commercially available transformation kit such as ogy).

The host of the transformant is, for example,
Candida Maltosa CMT101 (his5 :: HIS5, ade1,
ura3 :: URA3) can be preferably used. CMT101 is Candida maltosa CHAU1 (his5, ade1, ura3 / ura
3) (M. Ohkuma, et a1., Curr. Genet., Vol.23, 205,
(1993)) the histidine and uracil requirements of HIS
5, it was prepared by releasing the Ura3 gene by transformation.

(4) Promoter activity Using the obtained Candida maltosa transformant of pPL-CGCN4 ^{u0RFl, 2,3} , functional analysis of the novel promoter is carried out. Cultivation of the transformant is performed using a carbon source that can be assimilated by the transformant, and the β-galactosidase activity expressed in the cells is measured. The measurement is performed, for example, by Y. Mas
Method of uda et al. (Y. Masuda, et a1., Curr. Genet., vol.
25, 412, (1994)).

3. Expression Vector The expression vector of the present invention comprises the modified GCN4 of the present invention.
It is a vector containing a promoter and a gene to be expressed in the downstream region thereof in a manner capable of functioning. The vector can be constructed using an appropriate vector such as a plasmid, for example, as described in the above item (2). The functional aspect means an aspect in which the promoter of the present invention can exhibit a promoter activity for a gene to be expressed.

4. Transformant The transformant of the present invention can be obtained by transforming a host with the above vector. As a transformation method, as described in the above item (3), a known method such as a calcium method or an electroporation method may be used. The transformant can be selected using, for example, drug resistance or auxotrophy as an index. The host of the transformant of the present invention is not particularly limited as long as the promoter of the present invention can effectively function, but Candida yeast is preferable, and Candida maltosa is particularly preferable. Since the transformant of the present invention uses yeast as a host, it is highly safe and has a sugar chain addition function. Moreover, since the promoter of the present invention having a high promoter activity is included, highly efficient gene expression is possible.

[0046]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited by these examples. Example 1: Construction of a novel promoter Construction of a new promoter was carried out by using a primer for introducing a mutation and P
Carried out by CR. PCR was performed using KOD DNA polymerase (manufactured by TOYOBO) at 98 ° C for 2 minutes for 1 cycle, 98
It was carried out at 30 ° C. for 15 seconds at −46 ° C. for 2 seconds and −74 ° C. for 30 seconds at 74 ° C. for 1 minute.

First, a mutation was introduced into the translation initiation codon of uORF3. ORF31-FP (SEQ ID NO: 3) and ORF31-RP
(SEQ ID NO: 5) Using the C-GCN4 gene (SEQ ID NO: 1) as a template, a PCR reaction was carried out to prepare a target gene fragment uORF31. Similarly, ORF32-RP
Using the (SEQ ID NO: 4) and ORF32-FP (SEQ ID NO: 6) primers, a PCR reaction was performed using the C-GCN4 gene as a template to prepare an amplified DNA fragment uORF32.

Then, the above uORF31 and uORF32
Were digested with restriction enzymes SpeI, and the digested fragments were digested with DNA.
Connect with ligase. Using the ligated DNA fragment as a template, the ORF31-FP (SEQ ID NO: 3) and ORF3 described above were further prepared.
A PCR reaction was performed using a 2-RP (SEQ ID NO: 4) primer to prepare an amplified DNA fragment m-uORF3 in which the translation initiation codon ATG of uORF3 was replaced with AGT.

Furthermore, mutation was introduced into uORF1 and uORF2. Using the m-uORF3 as a template, ORF32-
A PCR reaction was performed using RP (SEQ ID NO: 4) and ORF23-FP (SEQ ID NO: 7) primers to obtain an amplified DNA fragment uORF.
23 was produced. In addition, ORF31-FP (SEQ ID NO: 3) and ORF2
Using 1-RP (SEQ ID NO: 8) primer, PCR reaction was carried out using C-GCN4 gene as a template, and amplified DNA fragment uOR
F21 was produced. Fabricated uORF21 and uORF2
3 was cleaved with restriction enzyme MunI, and the cleaved fragment was DN
Ligated with A ligase.

UORF21 and uOR thus connected
ORF32-RP using the DNA fragment composed of F23 as a template
(SEQ ID NO: 4) and ORF13-FP (SEQ ID NO: 9) primers were used for PCR reaction to obtain amplified DNA fragment uORF13.
To make. In addition, ORF31-FP (SEQ ID NO: 3) and ORF1
C-GCN4 using the 1-RP (SEQ ID NO: 10) primer
PCR reaction is performed using the gene as a template, and amplified DNA fragment uO
RF11 is produced. Fabricated uORF11 and uORF
13 is cut with restriction enzymes Spel and XbaI, respectively, and the cut fragments are ligated with DNA ligase.

UORF11 and uOR thus connected
ORF31-FP using the DNA fragment composed of F13 as a template
PCR was performed using (SEQ ID NO: 3) and ORF32-RP (SEQ ID NO: 4) primer, and an amplified DNA fragment m-uORF1,2 having mutations introduced in all the translation initiation codons of the three uORFs of the C-GCN4 gene. , 3 (SEQ ID NO: 2) could be prepared. In this fragment, the translation initiation codon ATG of uORF1 is converted to AGA, the translation initiation codon ATG of uORF2 is converted to CAA, and the translation initiation codon ATG of uORF3 is converted to AGT.

Example 2 Analysis of Promoter Function m-uOR, the novel promoter constructed in Example 1
The function of F1,2,3 (SEQ ID NO: 2) is β derived from the yeast Kluyveromyces lactis.
It was analyzed by utilizing the plasmid pPL1 containing the galactosidase gene (M. Ohkuma, et al., Biosci.
Biotech. Biochem., Vol.59, 1328, (1995)) (Fig. 2)

1) Promoter function of m-u ORFs 1, 2, and 3 First, pPL1 was cleaved with restriction enzymes Sa1I and SmaI, and the mu-u
ORFs 1, 2 and 3 were cut with a restriction enzyme Sa1I. These D
The NA fragments were ligated by DNA ligase, resulting in the construction of pPL-CGCN4 ^{uORFl, 2,3} . Next, the plasmid was added to Candida maltosa CMT1O1 (his5 :: HIS5, ad
e1, ura3 :: URA3) was transformed using the lithium acetate method (M. Takagi, et a1., J. Bacterio1ogy, vo1.167,
551, (1986)). CMT101 is Candida Maltosa C
HAU1 (his5, ade1, ura3 / ura3) (M. Ohkuma, et a1., Cu
rr, Genet., vol.23, 205, (1993)), the histidine- and uracil-auxotrophic requirements were eliminated by transforming the HIS5 and Ura3 genes. In this way, pPL-CG
Candida maltosa transformants of CN4 ^{uORFl, 2,3} were obtained.

Culture of the transformant was carried out in SD medium (manufactured by Difco).
Yeast Nitrogen Base w / o amino acid 0.17%, ammonium sulfate O.5%, and glucose 2%) were used at 30 ° C. The culture was inoculated into 10 ml of SD medium at 3% concentration,
After culturing for 5 hours, the β-galactosidase activity expressed in the cells was determined by the method of Y. Masuda et al. (Y. Masuda, et al., Cur.
r. Genet., vol.25, 412, (1994)).

2) As a control for comparison with a wild-type promoter, pPL1-CGC obtained by inserting a C-GCN4 promoter having no mutation introduced thereinto into pPL1.
N4 was prepared, and β-galactosidase in the transformant with the vector was measured. The PPL1-CGCN4 was prepared as follows. First, ORF31-FP (SEQ ID NO: 3) and ORF3
C-GCN4 using the 1-RP (SEQ ID NO: 5) primer
PCR was performed using the gene as a template to obtain an amplified DNA fragment. The obtained DNA fragment was cleaved with restriction enzyme Sa1I and ligated with pPL1 cleaved with restriction enzymes Sa1I and Smal using DNA ligase to obtain pPL1-CGCN4. The results are shown in Table 1.

[0056]

[Table 1]

As a reference, GAL promoter, P
The promoter activity when the GK promoter was inserted into a single copy vector having CEN and ARS, respectively,
It is summarized in Table 2 based on known literature.

[0058]

[Table 2]

3) Result The mutated GCN4 promoter has a higher expression level of the protein than the non-mutated promoter.
The promoter activity was nearly 100-fold. The activity was much higher than that of the known GAL promoter and PGK promoter.

[0060]

INDUSTRIAL APPLICABILITY According to the present invention, a promoter capable of expressing a useful gene constitutively and highly efficiently at the translation level in Candida yeast is provided without depending on induction conditions such as culture conditions and medium conditions. To be done.

[0061]

[Sequence list]                           SEQUENCE LISTING <110> KANEKA CORPORATION and JAPAN ENERGY CORPORATION <120> A new promoter <130> TKS-4689 <160> 10 <170> PatentIn Ver. 2.1 <210> 1 <211> 2684 <212> DNA <213> Candida maltosa <220> <223> C-GCN4 gene from Candida maltosa <400> 1 gatatcctcc gtttaacacc attgatatgt cgtatgccaa caataacgag ttcttacaca 60 atttacctga ttttcaacag gaaagaatcg tactcatctg ggaattacag aaacacgact 120 tgaatgtgaa aggaccgttt gttaaaaata ctgcaggatt gagtgtcaaa caaagtgaag 180 atttaccaga aagagtgtta gaaagacata gaggagagtt gctctgacga tgaacacaag 240 aagaagaact ccaccggtac tacgaatcaa gggaatctgg aaaaagatga cggtaaaaac 300 gataaaaagt ctcgggttcc aaaatggctc aaaatgaagt aaaacgtata aaatcaaata 360 tacaaacaaa caaacaaaca atcttgtcta gaattatttt cgggtttatt tttgcaaccc 420 ttaagttccg taagagcgga accggactaa ttacataaaa attatttagt tagttagtta 480 gttacacaga ataaataaat aacgggtgta acacgtgagc gcacgccatg acagatttgt 540 gtttaaattg aactgttaca gtttttcttc tttttacttt tcaattataa cgtcctttca 600 cttttgatta ctaattatga aagacaggtc cttatgtata gacagacaga tccataaaaa 660 atagaataat tgaggggtta taacggatga gtcataacgt tacttagcaa caaacaatga 720 agacgaagaa ttttattgcg ggaatttcat tgtgacagag agaaagaaaa gaaaaaaaaa 780 ttaagatttg atcaaataca cgactttgtt ttttttagcc tatgcatatg taatattatc 840 ccaccaataa tatcgtgttc acggcggaat ttctcctcgt atataaaact gtcctcgggt 900 acacgacgtg aaaatttttt tcacaagttt ccttttgaat actataaaaa tatctcactt 960 tttcctttac tccatgaaaa aaaaattttt tcagttccct tttttttttt cttcttcaat 1020 tatcaacaaa caaaacaaac aaaacaaacg aacctattaa ttaccttaaa tttttttaca 1080 actgattact tacattacta ttaattattt tttccccatt attgactatt acgtattatt 1140 agatatatcc actatgtctg cttaaattat tttattaaat ttacatattt ataaaaaaaa 1200 aagtttttat ttactatcca ttacagataa ataaaaaaaa agatcctcgc gctttgtttt 1260 gttaaaactc cttatattat tgcttaaaat gttgaaatag attacttatt atccccgtcc 1320 actatgacgt ttgttatcct aataccttaa attaaaaaga gaaaagaaaa gagattacac 1380 ccccctgcct agttattatt attattatta tccttaccgt ttattagttt tgttatttat 1440 tattatccta caaatttatc aattaagtta ttatcattaa aaatgtctgc tactactcct 1500 gctgtatacg aagattcttt atttgaatct caagatttat ttgctgctcc agctccacgt 1560 caaactgaag cttctttagc tgaaaaagac gttgaattag aattgatttc cgctttacca 1620 gcaatggaaa aatcaattaa cagagctcca tccccattcc aaatccactc cagtgttttg 1680 gattccgtgt ttagttcaaa catggatggg tctaatgaag ttgatcatac tcctatgttt 1740 gatgaattag attttatcat ggacggtgct aaagtcaatt cgaaagaaga ttggatctcc 1800 ttatttggag ttgaagaaac tggtgctact accactggtg acgcttctga tccaattctt 1860 tgtttagatg atgatgatgc acttgtacca aaagatgaac catgtttggg tgctttattg 1920 attgaaccat caccaaatgg attatcatct aacgatgaaa ccatttctgc tcctactact 1980 actgcctcca gtccagatat gtgtagcact gttgccacaa gtgttacttc tggtgctgtt 2040 aaaagaaagt ttagtgaagt tgcctatggt tcttcaaaag aacaatctca attgtttaca 2100 ccaaaccctt cttctacttt accaactcca atgttggatg ctaagccaag aaagagatct 2160 aaagttgatc atttgggttg tgttacttat tctaaaaaac aaagatctca accattgcaa 2220 ccaattgttg tttctggtat tgaagatcct gttgctttga aaagagctaa gaatacagaa 2280 gccgccagaa gatctagagc tcgtaaaatg gaaagaatga accaattgga agataaagtt 2340 gaagatttgg ttggtgaaaa acaagcttta caagatgaag ttgatagatt gaagagtttg 2400 ttgaccagtc atggtatact gttttaaaaa aaaaaggaaa actaaaaaaa aaaaaattaa 2460 aatataattg ttgcaaaaat ttactaaaaa aaaaaaagag aatattaatc attttagatt 2520 tatagtgttt atatataatt gaaaaataaa attattggtt ttattatcaa aagtttatta 2580 tcgtagatag ggaatacgtt ttgtgtacat aaatcttggt taggtttata accctaacgc 2640 acgatatttt tttttttttt tggtagattc cgatggaaga tatc 2684 <210> 2 <211> 857 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: modified C-GCN4 promoter from Candida maltosa <400> aggtccttat gtatagacag acagatccat aaaaaataga ataattgagg ggttataacg 60 gatgagtcat aacgttactt agcaacaaac aatgaagacg aagaatttta ttgcgggaat 120 ttcattgtga cagagagaaa gaaaagaaaa aaaaattaag atttgatcaa atacacgact 180 ttgttttttt tagcctatgc atatgtaata ttatcccacc aataatatcg tgttcacggc 240 ggaatttctc ctcgtatata aaactgtcct cgggtacacg acgtgaaaat ttttttcaca 300 agtttccttt tgaatactat aaaaatatct cactttttcc tttactccat gaaaaaaaaa 360 ttttttcagt tccctttttt tttttcttct tcaattatca acaaacaaaa caaacaaaac 420 aaacgaacct attaattacc ttaaattttt ttacaactga ttacttacat tactattaat 480 tattttttcc ccattattga ctattacgta ttattagata tatccactag atctgcttaa 540 attattttat taaatttaca tatttataaa aaaaaaagtt tttatttact atccattaca 600 gataaataaa aaaaaagatc ctcgcgcttt gttttgttaa aactccttat attattgctt 660 aaacaattga aatagattac ttattatccc cgtccactag tacgtttgtt atcctaatac 720 cttaaattaa aaagagaaaa gaaaagagat tacacccccc tgcctagtta ttattattat 780 tattatcctt accgtttatt agttttgtta tttattatta tcctacaaat ttatcaatta 840 agttattatc attaaaa 857 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> acgcgtcgac aggtccttat gtataga 27 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> agtatacagc aggagtagta gc 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> gactagtgga cggggataat aa 22 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> gactagtacg tttgttatcc taatacc 27 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> cccaattgaa atagattact tattatccc 29 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> cccaattgtt taagcaataa tataaggag 29 <210> 9 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> gctctagatc tgcttaaatt attttatta 29 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> gactagtgga tatatctaat aatacg 26

[0062]

[Sequence listing free text] SEQ ID No. 1: Candida
Maltosa-derived GCN4 gene SEQ ID NO: 2: modified Candida maltosa-derived GCN4 promoter SEQ ID NO: 3 to 10: primer

[Brief description of drawings]

FIG. 1 is a Candida maltosa C-GCN.
The outline of the structure of 4 genes is shown.

FIG. 2 shows an outline of the structure of a plasmid pPL1 using the β-galactosidase gene derived from the yeast Kluyveromyces lactis.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Masamichi Takagi             1-31-10 Sakaemachi, Fuchu-shi, Tokyo (72) Inventor Akinori Ota             57-2 Plaza, Saitama City, Saitama Prefecture F-term (reference) 4B024 AA20 CA03 CA04 CA05 DA12                       FA02                 4B065 AA73 AB01 BA02 CA60

Claims (13)

[Claims] 1. A modified GCN4 promoter obtained by introducing a mutation into at least one translation initiation codon existing between a transcription start point and a structural gene on a GCN4 gene derived from Candida yeast. 2. A modified G obtained by introducing mutations in all translation initiation codons existing between the transcription start point and the structural gene on the GCN4 gene derived from Candida yeast.
CN4 promoter. 3. The promoter according to claim 1, wherein the GCN4 gene derived from Candida yeast is the GCN4 gene derived from Candida maltosa. 4. The GCN4 gene has the following (a) or
The promoter according to claim 3, which comprises the DNA of (b). (a) DNA represented by SEQ ID NO: 1 (b) DNA hybridizing with a DNA having a nucleotide sequence complementary to the DNA represented by SEQ ID NO: 1 under stringent conditions and having GCN4 gene activity 5. The promoter according to claim 3, wherein the GCN4 gene consists of the DNA shown in SEQ ID NO: 1. 6. A base sequence represented by SEQ ID NO: 2,
The promoter according to any one of claims 1 to 5. 7. The method according to claim 1, wherein the promoter has a 50-fold or more improvement in protein expression level as compared to the wild-type GCN4 promoter due to mutation introduction. promoter. 8. A promoter comprising the following DNA (a) or (b): (a) DNA represented by SEQ ID NO: 2 (b) Hybridized with a DNA consisting of a nucleotide sequence complementary to the DNA represented by SEQ ID NO: 2 under stringent conditions and consisting of the DNA represented by SEQ ID NO: 2 DNA having promoter activity equivalent to that of the promoter 9. An expression vector comprising the promoter according to any one of claims 1-8. 10. A transformant containing the expression vector according to claim 9. 11. The transformant according to claim 10, wherein the host is yeast belonging to the genus Candida. 12. Production of a GCN4 promoter having improved promoter activity by introducing a mutation into at least one translation initiation codon existing between a transcription start point and a structural gene on a GCN4 gene derived from Candida yeast. Method. 13. A method for improving a promoter activity of a Candida yeast-derived GCN4 gene by introducing a mutation into at least one translation initiation codon existing between a transcription initiation point and a structural gene. .