JP2003164295A - System enabling high expression of gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for stably transferring a target gene into a host organism and expressing the gene at a high rate. <P>SOLUTION: A target gene to be expressed is connected to a pyruvic acid decarboxylase 1 promoter in a yeast Saccharomyces cerevisiae and the product is transferred into the genom of a host to effect the stable expression of the target gene at a high expression rate. In other words, the invention relates to a genetic fermentation method comprising the transfer of the target gene into a genom under the control of the promoter of a gene having an autoregulation mechanism or the promoter of a gene non-essential to the growth or fermentation of the host organism. The promoter may be a DNA having a promoter activity and containing a base sequence of the above promoter wherein 1-40 bases are deleted, substituted, or added, or a DNA having a promoter activity and hybridizable with a DNA having a sequence complimentary to the whole or a part of the base sequence of the above promoter under stringent condition. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for introducing and expressing a gene in a host organism using a genetic engineering technique.

[0002]

2. Description of the Related Art Production of substances in host organisms,
When modifying or analyzing the function of the host organism, genetic engineering techniques for introducing and expressing the same or different genes in the host organism are used. However, this genetic engineering method is still insufficient in terms of stability and high expression, and improvement is desired.

For example, when the host organism is the yeast Saccharomyces cerevisiae, 2
YEP type vectors using μm DNA are often used. The YEP vector can be introduced with a large number of copies of the gene, but its enzymatic activity may be reduced due to loss of the vector due to cell division, etc., and thus is not sufficient in terms of stability. In order to improve the enzyme activity, the vector contains a drug resistance marker, and this drug is added to the medium, or when the host strain is previously provided with an auxotrophic marker, the auxotrophic marker is added. It is possible to apply selective pressure by designing to include in the vector and using highly purified minimal medium (YNB, manufactured by Difco) as the medium, but in any case, the medium cost becomes high. There is a problem that it ends up.

On the other hand, a YIP vector utilizing homologous recombination is known as a chromosome transfer vector. This Y
The IP vector allows the transgene to stably exist on the genome depending on the design of the vector, but in general, the transgene cannot be highly expressed. For the reasons described above, there has been a demand in the art for a stable and low-cost method for introducing a gene into a host organism and highly expressing it.

[0005]

An object of the present invention is to provide a method for stably introducing a target gene into a host organism and highly expressing it.

[0006]

[Means for Solving the Problems] As a result of intensive research to solve the above problems, the present inventors have linked a target gene to be expressed to the pyruvate decarboxylase 1 promoter in the yeast Saccharomyces cerevisiae. ,
By introducing this into the genome of the host, it was found that the target gene can be stably highly expressed, and the present invention has been completed.

That is, the present invention is characterized in that a gene of interest is introduced into a genome under the control of a promoter of a gene having an autoregulation mechanism or a promoter of a gene not essential for growth or fermentation in a host organism. It is an expression method. The promoter has a deletion, substitution or addition of 1 to 40 bases in the nucleotide sequence of a promoter of a gene having an autoregulation mechanism or the nucleotide sequence of a promoter of a gene which is not essential for growth or fermentation in a host organism. DN containing a sequence and having promoter activity
May be A. Further, the promoter is a DNA consisting of a nucleotide sequence of a promoter of a gene having an autoregulation mechanism, or a sequence complementary to all or part of the nucleotide sequence of a promoter of a gene which is not essential for growth or fermentation in a host organism. It may be a DNA that hybridizes with the above under stringent conditions and that has a promoter activity.

In the present invention, the promoter of the gene having the above autoregulation mechanism includes the promoter of pyruvate decarboxylase 1 gene, and the promoter of the gene not essential for growth is the promoter of the gene encoding thioredoxin. Is mentioned. In this case, the host organism may be any of bacteria, yeasts, insects, animals or plants, and yeasts belonging to the genus Saccharomyces are particularly preferable. These host organisms mean individual organisms (excluding humans), tissues, and cells.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
In the living world, there are genes that have an autoregulation mechanism and genes that are not essential for growth and fermentation. The present inventors paid attention to this point and selected a promoter for such a gene in the method of introducing and expressing the gene. Therefore, the gene expression method of the present invention comprises introducing a target gene into a genome under the control of a promoter of a gene having an autoregulation mechanism or under the control of a promoter of a gene which is not essential for growth or fermentation in a host organism. Is characterized by. The outline of the method of the present invention is as follows.

1. Selection of promoter First, a promoter of a gene having an autoregulation mechanism or a promoter of a gene not essential for growth or fermentation of a host organism is selected. As the target host organism, any organism for which substance production and functional modification or functional analysis is desired can be used. For example, bacteria, yeasts, insects, animals, plants and the like can be mentioned.

(1) Gene promoter having an autoregulation mechanism In order to select a promoter of a gene having an autoregulation mechanism, first, a gene having an autoregulation mechanism is specified. The “autoregulation mechanism” is a gene in which a plurality of genes having the same function exist in the same organism, and usually at least one of them is expressed, but the rest of the genes are suppressed, and which is normally expressed. Means a mechanism in which the remaining genes are expressed and their functions are continued only when the cells become nonfunctional due to destruction. For example, the pyruvate decarboxylase gene (PD of yeast belonging to the genus Saccharomyces)
C) is a gene encoding an enzyme that decarboxylates pyruvic acid into acetaldehyde in the process of producing ethanol, and plays an important role in the fermentation process. Although PDC1, PDC5, and PDC6 exist in PDC, PDC1 normally functions, and PDC5 and PDC6 are suppressed by the action of PDC1. However, when a gene disruption or a mutation due to a drug is given to this PDC1 to inactivate the function of PDC1, the PDC5 gene is activated, and the ethanol production function of yeast is not lost. That is, the physiological expression system is almost the same as that of the parent strain.

A gene having an autoregulation mechanism can be identified by confirming whether or not a gene product protein is still expressed in a strain in which a gene is disrupted. The promoter of the gene having the autoregulation mechanism thus identified is selected. In the present invention, for example, a PDC1 promoter (hereinafter referred to as PDC1 promoter) can be selected.

(2) Promoter of a gene not essential for growth or fermentation To select a promoter for a gene not essential for growth or fermentation of a host organism, first, a gene not essential for growth or fermentation is specified. Here, “growth” means that the cells are still alive so that they can grow, and “fermentation” means that a substance such as alcoholic fermentation is produced.
Then, the "non-essential gene" means a gene which is not related to the growth and fermentation of the gene, the growth or fermentation or both of which are still maintained even if the gene is destroyed or inactivated.

A gene which is not essential for growth or fermentation can be specified by confirming whether or not the host organism continues to grow and ferment in a strain in which a gene is disrupted. Examples of such a gene include the TRX1 gene encoding thioredoxin which is present in most organisms. The TRX1 gene is involved in DNA replication, oxidative stress response, vacuolar inheritance, etc., but is not necessarily essential for growth or fermentation. That is, even if the TRX1 gene is destroyed or replaced in the host organism, the host organism can continue to grow or ferment. A promoter of a gene that is not essential for the growth or fermentation of the host organism thus identified is selected.

2. Preparation of target gene and promoter The above-selected promoter and target gene to be introduced into the host organism are prepared. In the present invention, the “target gene” means a gene that is desired to be expressed for substance production and functional modification or functional analysis, and may be either a homologous gene or a heterologous gene. For example, in the case of targeting substance production, a gene encoding a useful protein is preferable as the target gene, and examples of such useful protein include interferon, vaccine, hormone and the like. The target gene may be a gene encoding an enzyme, and examples thereof include a gene encoding lactate dehydrogenase that produces lactic acid from pyruvate.

For the preparation of the target gene and the above promoter, any technique known in the art can be adopted. For example, when the target gene and the above promoter are isolated from a source, they can be prepared by a method of synthesizing cDNA from RNA prepared by the guanidine isothiocyanate method. It can also be prepared by amplification by using genomic DNA as a template by PCR. The thus-obtained target gene and the above-mentioned promoter DNA can be used as it is, or if desired, by digesting with a restriction enzyme or adding a linker, depending on the purpose.

In the present invention, a DNA having a promoter activity and containing a sequence in which 1 to 40 bases are deleted, substituted or added in the base sequence of the promoter.
Can also be used as a promoter. The term “promoter activity” refers to the ability and function of producing a gene product of a target gene inside or outside the host when the target gene is linked to the downstream of the promoter in an expressible state and introduced into the host. Such a DNA has a promoter activity which is almost the same as that of a promoter having a full-length nucleotide sequence having no mutation (deletion, substitution or addition) under the same conditions as the above-mentioned conditions. It means that there is. For example, about 0.01 to 100 times, preferably about 0.5 to 20 times, the promoter activity of the full-length sequence,
More preferably, it is a DNA that maintains about 0.5 to 2 times the activity.

[0021] Such DNA is used for Molecular Cloning (Sa
mbrook et al. (1989) Cold Spring Harbor Lab. Press, Ne
w York) and the like. For example, based on the base sequence of a promoter of a gene having an autoregulation mechanism or the base sequence of a promoter of a gene that is not essential for growth or fermentation in a host organism, 1-40 bases are deleted from the base sequence. By the technique of artificially performing substitution or addition, for example, site-directed mutagenesis, mutants having different sequences can be prepared while maintaining promoter activity. For example, for site-directed mutagenesis in which 1 to 40 bases are replaced, see Proc. Natl. Acad. Sc.
i. USA 81 (1984) 5662-5666; WO85 / 00817 publication; Natur
e 316 (1985) 601-605; Gene 34 (1985) 315-323; Nuclei
c Acids Res. 13 (1985) 4431-4442; Proc. Natl. Acad.
Sci. USA 79 (1982) 6409-6413; Science 224 (1984) 14
A mutant can be obtained and used according to the technique described in 31-1433 or the like. In addition, a commercially available kit (Mutan-
These mutants can be prepared using G, Mutan-K (Takara Shuzo). Furthermore, error-prone polymerase chain reaction (error-prone PCR) is also known as a method for producing a mutant, and it is possible to introduce a mutation of 1 to several bases by selecting a condition with low stringency of replication. Yes (Cadwell, RC and Joyce, GF PCR Methods
and Applications 2 (1992) 28-33; Malboeuf, CM et
al. Biotechniques 30 (2001) 1074-8; Moore, GL and
Maranas CDJ Theor. Biol. 7; 205 (2000) 483-50
3).

A DNA probe having a sequence complementary to all or part of the nucleotide sequence of a gene promoter having an autoregulation mechanism or a gene promoter not essential for growth or fermentation in a host organism is used as a probe (100- (900 bases) and hybridized under stringent conditions to the same promoter as the DNA of the promoter of the gene that has an autoregulatory mechanism or the promoter of the gene that is not essential for growth or fermentation in the host organism. It is also possible to newly obtain and utilize a DNA having another base sequence having the function of (i.e., promoter activity). Here, the stringent condition means, for example, a sodium concentration of 10 to 300 mM, preferably 20 to 100 mM, and a temperature of 25 to 70 ° C, preferably 42 to 55 ° C.

Whether or not the mutant obtained as described above or the DNA obtained by hybridization has an activity as a promoter can be confirmed by the following method. That is, the promoter activity of the DNA obtained as described above is preferably that of various reporter genes such as luciferase gene (LUC), chloramphenicol acetyltransferase gene (CAT), β-galactosidase gene (GAL), etc. This can be confirmed by producing a vector linked to the downstream region, introducing the vector into the genome of the host using the vector, and measuring the expression of the reporter gene.

3. Introduction of the gene of interest and promoter Subsequently, the gene in which the autoregulation mechanism is present, or the gene which is not essential for growth or fermentation in the host organism is destroyed, and the gene of interest is introduced under the control of the promoter of this gene, or This gene replaces the gene of interest.

For example, the target gene isolated as described above and the promoter selected above are operably linked and introduced into the genome of the host organism. The term “operably linked” means that the target gene and the promoter are linked so that the target gene is expressed under the control of the promoter in a host organism into which the target gene is introduced. The target gene and the above promoter can be introduced using any method known in the art. For example, the gene of interest and the above promoter can be introduced into the genome of the host organism using a recombinant vector. The recombinant vector can be obtained by ligating (inserting) the target gene and the above promoter into an appropriate vector. The vector for inserting the target gene is not particularly limited as long as it can be integrated into the genome of the host organism, and examples thereof include plasmid DNA, bacteriophage DNA, retrotransposon DNA, yeast artificial chromosome DNA (YAC: yeast artificial DNA). chromosome)
And so on.

Examples of plasmid DNA include pRS403,
YI such as pRS404, pRS405, pRS406, pAUR101 or pAUR135
p-type E. coli-yeast shuttle vector, plasmid derived from E. coli (pBR322, pBR325, pUC18, pUC19, pUC118, pUC11
9, pTV118N, pTV119N, pBluescript, pHSG298, pHSG396
Or ColE plasmid such as pTrc99A, pACYC177 or pAC
P15A-based plasmids such as YC184, pMW118, pMW119, pMW21
8 or pMW101 and other pSC101-based plasmids), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, etc.), and the like.
n21A, EMBL3, EMBL4, λgt10, λgt11, λZAP), φX17
4, M13mp18, M13mp19 and the like. Examples of the retrotransposon include Ty factor. Examples of YAC vectors include pYACC2. To insert the target gene and the above promoter into the vector, first, the purified DNA is cleaved with an appropriate restriction enzyme, and then inserted into the restriction enzyme site or the multi-cloning site of the appropriate vector DNA, and ligated to the vector. Is adopted.

The target gene needs to be incorporated into a vector so that the function of the gene is exerted under the control of the promoter selected above. Therefore, in the recombinant vector, in addition to the selected promoter, target gene, terminator, cis element such as enhancer, splicing signal, poly
An A addition signal, a ribosome binding sequence (SD sequence), etc. can be linked. Alternatively, a selectable marker indicating that the vector is retained in the cells may be linked.
Examples of selectable markers include dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene and the like. Other marker genes include, but are not limited to, the tryptophan synthesis gene (TRP1 gene), and other marker genes such as URA3 gene with auxotrophy and AD
E2 gene, HIS3 gene, or G418 resistant gene having drug resistance can also be used.

Examples of the terminator sequence include the terminator gene of the glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH), but the present invention is not limited to this and may be a terminator sequence usable in the host organism. Anything can be used.

As described above, the recombinant vector can be prepared so as to be compatible with the expression of the target gene in the host organism. By transforming a host organism with this recombinant vector, the target gene can be expressed in the host organism under the control of the promoter selected above.

When a bacterium such as Escherichia coli is used as a host, the recombinant vector is capable of autonomous replication in the bacterium, and at the same time, a promoter, a ribosome binding sequence, a target gene,
It is preferably composed of a transcription termination sequence. Moreover, the gene which controls a promoter may be contained.

As Escherichia coli, for example, Escherichia
Escherichia coli K12, DH1 and the like.Examples of Bacillus subtilis include Bacillus subtilis.
btilis) and the like. The method for introducing the recombinant vector into the bacterium is not particularly limited as long as it is a method for introducing the DNA into the bacterium. Examples thereof include a method using calcium ions and an electroporation method.

When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe
e), Pichia pastoris and the like are used. As a method of introducing the recombinant vector into yeast,
It is not particularly limited as long as it is a method of introducing DNA into yeast, and examples thereof include electroporation method, spheroplast method,
Examples include the lithium acetate method.

When an insect or animal is used as a host, the recombinant vector can be introduced by, for example, the calcium phosphate method, lipofection method, electroporation method or the like. When a plant is used as the host, examples of the method for introducing the recombinant vector include the Agrobacterium method,
Particle gun method, PEG method, electroporation method, etc. are used.

When an insect, animal (excluding human) or plant individual is used as a host, the recombinant vector can be introduced according to a method for producing a transgenic animal or plant known in the art. For example, as a method for introducing a recombinant vector into an animal individual, a microinjection method for fertilized eggs, a method for introducing ES cells, a method for introducing cell nuclei introduced into cultured cells into fertilized eggs by nuclear transfer and the like can be mentioned. .

The host organism into which the recombinant vector has been introduced as described above is selected for the strain into which the target gene has been introduced under the control of the selected promoter. Specifically, a transformant is selected using the above selection marker as an index. PCR (polymerase chain reaction) is used to confirm whether the target gene is integrated under the control of the above promoter.
Method or Southern hybridization method. For example, prepare DNA from the transformant and
Design specific primers and perform PCR. After that, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis or capillary electrophoresis, and stained with ethidium bromide, SYBR Green solution, etc.,
Then, the introduced DNA can be confirmed by detecting the amplified product as one band. Alternatively, the amplification product can be detected by performing PCR using a primer that has been labeled with a fluorescent dye in advance. Furthermore, a method in which the amplification product is bound to a solid phase such as a microplate and the amplification product is confirmed by fluorescence or an enzymatic reaction can also be adopted.

As described above, the target gene is introduced into the genome under the control of the promoter of the gene having the autoregulation mechanism or the promoter of the gene not essential for the growth or fermentation of the host organism (genome integration), The gene of interest is expressed in the organism. The PDC1 promoter is a very strong promoter, so if you choose the PDC1 promoter,
Even if the target gene is introduced into the genome in a single copy,
It will be highly expressed. Further, since the original gene of the selected promoter is not essential for growth and fermentation, the host organism can continue growth and fermentation even if it is destroyed or replaced with the target gene, and the target gene can be expressed for a long period of time. You can

[0034]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. [Example 1] Construction of vector In this example, Bifidobacterium longum (Bifidobacterium long) was used as a target gene under the control of the promoter sequence of the pyruvate decarboxylase 1 gene (PDC1 gene) derived from Saccharomyces cerevisiae.
um) -derived lactate dehydrogenase gene (LDH gene) was used.

The chromosome-introduced vector newly constructed for this Example was named pBTRP-PDC1-LDH, and the details of this vector construction example are described below. The outline of this embodiment is shown in FIG. However, the procedure of vector construction is not limited to this. A promoter fragment (PDC1P) 971 of the PDC1 gene, which is a necessary gene fragment for vector construction.
The bp and the PDC1 gene downstream region fragment (PDC1D) 518 bp were isolated by PCR amplification using the genomic DNA of Saccharomyces cerevisiae YPH strain (Stratagene) as a template.

The genomic DNA of the Saccharomyces cerevisiae YPH strain is the Fast DNA Kit (Bio
(101 company), and the details were prepared according to the attached protocol. The DNA concentration is measured by a spectrophotometer Ultro spec 3000 (Amersh
am Pharmacia Biotech).

In the PCR reaction, Pyrobest DNA polymerase (Takara Shuzo Co., Ltd.), which is highly accurate for the amplified fragment, was used as an amplification enzyme. Saccharomyces cerevisiae YPH strain genomic DNA 50 ng / sample, primer DNA 50 pmol / sample, and Pyrobest DN prepared by the above method
0.2 units / sample of A polymerase was prepared in a total reaction volume of 50 μl. The reaction solution is a PCR amplification device Gene Amp PC
DN by R system 9700 (PE Applied Biosystems)
A amplification was performed. The reaction conditions of the PCR amplification device were 96 ° C. for 2 minutes, 25 cycles of (96 ° C. 30 seconds → 55 ° C. 30 seconds → 72 ° C. 90 seconds), and then 4 ° C. The PDC1P amplified fragment and the PDC1D amplified fragment were subjected to 1% TBE agarose gel electrophoresis to confirm the gene amplified fragment. The primer DNA used in the reaction
Is a synthetic DNA (Sawasde Technology Co., Ltd.), and the DNA sequence of this primer is as follows.

Amplification of PDC1P fragment-PDC1P-LDH-U (31mer, Tm value 58.3 ° C) Restriction enzyme Bam at the end
Add HI site: ATA TAT GGA TCC GCG TTT ATT TAC CT
A TCT C (SEQ ID NO: 1) ・ PDC1P-LDH-D (31mer, Tm value 54.4 ° C) Restriction enzyme Ec at the end
o Add RI site: ATA TAT GAA TTC TTT GAT TGA TTT G
AC TGT G (SEQ ID NO: 2) Amplification of PDC1D fragment ・ PDC1D-LDH-U (34mer, Tm value 55.3 ℃) Restriction enzyme Xh at the end
oI site added: ATA TAT CTC GAG GCC AGC TAA CTT CT
T GGT CGA C (SEQ ID NO: 3) -PDC1D-LDH-D (31mer, Tm value 54.4 ° C) Restriction enzyme Ap at the end
Add aI site: ATA TAT GAA TTC TTT GAT TGA TTT GA
C TGT G (SEQ ID NO: 4)

Each of the PDC1P and PDC1D gene amplified fragments obtained in the above reaction was purified by ethanol precipitation treatment, and then the PDC1P amplified fragment was digested with the restriction enzymes BamHI / EcoRI and
The PDC1D amplified fragment was subjected to restriction enzyme reaction with restriction enzymes XhoI / ApaI. The enzymes used below were all manufactured by Takara Shuzo. For a detailed manual of ethanol precipitation and restriction enzyme treatment, see Molecular Clon.
ing A Laboratory Manual second edition (Maniatis e
al., Cold Spring Harbor Laboratory press.1989).

A series of reaction operations in the construction of the vector were carried out according to a general DNA subcloning method.
That is, the restriction enzyme BamHI / EcoRI (Takara Shuzo) and the dephosphorylating enzyme Alkaline Phosphatase (BAP, Takara Shuzo) were applied to a pBluescriptII SK + vector (Toyobo Co., Ltd.), which was amplified by the PCR method and subjected to restriction enzyme treatment. PDC1P fragment
Ligation was performed by T4DNA Ligase reaction (Fig. 1A). T4 DN
For A Ligase reaction, LigaFast Rapid DNA Ligation Syst
em (Promega) was used, and the details followed the attached protocol.

Next, the solution subjected to the ligation reaction was transformed into competent cells. Escherichia coli JM109 strain (Toyobo Co., Ltd.) was used as the competent cells, and the details were performed according to the attached protocol. The obtained culture solution was spread on an LB plate containing 100 μg / ml of the antibiotic ampicillin and cultured overnight. The grown colonies were confirmed by a colony PCR method using the insert fragment primer DNA, and the plasmid DNA preparation solution by miniprep was confirmed by restriction enzyme treatment to isolate the target vector pBPDC1P vector (FIG. 1B). ).

LDH obtained by treating the pYLD1 vector constructed by Toyota Motor Corporation with the restriction enzymes EcoRI / AatII and the end-modifying enzyme T4 DNA polymerase.
The gene fragment was subcloned into the pBPDC1P vector which was also treated with the restriction enzyme EcoRI and the end-modifying enzyme T4 DNA polymerase in the same manner as described above to obtain pBPD.
A C1P-LDH I vector was prepared (Fig. 1C). In addition, the above
The pYLD1 vector was introduced into E. coli (name: "E. coli p
YLD1 "), the National Institute of Advanced Industrial Science and Technology, Patent and Biological Depositary Center (1-1, Higashi 1-1-chome, Tsukuba-shi, Ibaraki), under the Budapest Treaty as the deposit number (FERM BP-7423). October 26, 1999
Day). Subsequently, this vector was treated with XhoI / ApaI, and the amplified PDC1D fragment was ligated to prepare a pBPDC1P-LDH II vector (FIG. 2A). Finally, add the pBPDC1P-LDH II vector to Eco.
RV-treated with pRS404 vector (Stratagene) in Aa
TRP obtained by tII / SspI treatment and T4 DNA polymerase treatment
The 1-marker fragment was ligated to construct a chromosome-introduced pBTRP-PDC1-LDH vector as the final construct (FIG. 2B).

The nucleotide sequence was determined for confirmation of the constructed chromosome-introduced pBTRP-PDC1-LDH vector. ABI PRISM 310 Genetic Analyzer (PE Appli
ed Biosystems), and the details of the sample preparation method and instrument usage were in accordance with the manual attached to this device. The vector DNA used as a sample was prepared by the alkaline extraction method, and this was used as a GFX DNA Purification kit.
After column purification with (Amersham Pharmacia Biotech), spectrophotometer Ultro spec 3000 (Amersham Pharmacia
The DNA concentration measured by Biotech was used. As a comparative control, a YIP vector was constructed so that LDH (lactate devidogenase), which is a lactate producing gene, was introduced under the control of the PDC1 promoter.

Example 2 Introduction of Recombinant Vector into Host The yeast IFO2260 strain (strain registered in the Fermentation Research Institute), which is a host, is a tryptophan-requiring strain of 10 ml YP.
Culture in D medium at 30 ° C until logarithmic growth phase to collect bacteria and T
After washing with E buffer, 0.5 ml TE buffer, 0.5 ml, and 0.2 M lithium acetate were added, and shaking culture was performed at 30 ° C. for 1 hour. Then, pBTRP-PDC1-LDH treated with the restriction enzymes Apa1 and Spe1 was added.

After culturing the bacterial suspension of this plasmid at 30 ° C. for 30 minutes with shaking, 150 ml of 70% polyethylene glycol 4000 was added and well stirred. 42 after shaking culture at 30 ℃ for 1 hour
Heat shock was applied for 5 minutes at ℃. After washing the cells, the cells suspended in 200 ml of water were applied to a selective medium.

The obtained colony was isolated with a selective medium to obtain a colony, and LDH was downstream of the PDC1 promoter by PCR.
Has acquired the stock that has been introduced. Furthermore, spore formation was carried out in a sporulation medium, and diploidization was carried out by utilizing the homothallic property to obtain a strain in which the above vector was introduced into both diploid chromosomes. Yeast Saccharomyces
It was confirmed by PCR that S. cerevisiae was transformed with pBTRP-PDC1-LDH shown in FIG. 2 and introduced into the genome. The structure of the above vector on the genome is shown in FIG.

[Example 3] Confirmation of expression and stability of LDH gene The obtained transformant was inoculated into a YPD liquid medium (glucose 10%) so that the cell concentration was 1%, and the temperature was 30 ° C. After static culture for 2 days, the amount of lactic acid produced by the vector-non-introduced strain, the LDH-introduced strain with the YEP vector, and the pBTRP-PDC1-LDH-introduced strain was compared and examined. The results are shown in Table 1.
Shown in.

[0048]

[Table 1]

Lactic acid was not produced in the vector-non-introduced strain, whereas lactic acid was produced in LDH-introduced strains. Furthermore, the strain in which LDH was introduced under the control of the PDC1 promoter produced 2.5 times as much lactic acid as compared to the case where the YEP vector was introduced. In addition, for the purpose of confirming the stability of the trait introduced by this method, after subculturing 3 times on YPD plate, gene transfer and lactic acid production were examined by PCR. Lactic acid production was stopped in this system, whereas when LDH was expressed under the control of the PDC1 promoter, the same amount of lactic acid production was maintained as it was before subculture. In addition, since it was confirmed by PCR that there was no change in the structure on the genome,
The system for expressing LDH under the control of the PDC1 promoter of
It can be said that it exists stably and highly expresses a gene.

Example 4 Isolation of PDC1 Promoter Sequence Containing Mutant Sequence In this Example and the following Examples, 3 types of PDC1 promoter sequences having sequences differing by several bases were isolated and placed under this promoter. A chromosome-introduced vector designed to connect the LacZ gene was constructed. Using these vectors, a single copy of the promoter and the gene was introduced at the same position in the chromosome to prepare a transformed yeast. The β-galactosidase activity of each transformed yeast was measured,
The activity of three promoters was compared.

In the present embodiment, Saccharomyces
Cerevisiae pBTRP-PDC1-LDH introduced strain (strain produced in Example 2), IFO2260 strain (strain registered in Fermentation Research Institute) and YPH strain (Stratagene) genomic DNA
The PDC1 promoter sequence was isolated by the PCR amplification method using as a template. The method for preparing genomic DNA of each Saccharomyces cerevisiae (pBTRP-PDC1-LDH-introduced strain, IFO2260 strain, YPH strain) and the PCR amplification method were the same as in Example 1. The base sequence of the primer DNA used in the reaction is as follows.

Amplification of PDC1 promoter derived from pBTRP-PDC1-LDH-introduced strain ・ A restriction enzyme SalI site was added to PDC1 PrFrag-U2 (32mer, Tm value 64.4 ° C.) end: AAA TTT GTC GAC AAG GGT AGC CT
C CCC ATA AC (SEQ ID NO: 5) ・ PDC1 PrFrag-D2 (31mer, Tm value 61.1 ° C) Add restriction enzyme SalI site to the end: ATA TAT GTC GAC GAG AAT TGG GG
G ATC TTT G (SEQ ID NO: 6) Amplification of PDC1 promoter derived from IFO2260 strain and YPH strain ・ PDC1 PrFrag-U2 (32mer, Tm value 64.4 ° C) with restriction enzyme SalI site added: AAA TTT GTC GAC AAG GGT AGC CT
C CCC ATA AC (SEQ ID NO: 5) -PDC1 PrFrag-D (43mer, Tm value 62.5 ° C) Restriction enzyme at the end
SalI site added: TTT AAA GTC GAC TTT GAT TGA TTT
GAC TGT GTT ATT TTG CGT G (SEQ ID NO: 7)

Example 5 Construction of β-Galactosidase Analysis Vector Containing Mutant Promoter Sequence In this Example, a vector to which a reporter gene was ligated was constructed under the control of three isolated PDC1 promoter sequences. . The β-galactosidase gene (LacZ gene) was used as a reporter gene.

The chromosome-transferred vectors newly constructed for this example were used as pAUR-LacZ-T123PDC1 and pAUR-LacZ-OC2P.
They are named DC1 and pAUR-LacZ-YPHPDC1, and the details of vector construction examples are described below. The outline of this embodiment is shown in FIG. However, the procedure of vector construction is not limited to this.

A series of reaction operations in the construction of the vector followed the general DNA subcloning method. The pSV-β-Galactosidase Control Vector from Promega was cut out with a restriction enzyme to obtain a LacZ fragment, which was then treated with blunt ends.
The pAUR-LacZ vector was prepared. PAU thus constructed
The R-LacZ vector was treated with SalI (Takara Shuzo) and dephosphorylating enzyme Alkaline Phosphatase (BAP, Takara Shuzo). Next, three types of promoter sequences obtained in Example 4, that is, PDC1 derived from pBTRP-PDC1-LDH-introduced strain
Promoter (983bp), IFO2260 strain-derived PDC1 promoter (968bp), and YPH strain-derived PDC1 promoter (968b)
p) was treated with the restriction enzyme SalI (Takara Shuzo Co., Ltd.), and the T4 DNA Ligase was added to the pAUR-LacZ vector.
The reaction was linked. For T4 DNA Ligase reaction, Li
The gaFast Rapid DNA Ligation System (Promega) was used, and the details followed the attached protocol.

The obtained ligation reaction solution was used to transform competent cells, and the desired construction vector was obtained by the colony PCR method. The series of operations described above was performed in the same manner as in Example 1.

Nucleotide sequence analysis was carried out on the constructed vector, and the PDC1 promoter (98 pBTRP-PDC1-LDH-derived strain) was derived.
3bp), the IFO2260 strain-derived PDC1 promoter (968bp), and the YPH strain-derived PDC1 promoter (968bp) were compared in gene sequence. A comparison of this sequence is shown in FIG. The operation of the base sequence analysis was performed in the same manner as in Example 1.

The PDC1 promoter (983 bp) derived from the pBTRP-PDC1-LDH-introduced strain differs from the PDC1 promoter sequence (971 bp) obtained in Example 1 by 12 bases. Specifically, the promoter sequence of Example 1 was used. A restriction enzyme SalI site (GTCGAC) is added to both ends of the sequence.

Also, the PDC1 promoter derived from the IFO2260 strain (968
bp) is different from the PDC1 promoter sequence obtained in Example 1 by 30 bases.
The guanine (G) at position 861 of the promoter sequence of is replaced by cytosine (C), the cytosine (C) at position 894 is replaced by thymine (T), and the adenine (A) at position 925 is replaced by thymine (T). In addition, the 15-base sequence after the 972nd position (GAT
CCCCCAATTCTC) is added. Furthermore, the restriction enzyme SalI site (GT
CGAC) is added to the sequence.

The YDC strain-derived PDC1 promoter (968 bp) is
The PDC1 promoter sequence obtained in Example 1 is 37
Specifically, the cytosine (C) at the 179th position in the promoter sequence of Example 1 is thymine (T).
And the 214th adenine (A) becomes guanine (G) and 216
The guanine (G) is the adenine (A), the thymine (T) is the 271th is cytosine (C), the guanine (G) is the adenine (A), and the 490th adenine (A) is the guanine (A).
Is guanine (G), the 533rd cytosine (C) is thymine (T), and the 566th thymine (T) is cytosine (C).
And the 660th guanine (G) becomes cytosine (C) and 925
The adenine (A) at the th position is replaced by thymine (T), and the 15-base sequence after the 972th position (GATCCCCCAATTCT
C) is added. Furthermore, it is composed of a sequence in which a restriction enzyme SalI site (GTCGAC) is added to both ends of the promoter sequence of Example 1.

[Example 6] Introduction of a recombinant vector into a host To a tryptophan-requiring strain of a yeast IFO2260 strain (a strain registered in the Fermentation Research Institute), which is a host, 10 ml Y was added.
After culturing in PD medium at 30 ° C until the logarithmic growth phase, collecting cells and washing with TE buffer, add 0.5 ml TE buffer and 0.5 ml of 0.2M lithium acetate, and shake at 30 ° C for 1 hour. Culture was performed. Then, pAUR-LacZ-T123PDC1P and pAUR-LacZ-YPHPD treated with restriction enzyme Bst1107I (Takara Shuzo).
C1P and pAUR-LacZ-OC2PDC1P were added.

After culturing the suspension of this plasmid at 30 ° C. for 30 minutes with shaking, 150 μl of 70% polyethylene glycol 4000 was added and well stirred. After incubating this solution with shaking at 30 ° C for 1 hour, heat shock at 42 ° C for 5 minutes to remove the cells
Culture was performed in ml YPD medium at 30 ° C. for 12 hours. After washing the main culture, the suspension in 200 μl of sterilized water was applied to an aureovaticin selective medium. The concentration of aureovatisine added to the medium was 0.4 μg / ml. The obtained colonies were isolated in an aureovaticin selective medium, and PCR was performed on the obtained colonies to obtain the target strain.

Example 7 β in gene recombinant strain
Measurement of galactosidase activity β-galactosidase activity was measured for the above transformants and non-transformants. Each strain was cultured in 2 ml YPD liquid medium (glucose 2%) at 30 ° C. for 20 hours. These were collected, and 500 μl of 50 mM Tris-HCl and glass beads (42
5-600microns Acid Washed, SIGMA), and add 15
Vortex for minutes.

The supernatant of this solution was collected by centrifugation, and these β-galactosidase activities were measured. For the activity measurement, β-Galactosidase Enzyme Assay System (Promega) was used, and the details followed the attached protocol. ABS6
The activity value per 00 nm = 1.0 was determined, and the results are shown in FIG. 6 (before subculture) and FIG. 7 (after subculture).

From the above results, even if the PDC1 promoter sequence has an additional sequence of several tens of bases or a different sequence,
It became clear that it had a stable promoter activity. Therefore, it can be said that a promoter of a gene having an autoregulation mechanism or a promoter of a gene not essential for growth or fermentation in a host organism can be used even if it is not full length.

[0066]

INDUSTRIAL APPLICABILITY According to the present invention, a gene can be stably introduced and highly expressed without affecting the growth and fermentation of the host organism. Therefore, an effective means for substance production and functional modification or functional analysis is provided.

[0067]

[Sequence list]                      SEQUENCE LISTING <110> Toyota Jidosha Kabushiki Kaisha,       Toyota Central R & D Labs. <120> Gene Expression System <130> P02-0112 <150> JP 2001-286637 <151> 2001-09-20 <160> 7 <170> PatentIn Ver. 2.0 <210> 1 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 1 atatatggat ccgcgtttat ttacctatct c 31 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 2 atatatgaat tctttgattg atttgactgt g 31 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 3 atatatctcg aggccagcta acttcttggt cgac 34 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 4 atatatgaat tctttgattg atttgactgt g 31 <210> 5 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 5 aaatttgtcg acaagggtag cctccccata ac 32 <210> 6 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 6 atatatgtcg acgagaattg ggggatcttt g 31 <210> 7 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 7 tttaaagtcg actttgattg atttgactgt gttattttgc gtg 43

[0068]

[Sequence free text]

SEQ ID NO: 1 synthetic DNA SEQ ID NO: 2: Synthetic DNA SEQ ID NO: 3 synthetic DNA SEQ ID NO: 4: Synthetic DNA SEQ ID NO: 5: Synthetic DNA SEQ ID NO: 6: Synthetic DNA SEQ ID NO: 7: Synthetic DNA

[Brief description of drawings]

FIG. 1 is a construction diagram of a chromosome-introduced vector pBTRP-PDC1-LDH.

FIG. 2 is a construction diagram of a chromosome-introduced vector pBTRP-PDC1-LDH.

FIG. 3 is a diagram showing the genome structure of a strain obtained when the yeast Saccharomyces cerevisiae is transformed with the vector pBTRP-PDC1-LDH.

[Fig. 4] Chromosomal transfer vector pAUR-LacZ-T123PDC1
(A), pAUR-LacZ-OC2PDC1 (B) and pAUR-LacZ-YPHPD
It is a construction drawing of C1 (C).

FIG. 5: PBT1 promoter (983bp) derived from pBTRP-PDC1-LDH introduced strain, PDC1 promoter derived from IFO2260 strain (968b)
It is a figure which shows the comparison of p) and the gene sequence of PDC1 promoter (968bp) derived from YPH strain.

[Fig. 6] PBT1 promoter (983bp) derived from pBTRP-PDC1-LDH introduced strain, PDC1 promoter derived from IFO2260 strain (968b)
FIG. 3 is a diagram showing β-galactosidase activity before subculture in a transformant into which p) and a YDC strain-derived PDC1 promoter (968 bp) were introduced.

FIG. 7: PDC1 promoter (983 bp) derived from pBTRP-PDC1-LDH introduced strain, PDC1 promoter (968b) derived from IFO2260 strain
FIG. 3 is a diagram showing β-galactosidase activity after subculture in a transformant in which p) and a YDC strain-derived PDC1 promoter (968 bp) were introduced.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Osamu Osamu             1 Toyota Town, Toyota City, Aichi Prefecture Toyota Auto             Car Co., Ltd. (72) Inventor Noriko Hoya             1 Toyota Town, Toyota City, Aichi Prefecture Toyota Auto             Car Co., Ltd. (72) Inventor Yasuo Matsuo             1 Toyota Town, Toyota City, Aichi Prefecture Toyota Auto             Car Co., Ltd. (72) Inventor Norihiro Ishida             Aichi Prefecture Nagachite Town Aichi District             Ground 1 Toyota Central Research Institute Co., Ltd. (72) Masanori Hirai, Inventor             Aichi Prefecture Nagachite Town Aichi District             Ground 1 Toyota Central Research Institute Co., Ltd. (72) Inventor Katsuhiko Kitamoto             3-44-18 Kamigashida, Ushiku City, Ibaraki Prefecture F-term (reference) 4B024 AA20 BA08 CA04 DA12 EA04                       FA02 GA11 GA19 HA03 HA14                 4B065 AA21Y AA79X AA80Y AB01                       AC14 BA02 CA28

Claims (7)

[Claims] 1. A gene expression method, which comprises introducing a target gene into a genome under the control of a promoter of a gene having an autoregulation mechanism or a promoter of a gene not essential for growth or fermentation in a host organism. 2. The promoter has a deletion or substitution of 1 to 40 bases in the nucleotide sequence of a gene promoter having an autoregulation mechanism or the nucleotide sequence of a gene promoter not essential for growth or fermentation in a host organism. Alternatively, the gene expression method according to claim 1, which is a DNA containing an added sequence and having a promoter activity. 3. A sequence complementary to the nucleotide sequence of a promoter of a gene having an autoregulation mechanism, or the whole or part of the nucleotide sequence of a promoter of a gene not essential for growth or fermentation in a host organism. The method for expressing a gene according to claim 1, which is a DNA that hybridizes with the DNA consisting of 1) under stringent conditions and that has a promoter activity. 4. The gene expression method according to claim 1, wherein the promoter of a gene having an autoregulation mechanism is the promoter of pyruvate decarboxylase 1 gene. 5. The gene expression method according to claim 1, wherein the promoter of a gene not essential for growth is a promoter of a gene encoding thioredoxin. 6. The gene expression method according to claim 1, wherein the host organism is bacteria, yeast, insects, animals or plants. 7. The gene expression method according to claim 6, wherein the yeast belongs to the genus Saccharomyces.