JP2003144164A - Self-cloning type genetically modified practical yeast produced using a deletion selective plasmid and method for producing the same - Google Patents

Abstract

(57) [Summary] [Problem] The biggest factors that hinder the practical use of genetically modified yeast are the safety issues caused by eating yeast modified with genes derived from other species, and acceptance by consumers. There is no sense of security when it is done. In order to solve this problem, all useful genes to be introduced may be derived from yeast. SOLUTION: Unnecessary gene sequences such as gene sequences derived from other species may be removed after introduction of useful genes. If a useful gene derived from yeast is developed and introduced into a practical yeast and all other unnecessary gene sequences can be removed after introduction, a safe and useful self-cloning type genetically modified yeast is produced. be able to.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION
The present invention relates to a self-cloning genetically modified yeast using a removal-selection type plasmid and a method for producing the same.

[0002]

2. Description of the Related Art Yeast (Saccharomyces cerevisiae) is
It is used for familiar fermentation such as liquor and bread, and for the production of brewed foods, and is known to greatly affect the taste and aroma. Therefore, obtaining useful improved yeast varieties is very important in food development. The breeding of yeasts is carried out by various methods such as crossing yeasts of different types and inducing mutations by drugs and ultraviolet rays, and many useful yeasts obtained as a result have been put to practical use. With the recent development of gene manipulation technology, analysis of gene functions related to various brewing characteristics has also progressed. It is expected to improve yeast varieties by introducing or destroying these genes. However, although there are many practical examples of haploid laboratory yeasts, there is no gene recombinant yeast that has been put into practical use as diploid or more practical yeasts for industrial use.

[0003]

The biggest factor that hinders its practical application is the safety problem caused by eating yeast in which a gene derived from another organism has been recombined, and a sense of security when accepted by consumers. It is nothing. To solve this problem,
All useful genes to be introduced may be derived from yeast (self-cloning type). But there is another problem. Since there is no auxotrophic mutation in industrial yeast strains, it is unavoidable to use drug resistance genes as gene transfer markers.In addition, plasmids for gene transfer also contain bacterial gene sequences. It is that you are. With respect to these problems, all unnecessary gene sequences such as gene introduction markers and gene sequences derived from organisms of other species such as bacteria may be removed after the introduction of the useful gene.
If a useful gene derived from yeast can be developed and introduced into a practical yeast and all other unnecessary gene sequences can be removed after the introduction, a safe and useful self-cloning gene recombinant yeast can be produced. be able to.

[0004]

[Means for Solving the Problems] A problem in practical application of genetically modified yeast is that drug-resistant genes have to be used as gene transfer markers because industrial yeasts do not have auxotrophic mutations. That is, the plasmid for gene transfer contains the gene sequences of other species derived from bacteria. This problem can be solved if the unnecessary unnecessary gene sequences of other species can be removed after the introduction of the useful gene. In the present invention, unnecessary selective gene sequences are removed by using a selective deletion plasmid having a selective deletion marker.

By devising the structure of the removal-selective plasmid having the removal-selectable marker and the method for introducing it into the yeast chromosome, only the useful gene of interest remains on the chromosome when the removal-selectable marker is lost. , It becomes possible to remove all other unnecessary gene sequences. As shown in Table 1, although some deletion-selective markers have been present so far, they could only be used for yeast having a specific gene mutation (Rothstein, R. et al., Meth.
Enzymol., 194, 281-301, 1991; Sikorski, RS, Met
h. Enzymol., 194, 302-318, 1991; Alani, E. et a
l., Genetics, 116, 541-545, 1987). On the other hand, the removal technique of the present invention does not require a specific gene mutation and can be used for practical yeasts, and is a more versatile technique.

[0006]

[Table 1]

The present inventor has developed a technique for selectively removing unnecessary gene sequences for producing self-cloning recombinant yeast (Akada, R. et al., Mol. Ge.
n. Genet., 254, 267-274, 1997; J. Biosci. Bioen
g., 87, 43-48, 1999). For example, a growth inhibitory gene that inhibits yeast growth is conditionally induced to overexpress a gene in the downstream region, such as a galactose-inducible promoter (GAL1p, GAL7p, GAL10p, etc.), that is, ON of gene expression.
Combined with a promoter that can artificially control ON / OFF,
This is incorporated into a plasmid for yeast gene transfer. When this plasmid is introduced into yeast through a transformation operation and the yeast is allowed to undergo specific conditions under which a condition-inducible promoter is activated, the growth inhibitory gene is overexpressed, and until then, normal growth was achieved. Cells cause growth arrest. If the growth-inhibiting gene is eliminated from the yeast cell, growth arrest does not occur and the yeast cell can grow normally, so that it is possible to select a yeast from which the growth-inhibiting gene is removed.

[0008] After the completion of the gene manipulation system, its purpose is to apply it. Therefore, we considered applying this gene manipulation system to practical yeasts, especially to brewing yeasts. for that purpose,
First, a useful gene that changes the brewing characteristics is needed. Therefore, we paid attention to genes that produce high amounts of aroma components by increasing or decreasing the expression level of genes. Taking sake as an example, among the aromatic components, ethyl caproate (a scent of apples) and isoamyl acetate (a scent of pears) are the two major components of the sake's “Ginjo scent”. The value of the brewer's yeast produced is particularly high. FAS2, a gene involved in the biosynthetic pathway of ethyl caproate (Inokoshi, J., et al., Mol. Gen. Genet., 24
4, 90-96, 1994), a gene involved in the biosynthetic pathway of isoamyl acetate ATF1 (Yoshimoto, H., et al., J. Ferm. Bioen
g., Vol. 86, No. 1, 15-20. 1998) is already known (Table 2).

[0009]

[Table 2]

It is also known that among genes, the gene whose flavor, fragrance and the like are improved by destroying them (Table 2). These genes, by suppressing the gene expression level by the method of the present invention, the flavor and fragrance,
It is thought that it can be adjusted more delicately.

[0011] Although the examples using the ATF1 gene are described in the Examples, the present invention is not particularly limited as long as it is a gene of a practical yeast, especially a yeast for brewing, and has industrial utility. There is no. The main reason for paying attention to the ATF1 gene is that the ATF1 gene encodes acetyltransferase, which is a synthase of isoamyl acetate, and the effect is more direct. It has already been studied that isoamyl acetate-producing ability is altered by overexpression, suppression, or disruption of the ATF1 gene (Yoshimoto, H., et al.
l., J. Ferm. Bioeng., Vol. 86, No. 1, 15-20. 199
8), the present inventor also changed the expression level by substituting the promoter with the promoter of glyceraldehyde-3-phosphate dehydrogenase 3 (TDH3) known as one of the constitutively expressed promoters with the highest expression level, and the TDH3p- Of ATF1 sequence
It has been confirmed that 22.5 times higher production of isoamyl acetate (Wild-Type: 0.03 ppm, p305TDH3pATF1-5 introduced strain: 0.68 ± 0.22 ppm) was introduced into the W303-1A strain. However, for practical use, there remains a problem that unnecessary gene sequences of other species remain.

Therefore, only the yeast-derived sequence is present on the chromosome.
A method for constructing the TDH3p-ATF1 sequence was considered. In gene recombination using a deletion-selection type plasmid, homologous recombination at the time of introduction and homologous recombination at the time of removal selection will return to normal chromosomes unless they occur at different positions sandwiching the target sequence. Therefore, in order to construct the TDH3p-ATF1 gene sequence on the chromosome, it is not enough to combine the TDH3p-ATF1 gene sequence as it is with the elimination selection plasmid. TDH3
Up-ATF1p-TDH3p was introduced upstream of the promoter originally possessed by the ATF1 gene as a sequence capable of causing homologous recombination at the time of removal selection upstream of the p-ATF1 sequence.
-The ATF1 sequence was created and it was decided to combine it with the elimination selection plasmid. A significant difference is that the conventional technique (FIG. 1) is a mutant sequence consisting of at most several base pairs, whereas a second homologous recombination occurs across a promoter sequence consisting of several hundred base pairs. (Fig. 2). No such idea has ever been known.

According to the present invention, it has become possible to achieve the removal of unwanted other species genes after the insertion of a chromosome by using various removal selection type plasmids. In addition, all gene transfer markers can now be used for all yeasts. Therefore, the conventional gene transfer marker can be used only once in the same strain, so that the number of gene manipulations was limited, but without the restriction, the gene transfer marker can be used as many times as necessary. You can now operate. Since it is possible to use a marker for gene transfer that is easy to handle by introducing an auxotrophic mutation as a useful gene or disrupting the gene, it has been difficult to put it into practical use until now because there is no easy-to-use gene manipulation system. Or, even in yeasts that have not been put into practical use at all, it can be said that practical use will become much easier in the future. And, by utilizing useful genes related to brewing characteristics and brewing yeast breeding by gene recombination, genetic manipulation using a removal-selective plasmid can be used not only for research purposes but also for breeding of practical yeast for industrial use. Could be demonstrated.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described based on examples with reference to the drawings, but the present invention is not limited to these examples.

[0015]

Example 1 A PCR reaction for replication of the ATF1 promoter upstream sequence and introduction of a restriction enzyme site will be described. Using the chromosome extracted from Association No. 7 yeast (K7) as a template, ATF1-UPXBA and ATF1-UCSAL were used as primers (Table 3), and PCR reaction was performed using KOD DASH DNA Polymerase (TOYOBO). The composition of the reaction mixture is shown in Table 4. The PCR reaction cycle is [95 ℃, 1 min → 30 cycle × (95 ℃, 3
It was 0 sec → 56 ° C, 2 sec → 74 ° C, 2 min) → 4 ° C]. After the PCR reaction, the reaction solution was electrophoresed to separate and purify the desired 2.0 kb band. As a result, the upstream sequence of the ATF1 promoter was replicated over about 2.0 kb, and at the same time, the site of the restriction enzyme XbaI was introduced by the primer ATF1-UPXBA and the site of the restriction enzyme SalI was introduced by the primer ATF1-UCSAL.

[0016]

[Table 3]

[0017]

[Table 4]

The method for constructing the p305ATTDH3pATF1 plasmid is as follows:
It is as follows. A fragment (2 kb) obtained by digesting the sequence upstream of the ATF1 promoter synthesized by the PCR method with restriction enzymes XbaI and SalI, and p305TDH3pATF1-5 with restriction enzymes XbaI and SbaI.
Vector digested with alI and treated with Alkali Phosphatase
Ligation with (7.4 kb) was performed and E. coli was transformed with the Ligation solution to prepare plasmid p305ATTDH3pATF1. p
The GG119TDH3pATF1 plasmid uses pGG119 as a restriction enzyme XbaI.
The vector (7.4 kb) treated with Alkali Phosphatase after digestion with Sac and SalI and p305ATTDH3pATF1 were treated with restriction enzymes XbaI and Xho.
Ligation with the fragment (3.8 kb) cut with I was performed, and E. coli was transformed with the Ligation solution to obtain plasmid pG.
G119TDH3pATF1 was produced (Fig. 3).

[0019]

Example 2 As a method for transforming yeast, the One-Step transformation method based on the method by Chen et al. (Chen, DC, Curr. Genet. 21, 83-84, 1992) was used.

The pGG119TDH3pATF1 plasmid was cloned into the restriction enzyme S
1 cut in ORF (Open Reading Frame) of ATF1 by phI
And cerulenin-containing medium (MM + cer 1.0 μg / ml medium)
Was transformed into K7 strain. With drug resistance selection,
Transformants that showed resistance to both MM + cer 1.0 μg / ml medium and cycloheximide-containing medium (MM + cyc 0.5 μg / ml medium) were selected as candidates for the chromosome integration strain. One of the candidate strains, RAK1809, which was later confirmed to be a chromosome integrant, was streaked in a drug medium and statically cultured at 28 ° C for 3 days. The results are shown in Fig. 4.

When a combination of TDH3-1 and ATF1-UCSAL is used as the PCR primer, when the template is a plasmid, the C-terminal of the ATF1 ORF is deleted and ATF1-UCSAL cannot be annealed.
In the case of the chromosome of e, since the TDH3 promoter sequence is not present near ATF1, the TDH3p-ATF1 sequence cannot be synthesized by PCR (Fig. 5A). In the case of chromosomal integrant, since the C-terminal of ATF1 ORF is complemented, TDH3p
-ATF1 sequence (2.3 kb) can be synthesized. Using the extracted chromosome as a template, TDH3-1 and ATF1 as primers
-The introduction of the TDH3p-ATF1 sequence was confirmed depending on whether the 2.3 kb band was synthesized when the PCR reaction was performed using APA (Fig. 5B).

The PCR reaction for confirming the introduction of the TDH3p-ATF1 sequence was carried out by using KDH DASH DNA Pol with TDH3-1 and ATF1-APA (Table 5) as primers and the extracted chromosome as a template.
PCR reaction using ymerase (TOYOBO) was performed. Table 6 shows the composition of the reaction mixture. PCR reaction cycle is [94 ℃,
1 min → 25 cycle × (94 ℃, 20 sec → 54 ℃, 2 sec
→ 74 ℃, 2 min) → 4 ℃].

[0023]

[Table 5]

[0024]

[Table 6]

Chromosomes were extracted from the candidates for chromosomal integrants and used as a template. When PCR was performed using TDH3-1 and ATF1-APA as primers, confirmation was made by strains RAK1807, RAK1808 and There was RAK1809 (Figure 5B). Obtained chromosome integrants RAK1807, RAK1
For 808 and RAK1809, removal selection was performed in YPgal medium. The strains obtained by elimination selection were examined for drug resistance.
The results of streaking each strain in a drug medium and culturing at 28 ° C. for 3 days were shown (FIG. 4). The strains that had been selected for elimination had no drug resistance, and like Wild-Type, MM + cer
It could not grow in 1.0 μg / ml medium or MM + cyc 0.5 μg / ml medium. This means that the drug resistance marker was removed along with the removal selection marker.

Regarding the strains after elimination selection, which had lost the drug resistance, they were confirmed by the PCR method using TDH3-1 and ATF1-APA as primers as in the case of the chromosome integrant. A band of the desired size was detected in the strain. The one in which the band was not detected was one in which the homologous recombination at the time when the unnecessary sequence was removed occurred in the ORF of ATF1 and returned to the wild type, and the one in which the unnecessary sequence was removed was detected. It is considered that homologous recombination occurred within the sequence of up-ATF1p as desired and the TDH3p-ATF1 sequence was constructed on the chromosome. A total of 11 strains were confirmed by the PCR method, and finally RAK1857, RAK1858 and RAK1861 were confirmed.
Bands of the target size were detected in 3 strains (Fig. 5).
B).

When the extracted chromosome was treated with two kinds of restriction enzymes, AflII and NaeI, NaeI was detected upstream of ATF1 gene, AflI.
I can cut downstream of the ATF1 gene, and in the chromosomal integrant, the 4.9 kb band of Wild-Type and the 16.1 kb band indicating insertion of the full-length plasmid sequence, Wi
A 4.9 kb band of ld-Type and a 5.6 kb band showing the state in which the TDH3p-ATF1 sequence was introduced should be detectable. When the extracted chromosomes were treated with restriction enzymes AflII and NaeI (corresponding to 10 μl of chromosomal DNA solution), Southern blot hybridization using ATF1 probe confirmed that all of the strains had a band slightly larger than 10 kb. Were observed (Fig. 6), and the chromosomal integrant RAK180
In 7, RAK1808 and RAK1809, even larger bands were detected. Selected strain RAK1857, which removes RAK1809
The band disappeared in RAK1858 and RAK1861. Regarding this result, a band with a size slightly larger than 10 kb was not cleaved by the restriction enzyme NaeI, and the restriction enzyme AflII
This is in agreement with the size of 11.6 kb when the site about 6.7 kb upstream of NaeI was cleaved. Therefore, it was considered that the cleavage with NaeI was not successful. Therefore, the band detected only in the chromosomal integrant was explained to be a band of about 23 kb in which the full length 11.2 kb of the plasmid was inserted into 11.6 kb, and since the band disappeared completely after elimination selection, It was found that the chromosomal integrant was successfully obtained and the strain was selected for removal.

When the TDH3p-ATF1 sequence is introduced, a new SalI restriction enzyme site is generated immediately upstream of the TDH3 promoter. When the extracted chromosome is treated with three types of restriction enzymes, AflII, NaeI, and SalI, Wild-Type is only 4.9 kb when it can be completely cut, and 13.5 kb, 4.9 kb, 2.6 kb in the chromosome integrant, removal selection When the TDH3p-ATF1 sequence was introduced later, the 4.9 kb and 2.6 kb bands should be detectable. The same extracted chromosome as that used in FIG. 6 was treated with restriction enzymes AflII and NaeI (corresponding to 5 μl of chromosomal DNA solution) was further treated with restriction enzyme SalI, and Southern blot hybridizatio using ATF1 probe was used.
When detected by n, Wild-Type was only around 6 kb and 6 in chromosome integrants RAK1807, RAK1808 and RAK1809.
Around kb and around 2.6 kb of interest, and two bands of 10 kb or more, 6 in the strains RAK1857, RAK1858 and RAK1861 after removal selection
Bands around kb and around the target 2.6 kb were detected (Fig. 7). Since the SalI site is located 1.2 kb upstream of the NaeI site on the chromosome, this result can also be explained by considering that the cleavage by NaeI was not successful. Objective
A band around 2.6 kb was not detected in Wild-Type but was detected in the chromosome integrant and the strain after the removal selection, which indicates that the TDH3p-ATF1 sequence was introduced into these chromosomes. However, in RAK1807, the band around the desired 2.6 kb is very thin. The band of RAK1807 was thin even when confirmed by the PCR method. RAK1807 may have some mutation at the SalI site, that is, at the site where primer TDH3-1 anneals.

PGG119TDH3pATF1 is commercially available pBLUESCRIPT K
This is a plasmid derived from S +. PBLUESCR after removal selection
If there is no remaining sequence complementary to IPT KS +, it can be said that unnecessary sequence deletion is complete. KS + vect for the same extracted cells treated with restriction enzymes AflII and NaeI (corresponding to 5 μl of chromosomal DNA solution) as used in FIG.
When detected by Southern blot hybridization using or probe, the chromosome integrants RAK1807, RAK
A large band of about 23 kb, which is considered that the full-length plasmid was inserted into the chromosome only in 1808 and RAK1809, was detected, and strains RAK1857, RAK1858 and RAK1861 after removal selection were W
Similar to ild-Type, no band was detected (Fig. 8). Therefore, it was proved that no unnecessary DNA sequence remained in the strain obtained by the elimination selection.

Disappearance of drug resistance described above, confirmation by PCR method, Southern blot hybridizat using two kinds of probes
Based on the results of confirmation using ion, etc., TDH3p-AT was put on the chromosome without leaving any unnecessary gene sequences of other species on the practical yeast chromosome.
It was confirmed that only the F1 sequence was constructed. As a result, a technique has been completed in which expression of any gene on the chromosome of a practical yeast can be regulated by using only yeast-derived genes without leaving any unnecessary gene sequences of other species.

[0031]

INDUSTRIAL APPLICABILITY According to the present invention, a self-cloning gene recombinant practical yeast produced by using a removal-selection type plasmid is used to remove only yeast-derived genes without leaving any unnecessary gene sequences of other species. By using this, a technique capable of regulating the expression of all genes on the chromosome of a practical yeast has been completed.

[Brief description of drawings]

FIG. 1 is a diagram showing a method of using a deletion-selection type plasmid according to a conventional technique.

FIG. 2 is a diagram showing a method for constructing a TDH3-ATF1 sequence on a chromosome with only yeast-derived sequences.

FIG. 3 is a diagram showing a method for constructing a pGG119TDH3pATF1 plasmid.

FIG. 4 shows drug resistance of pGG119TDH3pATF1 chromosomal integrant and strains after selection for elimination.

FIG. 5 is a diagram showing confirmation of a TDH3p-ATF1 sequence on a chromosome by a PCR method.

FIG. 6 is a diagram showing confirmation by Southern blot hybridization of pGG119TDH3pATF1 chromosomal integrant and strain after elimination selection.

FIG. 7 Southern blot hy of TDH3p-ATF1 sequence on chromosome
It is a figure which shows the confirmation by bridization.

FIG. 8 is a diagram showing confirmation of pGG119TDH3pATF1 chromosome integrant and complete loss of unnecessary sequences by Southern blot hybridization.

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Claims (9)

[Claims] 1. A deletion-selecting plasmid, characterized in that the promoter region of a yeast gene containing the upstream region of the promoter is replaced with or inserted into another promoter of expression-regulatable yeast origin. A self-cloning type recombinant genetic yeast produced by using. 2. A self-cloning type prepared by using the removal-selection-type plasmid according to claim 1, wherein another yeast-derived promoter whose expression can be regulated is an overexpression type or an expression suppressing type. Practical recombinant yeast of. 3. A self-cloning gene recombinant practical yeast produced by using the removal-selection type plasmid according to claim 1, wherein the expression-regulatable promoter is an overexpression type. 4. A yeast AT containing an upstream region of a promoter
A self-cloning gene recombinant practical yeast produced by using the removal-selection plasmid according to any one of claims 1 to 3, wherein the promoter region of the F1 gene is replaced by a yeast-derived TDH3 promoter. 5. The self-cloning prepared using the removal-selection plasmid according to claim 1, wherein the gene sequence for removal selection has a GIN11M86 sequence downstream of the galactose-inducible promoter (GALp). Type genetically modified yeast. 6. A removal-selection plasmid characterized in that the promoter region of a yeast gene containing the upstream region of the promoter is replaced with or inserted into another promoter of yeast expression-regulatable expression. A method for producing a self-cloning gene recombinant practical yeast, which comprises transforming the practical yeast and then performing removal selection. 7. The expression-regulatable promoter is an overexpression type or an expression-suppressing type.
The method for producing a self-cloning type recombinant recombinant yeast according to 1. 8. The method for producing a self-cloning gene recombinant practical yeast according to claim 6 or 7, wherein the expression-regulatable promoter is an overexpression type. 9. A method in which a plasmid gene is integrated into a yeast chromosome by homologous recombination at the time of transformation with a removal-selection type plasmid, and then other species genes are selectively removed by homologous recombination at the time of removal selection treatment. 9. The method for producing the self-cloning gene recombinant practical yeast according to any one of claims 6 to 8.