JP2013021940A - Method for producing gene knockout yeast - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a production method of gene knockout yeast which is capable of conveniently obtaining a gene knockout yeast which has been obtained only by using a special apparatus or a much time-consuming method due to occurrence of growth inhibition due to gene knockout.SOLUTION: There is disclosed a production method of gene knockout yeast comprising a first step in which an alternative metabolite production path and compensatory path are constructed for host yeast, a second step in which gene knockout causing the growth inhibition of the host yeast is carried out, and a third step in which the compensatory path is dropped out.

Description

  The present invention relates to a yeast production method capable of efficiently producing useful chemical substances other than ethanol. More specifically, it is possible to easily obtain a gene-deficient yeast that can be obtained only by a special device or a method that requires a lot of time, because growth inhibition occurs when the gene is deficient in order to reduce ethanol production ability. The present invention relates to a method for producing a gene-deficient yeast.

  Since yeast produces ethanol with high efficiency in the presence of a high concentration of glucose, it has been conventionally used as a production strain for sake, beer and the like. In recent years, it is considered promising as a substance-producing bacterium using biomass as a raw material because of its high growth ability, glucose utilization, acid resistance, and the like.

  When a substance other than ethanol is produced using yeast, a pyruvate decarboxylase (abbreviated as PDC) gene or alcohol dehydrogenase (ADH) in the ethanol production pathway is used to reduce the productivity of ethanol produced in large quantities. (It is abbreviated.) It is desirable to destroy genes such as genes. However, it is known that, when these genes are to be deleted, particularly when a plurality of genes are deleted at the same time, host growth and fermentation inhibition occur (Patent Document 1: JP 2003-500062). , Non-Patent Document 1: Hohmann, S. et al. (1990) EUR. J. Biochem., 188, p615-621), obtaining a strain capable of producing the desired substance by modifying a metabolic gene of yeast It was an obstacle.

  In general, as a gene deletion method of yeast, a simple and highly efficient gene replacement method by homologous recombination is used (Non-patent Document 2: Baudin, A. et al. (1993) Nucleic Acids Res., 21, p3329-3330, Non-Patent Document 3: Brachmann, C.B. et al. (1998) Yeast, 14, p115-132). However, when a gene-deficient strain causes growth inhibition, simple gene replacement using homologous recombination has a problem that it is difficult to obtain a mutant strain by selection on a plate. In the conventional method, spores are formed and separated (Patent Document 2: JP-T-2001-516484, Patent Document 3: JP-A 2006-6271) or by continuously culturing in a medium having a different composition. Although evolutionary engineering (Patent Document 4: Japanese Translation of PCT International Publication No. 2006-525025) has obtained mutant strains, a special device such as a manipulator is required, and it takes a very long time until the desired strain is obtained. There was also a difficulty of requiring.

Special table 2003-500062 gazette Special table 2001-51658 gazette JP 2006-6271 A Special table 2006-525005 gazette

Hohmann, S. et al. (1990) Eur. J. Biochem., 188, p615-621. Baudin, A. et al. (1993) Nucleic Acids Res., 21, p3329-3330. Brachmann, C. B. et al. (1998) Yeast, 14, p115-132.

  An object of the present invention is to easily obtain a gene-deficient yeast that can be obtained only by a special apparatus or a method that requires a lot of time, because growth inhibition occurs when a gene is deficient in order to reduce ethanol production ability. Another object is to provide a method for producing a gene-deficient yeast.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor has established a first step of constructing an alternative metabolite production pathway and a compensation pathway for the host yeast, and a gene that causes growth inhibition of the host yeast. In order to complete the present invention, it has been found that a yeast strain deficient in a gene causing growth inhibition can be obtained by performing a treatment including a second step of performing deletion and a third step of removing the compensation pathway. It came.

That is, the present invention includes the following [1] to [16].
[1] A first step of constructing an alternative metabolite production pathway and a compensation pathway for the host yeast, a second step of deleting a gene that causes growth inhibition of the host yeast, and dropping the compensation pathway A method for producing a gene-deficient yeast, comprising a third step.
[2] The method for producing a gene-deficient yeast according to [1], wherein the alternative metabolite is malic acid and / or succinic acid.
[3] The step of constructing the alternative metabolite production pathway is a step of introducing and / or amplifying a malate dehydrogenase (MDH) gene and / or a malate transporter (MAE) gene in [1] or [2] A method for producing the gene-deficient yeast as described.
[4] The method for producing a gene-deficient yeast according to [3], wherein the malate dehydrogenase (MDH) is encoded by at least one gene selected from the group consisting of MDH1 gene, MDH2 gene and MDH3 gene.
[5] The method for producing a gene-deficient yeast according to [3], wherein the malate transporter (MAE) is encoded by the mae1 gene.
[6] The method for producing a gene-deficient yeast according to any one of [1] to [5], wherein the compensation path construction step is a step of introducing the same gene as the gene to be deleted in the second step.
[7] In any one of [1] to [6], in the step of constructing the compensation pathway, a plasmid having a selectable marker that is lost in the third step to enable functional loss is used. A method for producing a gene-deficient yeast.
[8] The method for producing a gene-deficient yeast according to [1], wherein the gene to be deficient is a gene in an ethanol biosynthesis pathway.
[9] The method for producing a gene-deficient yeast according to [8], wherein the ethanol biosynthetic pathway gene is a gene encoding pyruvate decarboxylase (PDC) and / or alcohol dehydrogenase (ADH).
[10] The method for producing a gene-deficient yeast according to [9], wherein the pyruvate decarboxylase (PDC) is encoded by at least one gene selected from the group consisting of a PDC1 gene, a PDC5 gene and a PDC6 gene. .
[11] The gene deficiency according to [9], wherein the alcohol dehydrogenase (ADH) is encoded by at least one gene selected from the group consisting of an ADH1 gene, an ADH2 gene, an ADH3 gene, an ADH4 gene, and an ADH5 gene Yeast production method.
[12] The method for producing a gene-deficient yeast according to any one of [1] to [11], wherein the second step is performed using a gene disruption method by gene targeting using a PCR product.
[13] The method for producing a gene-deficient yeast according to any one of [1] to [12], wherein the host yeast belongs to the genus Saccharomyces.
[14] The method for producing a gene-deficient yeast according to [13], wherein the host yeast is Saccharomyces cerevisiae.
[15] Production of the gene-deficient yeast according to any one of [1] to [14], wherein the strain is obtained in at least one of the first to third steps by selection on a plate containing a selection marker. Method.
[16] The method for producing a gene-deficient yeast according to [15], wherein the selection marker is a drug resistance marker or an auxotrophic marker.

  According to the method of the present invention, rapid growth inhibition due to a gene deficiency of the host yeast can be prevented by the action of the compensation pathway, and a strain deficient in the target gene can be easily obtained without requiring a special device or a long time. According to the method of the present invention, it is possible to obtain a strain deficient in one or a plurality of genes that causes growth inhibition of host yeast by simple selection using homologous recombination and plates, and using a special apparatus, spores can be obtained. Compared to the conventional method of forming and separating, there are significant advantages in terms of time and money. Moreover, the recombinant yeast obtained by the present invention is useful as a producing bacterium for producing useful chemical substances such as organic acids.

Deletion of the ADH1 gene by gene targeting and the confirmation process are shown.

Hereinafter, the present invention will be described in detail.
The method for producing a gene-deficient yeast of the present invention comprises a first step of constructing an alternative metabolite production pathway and a compensation pathway for the host yeast, and a second step of performing a gene deficiency that causes growth inhibition of the host yeast. And a third step of dropping the compensation path.

  In this specification, “gene deficiency” refers to a state in which a gene originally loses its function, a part or all of the base sequence of the gene is deleted, and the base sequence of the gene is replaced with another base sequence. It is caused by. Such a state may occur naturally, but in the present invention, it means that an arbitrary gene is artificially deleted depending on the purpose.

  As used herein, “growth inhibition” refers to a state in which the growth rate of genetically modified yeast is significantly reduced compared to the wild type strain, or a state in which growth is no longer possible. A gene causing growth inhibition refers to a gene in a major metabolic pathway in which growth of a host is inhibited when one or more genes are deleted. For example, glycogen synthase, glucose-1-phosphate uridiri Transferase, glycogen phosphorylase, phosphoglucomutase, glucokinase, hexokinase, glucose-6-phosphatase, glucose phosphate isomerase, glucosamine phosphate isomerase, phosphofructokinase, hexose diphosphatase, fructose bisphosphate aldolase, triose phosphate isomerase, glycerol -3-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, bisphosphoglyceromutase Bisphosphoglycerate phosphatase, phosphoglycerate kinase, phosphoglyceromutase, enolase, phosphoenolpyruvate carboxykinase, pyruvate carboxylase, pyruvate kinase, lactate dehydrogenase, pyruvate decarboxylase, alcohol dehydrogenase, malate dehydrogenase, malate synthase And genes encoding enzymes such as isocitrase, fumarate hydratase, succinate dehydrogenase, succinyl-CoA synthetase, oxoglutarate dehydrogenase, isocitrate dehydrogenase, aconitate hydratase, and citrate synthase.

  Among them, a group of genes in the biosynthetic pathway of ethanol is preferable as a deficient target of the present invention, since growth inhibition due to the deletion is remarkable, and pyruvate decarboxylase (abbreviated as PDC) and alcohol dehydrogenase (abbreviated as ADH). ) Is preferable as a deletion target. Particularly preferred are PDC1, PDC5, PDC6, ADH1, ADH2, ADH3, ADH4 and ADH5 genes (Sacccharomyces Genome Database System Name: YLR044C, YLR134W, YGR087C, YOL086C, YOL086C, YOL086C, YOL086C, YOL086C, YOL0486C, YOL086C, YOL086C, YOL086C, YOL086C, YOL086C, YOL0486C, YOL086C, YOL086C, YOL0308C, YOL086C, YOL086C, YOL086C, YOL087C, YOL086C, YOL086C, YOL086G,

  The yeast to be gene-deficient in the present invention is not particularly limited as long as it is a gene-deficient yeast.For example, the genus Saccharomyces, the genus Kluyveromyces, the genus Schizosaccharomyces, Examples include yeasts such as the genus Zygosaccharomyces, the genus Yarrowia, the genus Hansenula, the Pichia genus, the Candida genus, among which the genus Saccharomyces is preferred, and the genus Saccharomyces is preferred. Saccharomyces cerevisiae) is particularly preferred. Moreover, since it becomes easy to select a transformant when a gene is introduced by transformation, a strain having auxotrophy is preferable.

  In the present specification, the “alternative metabolite” refers to a metabolite that can take a redox balance in the cells instead of the metabolite of the target gene when the target gene to be deleted is deleted. For example, alternative metabolites of ethanol include lactic acid, acrylic acid, acetic acid, pyruvic acid, oxaloacetic acid, malic acid, glyoxylic acid, 3-hydroxypropionic acid, fumaric acid, succinic acid, itaconic acid, levulinic acid, adipic acid, Examples include organic acids such as ascorbic acid and citric acid, and alcohols such as 1-propanol, 2-propanol, 1-butanol, isobutanol, 2-methyl-1-butanol, and 3-methyl-1-butanol. Among these, lactic acid, malic acid, and succinic acid are preferable as alternative metabolites, and malic acid and succinic acid are more preferable.

  In this specification, “construction of an alternative pathway” means that a metabolic pathway gene that leads to an alternative metabolite in the first step of the present invention is introduced into a host yeast in advance, and a function for producing these metabolites is given. is there. For example, lactate dehydrogenase, citrate synthase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, pyruvate dehydrogenase, pyruvate carboxylase, malate synthase, isocitrase, malate dehydrogenase, fumarase, fumarate reductase, succinate dehydrogenase, A gene encoding a malate transporter or the like can be used. Among them, it is preferable to introduce and / or amplify genes such as malate dehydrogenase, malate transporter, etc., in which an alternative pathway in which the production amount of malic acid and succinic acid as an alternative metabolite is increased, MDH1, MDH2, One or more of the MDH3 gene (Sacccharomyces Genome Database Systemic Name: YKL085W, YOL126C, YDL078C) and the mae1 gene (NCBI accession number: NM_001020205) are preferably introduced and / or amplified.

  The gene to be introduced can be isolated by a known method such as PCR using chromosomal DNA as a template. Alternatively, it is designed based on the base sequence of the gene used in the present invention, using as a template total nucleic acid prepared by a conventional method, cDNA obtained by RT-PCR from total RNA, or a commercially available cDNA library. Can be obtained by a PCR method using a primer. Moreover, it can also carry out by well-known methods, such as hybridization, from a chromosomal DNA library.

  Any vector can be used for constructing the alternative pathway as long as it is replicated and stably maintained in the yeast into which the gene is introduced, such as a chromosomal integration plasmid, single copy plasmid, A copy-type plasmid or the like can be used. Examples thereof include the YI, YC, and YE types of the pRS series and the p4XX series (Dominik et al., (1995) Gene. 156: p.119-122). In order to incorporate an arbitrary gene into a vector, for example, a DNA fragment containing the gene may be cleaved with an appropriate restriction enzyme, inserted into a restriction site of the vector DNA or a multicloning site in-frame, and ligated. The expression vector used in the present invention preferably contains a selectable marker indicating that the vector is retained in the cell.

  The promoter used for the functional expression of the introduced gene may be any as long as it can induce the expression of the gene under its control in the yeast into which the expression vector is introduced. For expression in yeast, known promoters such as glyceraldehyde 3-phosphate dehydrogenase promoter, PH05 promoter, MFα1 promoter, ADH promoter, TEF promoter, and CYC1 promoter can be used. Of these, the glyceraldehyde 3-phosphate dehydrogenase promoter is most preferred. As the terminator, known terminators such as ENO1 terminator, GAL10 terminator, GAPDH terminator, ADH terminator, CYC1 terminator, and the like can be used.

  In the present specification, “construction of compensation pathway” corresponds to the step of introducing a gene that expresses a function equivalent to the target gene to be deleted in the second step of the present invention. In other words, it means that a target gene that is identical to or equivalent to the target gene to be gene-deficient is expressed to make a state that complements the function of the target gene by expressing the gene, and specifically, A gene capable of expressing the same or equivalent function as the target gene is introduced into yeast and expressed. For example, when it is desired to delete the ADH1 gene on the yeast genome, a compensation pathway can be constructed by linking the ADH1 gene isolated by PCR or the like to an expression plasmid and introducing it into the yeast by transformation. The introduced gene is preferably the same as the gene to be deleted, but it may be a different gene or may be derived from another organism as long as it has the same function. Moreover, the gene to be introduced may be one kind or plural kinds.

  The vector used for construction of the compensation pathway is preferably a plasmid. Anything can be used as long as it is autonomously replicated in a plasmid state in yeast. For example, vectors such as the YC and YE types of the pRS series and the p4XX series (Dominik et al., (1995) Gene. 156: p.119-122) can be mentioned. In order to incorporate an arbitrary gene into a vector, for example, a DNA fragment containing the gene may be cleaved with an appropriate restriction enzyme, inserted into a restriction site of the vector DNA or a multicloning site in-frame, and ligated. It is preferable that the plasmid for constructing the compensation pathway has a selection marker that can be lost later to enable loss of function.

  In order to transform a host yeast using the gene used in the present invention or a recombinant vector containing the same, generally used gene transfer methods such as the competent cell method, the calcium phosphate method, and the electroporation method are used. The lipofection method, particle gun method, polyethylene glycol (PEG) method, lithium chloride method, Agrobacterium method, protoplast fusion method and the like may be used. The selection of transformants can be performed according to a conventional method, but can usually be performed using a selection marker incorporated into the used recombinant vector.

  There are no particular restrictions on the order in which the alternative metabolite production pathway and the compensation pathway are constructed in the first step of the present invention. That is, a compensation path can be constructed after construction of an alternative metabolite production path, or an alternative metabolite production path can be constructed after construction of a compensation path. It is also possible to simultaneously construct a compensation route and an alternative metabolite production route.

  Confirmation that the gene has been introduced into yeast can be carried out using PCR, Southern hybridization, Northern hybridization, Western blotting, or the like. For example, a plasmid may be extracted from transformed yeast, and PCR amplification may be performed using a gene-specific primer. The amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, cybersafe (SYBR Safe), etc., and the amplified product is detected as a single band, It can be confirmed that the target gene has been introduced.

  The construction of the alternative pathway and the compensation pathway in the first step of the present invention is aimed at avoiding the growth inhibition of yeast caused by the loss of the function of the target gene on the chromosome. That is, even if the target gene on the chromosome is lost in the subsequent second step and its function is lost, the gene on the plasmid introduced as a compensation pathway compensates for its function, so that it can grow even if the target gene is lost. Can be maintained. Further, even if the plasmid introduced as a compensation route in the subsequent third step is dropped, a metabolite that can achieve a redox balance in the microbial cells is produced by constructing an alternative route in the first step. This means that a yeast that can grow even if the target gene is deficient is obtained. When the first step is not performed, or when only one of the alternative route construction and the compensation route construction in the first step is performed, growth inhibition occurs in the second or third step in any case, The target yeast cannot be obtained.

  In the first step, the second step and the third step described later in the present invention, a general drug resistance gene or an auxotrophic complementary gene can be used as a marker gene. For example, known drug resistance marker genes such as G418 resistance gene, aureobasidin resistance gene, kanamycin resistance gene, neomycin resistance gene, hygromycin resistance gene, phleomycin resistance gene, tetracycline resistance gene, zeocin resistance gene, ampicillin resistance gene are used. can do. Alternatively, a known auxotrophic marker gene that complements auxotrophy such as uracil requirement, lysine requirement, leucine requirement, histidine requirement, tryptophan requirement, methionine requirement, biotin requirement, and adenine requirement is used. be able to.

  The gene deletion in the second step of the present invention can be performed using gene replacement by general homologous recombination. For example, a gene disruption method by gene targeting using a PCR product (one-step gene disruption), a gene disruption method (two-step gene disruption) in which a marker gene is recovered after introducing a PCR product or a plasmid, and the like can be mentioned. In either case, gene deletion is possible by replacing the target gene with a marker gene by homologous recombination.

  The marker gene is introduced into the host yeast in the form of a vector having a homologous sequence with the chromosomal DNA of the host yeast at the 5 ′ end and 3 ′ end thereof, and the homologous sequence and the host chromosomal DNA undergo homologous recombination. Is introduced into the chromosome of the host yeast by insertion or substitution, whereby the target gene is deleted. By designing a homologous sequence based on chromosomal DNA of a host yeast whose base sequence is clear, a marker gene can be introduced at an arbitrary position on the chromosome and an arbitrary target gene can be deleted.

  The vector for introducing the marker gene into the host yeast may be a linear DNA fragment, or may be circular like a plasmid. The vector can be introduced using a known gene introduction technique. For example, a competent cell method, a calcium phosphate method, an electroporation method, a lipofection method, a particle gun method, a polyethylene glycol (PEG) method, a lithium chloride method, an Agrobacterium method, a protoplast fusion method and the like may be used.

  In the present invention, it can be confirmed by a known method that the target gene has been deleted. For example, confirmation based on the phenotype of the target gene or PCR can be performed on a region on a chromosome sandwiching the target gene, and confirmation can be performed based on the size of the amplified fragment. Moreover, you may detect using a probe specific to a target gene.

  The elimination of the compensation pathway constructed in the first step, which is the third step in the present invention, that is, the elimination of the plasmid is carried out without applying a selective pressure by the selection marker in the plasmid used for construction of the compensation pathway. Culturing can be performed by a method that utilizes the fact that the plasmid naturally falls off when the yeast divides. Since the screening for the strain with the missing plasmid can be carried out by positive screening in which only the dropped strain can survive, the plasmid having the Ura3 and Lys2 markers and 5-fluoroorotic acid (FOA) as the above-mentioned auxotrophic complementary gene can be used. ) And α-aminoadipic acid (αAA) are preferably used.

  The medium used for yeast culture contains glucose as a carbon source and a nitrogen source that can be assimilated by the recombinant yeast obtained in the present invention, inorganic salts, vitamins, etc., and can efficiently culture recombinant yeast. If so, either a natural medium or a synthetic medium may be used. Any of static culture, shaking culture, and deep aeration-agitation culture may be used, but shaking culture is preferably used. The culture temperature is usually 15 to 40 ° C.

  Hereinafter, although an example, a reference example, and a comparative example explain the present invention still in detail, the present invention is not limited to these.

Example 1
Step 1) Construction of Alternative Route and Compensation Route Example 1-1, Reference Examples 1-2: Construction of Alternative Route (1) Isolation of MDH3 Gene and Preparation of Plasmid Containing MDH3 Gene Saccharomyces cerevisiae BY4742 Chromosomal DNA of the strain was prepared using QIA amp DNA Mini kit (manufactured by QIAGEN) as described in the attached instructions. Hereinafter, all the preparations of genomic DNA were in accordance with the description in the manual. A PCR reaction was performed using the obtained chromosome as a template and DNA having the nucleotide sequences shown in SEQ ID NOS: 1 and 2 as primers, and a DNA fragment containing about 1.0 kbp MDH3 gene was obtained. PCR reaction was performed using PrimeSTAR HS DNA Polymerase (manufactured by Takara Bio Inc.) according to the description in the attached manual. Hereinafter, all PCR methods were in accordance with the description in the manual. The chain length of the DNA fragment was confirmed by electrophoresis of the PCR reaction product on a 0.9% agarose gel. Using the obtained DNA fragment and Zero Blunt (registered trademark) TOPO (registered trademark) Cloning Kit (manufactured by Invitrogen), cloning was performed according to the description of the attached manual. Treat the resulting plasmid and p413GPD vector with SpeI and SmaI, respectively, and perform electrophoresis on 1.0% agarose gel prepared using SeaPlaque (registered trademark) GTG (registered trademark) agarose (manufactured by CAMBREX). The SpeI-SmaI fragment was isolated and purified. After that, ligation was performed using Takara ligation mix (manufactured by Takara Bio Inc.) to obtain a target plasmid in which the MDH3 gene was ligated to p413GPD. When the base sequence of the inserted DNA fragment was examined using a DNA sequencer, it had DNA having the base sequence represented by SEQ ID NO: 3.

(2) Isolation of the mae1 gene and preparation of a plasmid having the mae1 gene The cDNA library of Schizosaccharomyces pombe (Bio Academia) is used as a template and has the nucleotide sequences shown in SEQ ID NOs: 4 and 5. A PCR reaction was performed using DNA as a primer to obtain a DNA fragment of about 1.3 kbp containing the mae1 gene. The chain length of the DNA fragment was confirmed by electrophoresis of the reaction product on a 0.9% agarose gel. Using the obtained DNA fragment and Zero Blunt (registered trademark) TOPO (registered trademark) Cloning Kit (manufactured by Invitrogen), cloning was performed according to the description in the attached instruction manual. Treat the resulting plasmid and p415GPD vector with SpeI and PstI, respectively, and perform electrophoresis on 1.0% agarose gel prepared using SeaPlaque (registered trademark) GTG (registered trademark) agarose (manufactured by CAMBREX). The SpeI-PstI fragment was isolated and purified. After that, ligation was performed using Takara ligation mix (manufactured by Takara Bio Inc.) to obtain a target plasmid in which the mae1 gene was ligated to p415GPD. When the nucleotide sequence of the inserted DNA fragment was examined using a DNA sequencer, it had DNA having the nucleotide sequence represented by SEQ ID NO: 6.

(3) Introduction of MDH3 and mae1 genes into yeast lacking PDC1 gene As a transformation host, a commercially available gene-deficient strain lacking the PDC1 gene of Saccharomyces cerevisiae BY4742 (purchased from Open Biosystems, Inc. ΔPDC1 strain).
Using the plasmid prepared in (1) and (2) above, the ΔPDC1 strain was transformed by the transformation method described in Chen D, Yang B, Kuo T. (1992). Curr Genet 21: 83-84. Thus, the transformant 1 of Example 1-1 was obtained.
As the transformant selection medium, an agar medium to which an amino acid was added if necessary to an SD agar medium having the following composition was used. That is, transformant 1 grows by culturing at 30 ° C. for 3 days using SD (Ura, Lys) agar medium (SD agar medium supplemented with uracil 20 mg / L and lysine hydrochloride 30 mg / L). It was obtained by selecting colonies to be selected.
SD agar composition:
East Nitrogen Base (Difco) 6.7g / L
Glucose 20g / L
Agar 20g / L

(4) Culture results of transformant When the obtained transformant 1 was prepared in the SC liquid medium having the composition shown below, a test tube culture test was performed using the liquid medium prepared by removing the amino acid corresponding to the selection marker. went. That is, SC (-His, -Leu) liquid medium prepared without adding histidine and leucine was used as transformant 1 of Example 1-1. For reference, untransformed ΔPDC1 strain (Reference Example 1) and BY4742 (Reference Example 2) were cultured side by side. In Reference Examples 1 and 2, a normal SC liquid medium was used. Table 1 shows the plasmids, agar medium and liquid medium used in Example 1-1 and Reference Examples 1-2.
One platinum ear from each plate was inoculated into 5 mL of each medium and cultured overnight at 30 ° C., and this was used as a preculture solution. The obtained preculture solution was inoculated into 5 mL of SC liquid medium in a new test tube so that the initial turbidity (OD660) was 0.05, and cultured at 30 ° C. OD660 was measured with a turbidimeter (miniphot 518R, manufactured by TAITEC). The contents of ethanol, pyruvic acid, malic acid and succinic acid in the culture supernatant were quantified using liquid chromatography (manufactured by Shimadzu Corporation) under the following conditions. The results are shown in Table 2.

SC liquid medium composition:
East Nitrogen Base (Difco) 6.7g / L
Glucose 20g / L
Adenine sulfate 20mg / L
Uracil 20mg / L
Tryptophan 20mg / L
Histidine hydrochloride 20mg / L
Arginine hydrochloride 20mg / L
Methionine 20mg / L
Tyrosine 30mg / L
Leucine 60mg / L
Isoleucine 30mg / L
Lysine hydrochloride 30mg / L
Phenylalanine 40mg / L
Glutamic acid 100mg / L
Aspartic acid 100mg / L
Valine 150mg / L
Threonine 200mg / L
Serine 400mg / L
Liquid chromatography analysis conditions:
Sample: culture supernatant,
Column: Shodex SH1011 (300 × 8.0 mm, manufactured by Showa Denko KK),
Mobile phase: 20 mM sulfuric acid,
Flow rate: 0.6 ml / min,
Column temperature: 60 ° C.
Detection: RI detector.

  As shown in Table 2, Saccharomyces cerevisiae of Example 1 produced more organic acid than Reference Examples 1 and 2, and the construction of an alternative route was confirmed.

Example 1-2: Construction of Compensation Pathway (5) Isolation of ADH1 Gene and Preparation of Plasmid Containing ADH1 Gene Chromosomal DNA of Saccharomyces cerevisiae BY4742 strain is used as a template and represented by SEQ ID NOs: 7 and 8 A PCR reaction was performed using DNA having a base sequence as a primer to obtain a DNA fragment of about 1.0 kbp containing the ADH1 gene. The chain length of the DNA fragment was confirmed by electrophoresis of the reaction product on a 0.9% agarose gel. Using the obtained DNA fragment and Zero Blunt (registered trademark) TOPO (registered trademark) Cloning Kit (manufactured by Invitrogen), cloning was performed according to the description of the attached manual. By subjecting the obtained plasmid and the p426GPD vector to restriction enzyme treatment with ClaI and SalI, and electrophoresis on 1.0% agarose gel prepared using SeaPlaque (registered trademark) GTG (registered trademark) agarose (manufactured by CAMBREX). The ClaI-SalI fragment was isolated and purified. After that, ligation was performed using Takara ligation mix (manufactured by Takara Bio Inc.) to obtain a target plasmid in which the ADH1 gene was ligated to p426GPD. When the nucleotide sequence of the inserted DNA fragment was examined by a DNA sequencer, it had DNA having the nucleotide sequence represented by SEQ ID NO: 9.

(6) Introduction of ADH1 gene into transformant 1 The plasmid prepared in (5) above is transformed according to Chen D, Yang B, Kuo T. (1992). Curr Genet 21: 83-84. To the transformant 1 of Example 1-1. As the medium, an SD (Lys) agar medium obtained by adding 30 mg / L lysine hydrochloride to an SD agar medium was cultured at 30 ° C. for 3 days, and transformant 2 of Example 1-2 shown in Table 3 was obtained. did.

  A plasmid was extracted from the transformant 2 of Example 1-2, and it was confirmed by PCR that the ADH1 gene was introduced. That is, the construction of the compensation path in Example 1-2 was confirmed.

Step 2) Deletion of gene causing growth inhibition (7) Preparation of DNA fragment for deletion of ADH1 gene PCR reaction is performed using chromosomal DNA of Saccharomyces cerevisiae BY4742 strain as a template, and homologous recombination sequence on the 5 'end side As ADH1 UP (−229 to −1), each DNA fragment of ADH1 DN (1048 to 1336) as a 3′-end homologous recombination sequence was amplified. The primers of SEQ ID NOs: 10 and 11, and SEQ ID NOs: 12 and 13 were used as PCR primers, respectively. Similarly, the gene fragment of the LYS2 marker was amplified using the pRS317 vector as a template and the primers of SEQ ID NOs: 14 and 15.
After purifying these fragments, the fragments were mixed and subjected to a second PCR using the primers of SEQ ID NOs: 10 and 13, to obtain a fragment of about 5.4 kbp. The finally obtained DNA fragment is referred to as “ADH1 gene-deficient DNA fragment”.

Example 1-3, Comparative Examples 1-3:
(8) Preparation of ADH1 gene-deficient yeast Examples obtained in (3) and (6) above using the gene disruption method by gene targeting using the ADH1 gene-deficient DNA fragment prepared in (7) above. Transformant 1-1 of 1-1, transformant 2 of Example 1-2, ΔPDC1, and BY4742 were transformed. As the transformant selection medium, the agar medium described in Table 4 was used for 3 days at 30 ° C., and four types of transformants, namely transformant 2 to transformant 3 of Example 1-3 were used. Transformant 1 to Transformant 4 (Comparative Example 1), ΔPDC1 from Transformant 5 (Comparative Example 2), and BY4742 from Transformant 6 (Comparative Example 3).

(9) Confirmation of gene deletion Genomic DNA was prepared from the four types of transformants obtained in (8) above. Subsequently, whether or not the ADH1 gene-deficient DNA fragment was integrated into the genome was confirmed by PCR. In this PCR, it was confirmed whether the target sequence was extended to the expected length using three sets of primers. That is, the primers of SEQ ID NOS: 10 and 13 are used as primers that extend the full length of the homologous recombination part of the ADH1 gene, and the primers that extend from the middle of the LYS2 marker to the homologous recombination sequence on the 3 ′ end side of the ADH1 gene. The primers of SEQ ID NOs: 13 and 16 were used, and the primers of SEQ ID NOs: 13 and 17 were used as primers that extend from the middle of the ADH1 gene to the homologous recombination sequence on the 3 ′ end side of the ADH1 gene (see FIG. 1).
The results of confirmation PCR and ADH1 gene deletion are shown in Table 5.

As shown in Table 5, since growth inhibition does not occur with the single deletion of the ADH1 gene, the ADH1 gene deletion of BY4742 in Comparative Example 3 is possible, but the ADH1 gene deletion when ΔPDC1 is used as the host in Comparative Example 2, that is, Since double inhibition of the PDC1 gene and the ADH1 gene results in growth inhibition, it is technically difficult to obtain a double-deficient strain in Comparative Example 2 in which an alternative route and a compensation route are not constructed. In Comparative Example 1, an alternative route is constructed, but since a compensation route is not constructed, a double-deficient strain cannot be obtained as in Comparative Example 2.
On the other hand, as in Example 1-3, a transformant in which an alternative pathway (introduction of MDH3 gene and mae1 gene) and a compensation pathway (introduction of a plasmid in which the ADH1 gene is linked to a p426GPD vector) has been constructed is used. This indicates that the ADH1 gene deficiency is possible.

Step 3) Deletion of gene of compensation pathway (10) Deletion of plasmid The transformant 3 of Example 1-3 obtained in (8) above was prepared without adding histidine, leucine and lysine (- His, -Leu, -Lys) liquid medium was used for 3 days. Then, when it apply | coated to SC (-His, -Leu, -Lys) agar medium containing 5-FOA 1mg / L, the colony appeared with the frequency of about 1.3 * 10 < ^-5 >. The obtained colonies are transferred to both SC (+ Ura, -His, -Leu, -Lys) agar medium containing uracil and SC (-Ura, -His, -Leu, -Lys) agar medium containing no uracil, Plated overnight at 30 ° C. Among the transferred colonies, a strain that grew only on the SC (+ Ura, -His, -Leu, -Lys) agar medium and did not grow on the SC (-Ura, -His, -Leu, -Lys) agar medium, It was selected as a strain of Example 1 in which the plasmid (p426GPD vector linked with the ADH1 gene) was dropped. In the strain of Example 1, the uracil requirement is restored, and both the PDC1 gene and the ADH1 gene are deleted.

  The recombinant yeast obtained by the present invention is useful as a producing bacterium for producing useful chemical substances such as organic acids, and is useful in the technical field of producing organic acids using saccharides as a raw material.

Claims (16)

  A first step of constructing an alternative metabolite production pathway and a compensation pathway for the host yeast, a second step of deleting a gene that causes growth inhibition of the host yeast, and a third step of dropping the compensation pathway A method for producing a gene-deficient yeast, comprising a step.   The method for producing a gene-deficient yeast according to claim 1, wherein the alternative metabolite is malic acid and / or succinic acid.   The gene-deficient yeast according to claim 1 or 2, wherein the step of constructing the alternative metabolite production pathway is a step of introducing and / or amplifying a malate dehydrogenase (MDH) gene and / or a malate transporter (MAE) gene. Manufacturing method.   The method for producing a gene-deficient yeast according to claim 3, wherein the malate dehydrogenase (MDH) is encoded by at least one gene selected from the group consisting of MDH1 gene, MDH2 gene and MDH3 gene.   The method for producing a gene-deficient yeast according to claim 3, wherein the malic acid transporter (MAE) is encoded by the mae1 gene.   The method for producing a gene-deficient yeast according to any one of claims 1 to 5, wherein the step of constructing the compensation pathway is a step of introducing the same gene as the gene to be deleted in the second step.   The gene-deficient yeast according to any one of claims 1 to 6, wherein a plasmid having a selection marker that can be lost in the third step is used in the step of constructing the compensation pathway. Production method.   The method for producing a gene-deficient yeast according to claim 1, wherein the gene to be gene-deficient is a gene in the ethanol biosynthesis pathway.   The method for producing a gene-deficient yeast according to claim 8, wherein the ethanol biosynthetic pathway gene is a gene encoding pyruvate decarboxylase (PDC) and / or alcohol dehydrogenase (ADH).   The method for producing a gene-deficient yeast according to claim 9, wherein the pyruvate decarboxylase (PDC) is encoded by at least one gene selected from the group consisting of a PDC1 gene, a PDC5 gene and a PDC6 gene.   10. The gene-deficient yeast according to claim 9, wherein the alcohol dehydrogenase (ADH) is encoded by at least one gene selected from the group consisting of ADH1 gene, ADH2 gene, ADH3 gene, ADH4 gene and ADH5 gene. Method.   The method for producing a gene-deficient yeast according to any one of claims 1 to 11, wherein the second step is performed using a gene disruption method by gene targeting using a PCR product.   The method for producing a gene-deficient yeast according to any one of claims 1 to 12, wherein the host yeast belongs to the genus Saccharomyces.   The method for producing a gene-deficient yeast according to claim 13, wherein the host yeast is Saccharomyces cerevisiae.   The method for producing a gene-deficient yeast according to any one of claims 1 to 14, wherein the strain is obtained in at least one of the first to third steps by selection on a plate containing a selection marker.   The method for producing a gene-deficient yeast according to claim 15, wherein the selection marker is a drug resistance marker or an auxotrophic marker.

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