JP2785084B2 - Method for producing squalene-producing yeast and novel yeast produced - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a squalene-producing microorganism by genetic manipulation, and a novel microorganism produced thereby.

[0002] Squalene is mainly used as a raw material of squalane used for base oils of cosmetics and the like.
It is also used as a health food, but its effects are said to be good for hypertension, stomach pain, liver disease, rheumatism, duodenal ulcer, arthritis, stomatitis, hemorrhoids, various dermatitis, stiff shoulders, burns, and hangovers. I have.

[0003] The present invention provides a method for producing a novel microorganism and a microorganism having a high productivity of squalene for producing squalene having such a physiologically active or medical effect.

[0004]

2. Description of the Related Art Squalene is contained in the organelles of various animals and plants, but most is found in the liver of sharks living in the deep sea. The squalene is used in the base oils for cosmetics and health foods mentioned above, and because of its high price and limited supply, it can be used in industrial applications such as other resin plasticizers, magnetic tapes, and precision mechanical lubricating oils. Usage has not been achieved. Therefore, in the future, there is a demand for a method of producing natural squalene that can stably supply without requiring a method of capturing organisms from the natural world where supply is unstable at such a high price.

[0005] Thus, some production of natural squalene by microorganisms has been devised. For example, a method using a filamentous fungus of the genus Mortierella (JP-B-59-203)
No. 56). However, this method has a drawback that productivity is low in terms of culturing filamentous fungi. In addition, a method using bacteria (Japanese Patent Application Laid-Open No. 61-212290) has been proposed, but this method also has a maximum content of 1.4 bacteria per cell.
As low as 8 mg / g cells, it has not yet been used industrially.

[0006] In addition to squalene, in general, the production of intermediate substances in the essential metabolic pathway of microorganisms is extremely low only by using wild-type microorganisms, and the productivity is increased by artificial mutation. There are methods such as producing a mutant strain.

[0007]

However, the use of wild-type microorganisms has low productivity, and in order to increase these productivity, as described above, the use of a physical or chemical artificial mutagen source is required. A mutant was being produced.
Although this technique can easily and sometimes rarely yield better microorganisms than expected, on the other hand, in many cases it is not possible to obtain at all. Such a method generally has a very low probability of obtaining a target microorganism.

[0008] Furthermore, the microorganisms produced by the mutation cannot stably retain the obtained traits on an industrial production scale, and may cause reversion and maintain the productivity. Above all, even if a method for producing a mutant strain by artificial mutation is used, it is difficult to stably obtain a desired squalene-producing microorganism. Furthermore, it is possible to estimate how much the microorganism that has been mutated can increase its production capacity,
It is almost impossible to convert the mutations that give rise to other cognate, potential wild-type microorganisms. It is not suitable for carrying out a breeding method based on the method.

[0009]

SUMMARY OF THE INVENTION In order to make up for many of these drawbacks, the present invention has examined and studied metabolic pathways of squalene in eukaryotes, particularly in baker's yeast, and has found that the present invention is based on the use of genetic engineering. It was found that the objectives of the present invention could be achieved, and the present invention was finally completed. Hereinafter, the present invention will be described in detail.

For example, squalene in yeast is one of the intermediates in the ergosterol biosynthesis system, one of its essential metabolic pathways. Squalene is acetyl C
It is converted to squalene via mevalonic acid starting from oA, and then converted to squalene 2,3-epoxide by an enzyme called squalene epoxidase.
Then, the cyclization of this compound finally leads to ergosterol. If a metabolic intermediate is to be produced, various methods may be used to block the biochemical reactions of the metabolic pathway after the intermediate.

In the present invention, squalene is used as a method for stably and surely bringing a metabolic pathway to a blocking direction.
Incorporating genetically safe foreign DNA into metabolic genes on the chromosome of the microorganism that is trying to produce squalene, thereby disrupting, altering and / or removing the gene for the squalene consuming squalene epoxidase enzyme And lower the metabolic mechanism of squalene consumption. That is, the objective can be achieved by performing homologous recombination gene manipulation.

When the gene of interest is unknown when performing the present genetic manipulation, the chromosomal DNA of the microorganism to be subjected to is oversized (for example, 1 Kbp or less) so as not to function as one gene. Cleavage is performed using a sonic generator or a nuclease. After the modified small fragment of the DNA is modified so that its end matches the insertion site of the vector, it is randomly inserted into the insertion site of the chromosome integration vector (for example, pIA02) and ligated. This consolidated body was introduced into E. coli, library (hereinafter referred to as a gene bank) of DNA fragments through the E. coli to create made, by introducing a microorganism to try to production to recover the vector,
Transformation may be performed and a desired transformed microorganism may be selected.

The squalene accumulation of the target transformed microorganism is confirmed by the difference in growth between cultivation in the presence and absence of sterol, the incorporation of sterol into the cells during growth, and the analysis of the squalene produced. Can be sorted out.

Further, in order to confirm whether predetermined DNA has been introduced into the obtained microorganism, the DNA may be detected by Southern hybridization. Further, the DNA in which the homologous recombination has occurred is based on the indicator gene (replication region and ampicillin resistance gene) specific to the introduced vector.
After being cut with an appropriate restriction enzyme and ligated, it is introduced into E. coli and cultured. The gene can be easily identified by isolating the plasmid from the ampicillin-resistant colony and determining the nucleotide sequence according to a restriction enzyme map or a conventional method.

By performing the above-mentioned operations, the desired microorganism having a high squalene-producing ability can be freely prepared by genetic manipulation. The present invention will be described in more detail below.

According to the present invention, Saccharomyces cerevisiae strains E13 and E16, for example, have been newly prepared as novel microorganisms having high squalene-producing ability obtained by genetic engineering, which is one of the objects of the present invention. Well-known yeast Saccharomyces cerevisiae SHY3 (ATCC 44971) [Gene, 8, 1
7 to 24, (1979)] as parent strains, each of which is a novel mutant prepared by the method described below.
No. 2600 (FERM P-12600) and No. 12601 (FERM P-12601).

(1) [Insertion of the gene bank consisting of plasmid pIA02 and SHY3 chromosome fragment into the yeast Saccharomyces cerevisiae SHY3 (hereinafter referred to as SHY3) chromosome] Plasmid pIA02 prepared by the method described below
And a gene bank consisting of the SHY3 chromosome fragment is prepared and inserted into the SHY3 chromosome.

The gene bank used herein is a self-ligating fragment of about 6.7 kilobase pairs (Kbp) of the two fragments obtained by cutting the well-known plasmid YEp13 with Sa1I to obtain a chromosomal integration vector. pIA02 (to be described later) was newly constructed, and a fragment obtained by randomly cutting a small fragment of 1 kbp or less of the chromosomal DNA of yeast was inserted into the PvuII cleavage site. These are prepared by the following steps.

A. [Gene bank consisting of plasmid pIA02 and SHY3 chromosome fragment]

The shuttle vector pIA02 for chromosome integration in yeast is prepared as follows. First, a well-known shuttle vector YEp13 (Gene, 8, 121
(1979)) and cut with SalI to give two fragments of about 4 Kbp and about 6.7 Kbp. The approximately 4 Kbp fragment contains a 2 μm ORI (2 μm DNA) replication region of the shuttle vector YEp13 in yeast. on the other hand,
The larger fragment of about 6.7 Kbp contains the LEU2 gene as a marker for transformation into yeast and the ampicillin resistance gene A as a marker for transformation into E. coli.
mpR and the replication region ori in Escherichia coli. In addition, this fragment has PvuII having a unique restriction site that does not cleave each of the above essential sequences, and can be used as a cloning site. Plasmid pIA02 was prepared by fractionating the larger fragment of about 6.7 Kbp by agarose gel electrophoresis, recovering it from the gel, and self-ligating. Therefore, a plasmid obtained by self-ligating the larger fragment is extremely effective as a chromosome-integrated shuttle vector in a target yeast. The construction of brasmid pIA02 and its restriction map are shown in FIG.

(B) Preparation of minute fragments of yeast chromosomal DNA Yeast chromosomal DNA was prepared by a known method [Methods in
Cell Biology, pp. 39-44, Acad.
emic Press, N.M. Y (1975)], and cut into small pieces using a nuclease or an ultrasonic crusher. After confirming by agarose gel electrophoresis that the DNA fragment was cut to a small size of 1 Kbp or less, the terminal was treated with T4 DNA polymerase or Mung.
Smoothing is performed using Bean Nuclease or the like.

(C) Preparation of a Gene Bank Consisting of Plasmid pIA02 and SHY3 Chromosome Fragment Plasmid pIA02 is cut with PvuII, and the cut end is phosphorylated with alkaline phosphatase to prevent self-ligation.

A mixture of minute fragments of yeast chromosomal DNA whose ends have been blunted is mixed with the above-mentioned pIA02 and randomly ligated to obtain a ligated mixed DNA.

B. [Introduction of Gene Bank into SHY3] The ligated mixed DNA obtained as described above was once introduced into Escherichia coli and spread on a nutrient agar medium containing ampicillin.
00. These colonies are cultured about every 400 colonies, the amount is increased, the plasmid is recovered, and a gene bank is obtained.

In the above operation, since introduction of DNA into yeast requires several μg or more, the amount was increased. This is introduced into SHY3 to obtain a homologous recombinant transformant in which the DNA of the gene bank has been integrated into the chromosome.

The gene bank is introduced into SHY3 by a method known per se (for example, the method described in JP-A-59-28478). For example, SH
After culturing Y3 using a YPD medium, the cells are collected and suspended in a buffer. After adding a metal ion such as lithium, rubidium or cesium thereto, the above-mentioned ligated mixed DNA and aqueous solution of polyethylene glycol are added, and then heat-treated at about 40 ° C. for a short time and washed with sterilized water or the like. (This method is particularly preferable as a method for introducing DNA into yeast because it does not require protoplast treatment of the cells.)

The cells thus treated are suspended in sterilized water or the like, ergosterol is added thereto, and the cells are spread on a minimal agar medium containing no leucine and cultured. (The transformed cells complement leucine biosynthesis deficiency of the parent strain by the action of one of the leucine synthase genes (LEU2) on the introduced plasmid, and biosynthesize leucine even in a medium without leucine. Cells that grew and were not transformed cannot grow on a medium without leucine because they cannot synthesize leucine.)

(2) [Isolation of ergosterol-requiring strain that accumulates squalene]
The ergosterol-requiring strains are isolated by transferring each to a YPD agar medium to which ergosterol is added and a YPD agar medium to which ergosterol is not added, observing the requirement for ergosterol.

The isolated ergosterol-requiring strain is cultured in a nutrient medium supplemented with sterol, and squalene is identified and the accumulated content is analyzed. Squalene can be identified by mass spectrometry or nuclear magnetic resonance spectrum, and squalene can be quantified by gas chromatography, thin-layer chromatography, or the like.

The strains in which squalene accumulation was observed were further analyzed based on the presence or absence of sterol uptake, the ability to recover growth in the presence of sterols, and the analysis by Southern hybridization using DNA recovered from the proliferating cells. Transformants are distinguished.

[0031]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples unless it exceeds the gist thereof.

However, the operations in the following examples were performed by the following methods I to X, unless otherwise specified.

I [Microfragmentation of Yeast Chromosome] Yeast chromosome DNA was prepared by the above-mentioned well-known method.
The prepared DNA solution is reduced in size to 1 by using a deoxyribonuclease (hereinafter referred to as DNase I), an ultrasonic generator or a restriction enzyme that recognizes four bases.
Prepare a fragment of Kbp or less. The size of the DNA fragment is
Confirm by agarose gel electrophoresis.

II [Smoothing treatment of DNA fragment] The DNA fragment prepared as described above was subjected to DNA Blu
DNting Kit (manufactured by Takara Shuzo Co., Ltd.) and used in accordance with the method described in the catalog, and having a blunted end.
Create the A fragment.

III [Cleavage and Recovery of DNA by Restriction Enzyme] The buffer for cleavage by restriction enzyme is Universal
DNA is cut and recovered using Buffer Set (manufactured by Takara Shuzo) according to the method described in the catalog.

IV [Treatment of Plasmid DNA with Alkaline Phosphatase (BAP Treatment)] In order to prevent self-ligation of the plasmid by the ligase reaction, a fragment of the plasmid DNA digested with the restriction enzyme is treated with BAP prior to the ligase reaction.

About 0.1 μg of the plasmid DNA cleaved with the restriction enzyme was added to a 1 M Tris-HCl buffer (pH 8.0).
With 0.1 Unit of BAP at 55 ° C. for 30 minutes. Thereafter, phenol treatment is repeated three times to inactivate BAP, and the plasmid is purified by an ethanol precipitation method.

V [ligation with T4 DNA ligase] The plasmid DNA and the chromosomal DNA fragment to be ligated
The ligation is performed using NA Ligation Kit (Takara Shuzo) according to the method described in the catalog.

VI [Transformation of Escherichia coli] The DNA solution after completion of the ligation reaction with ligase is col
Using i (Escherichia coli) HB101 competent cell (manufactured by Takara Shuzo Co., Ltd.), transformation is performed according to the method described in the catalog.

VII [Separation and Purification of Plasmid] E. coli HB101 transformant colonies of about 4
Every 100 colonies were scraped and 100 ml of L. Bro
th (1% bactotripton, 0.5% yeast extract, 1
% Sodium chloride, pH 7.2) with ampicillin 1
The cells were cultured at 37 ° C at 00 µg / ml for 2 to 6 hours. After collecting the cultured cells, a well-known lysozyme, alkali, SDS
The plasmid is separated and purified according to the treatment method.

VIII [Yeast Transformation Method] The yeast cells were aerobically aged at 22 ° C. in a YPD medium (1% yeast extract, 2% peptone, 2% glucose, pH 5.5).
When the absorbance at 550 nm of the cell culture reaches a range of 0.8 to 1.0, the cells are aseptically collected.

[0042] The collected cells were added to 10 ml of TE buffer solution (1
0 mM Tris-HCl, 1 mM EDTA-2Na, pH
After washing in 7.5), 1 ml of 0.1 M lithium acetate TE buffer is added, and the mixture is gently shaken at 22 ° C. for 1 hour. 0.1 ml and 10 μg of this shaken solution
The above gene bank DNAs are mixed and left at 22 ° C. for 30 minutes. Then 40% polyethylene glycol 40
After adding 0.7 ml of 00TE buffer and leaving still for 1 hour, a heat treatment is performed at 45 ° C. for 10 minutes.

The cells are seeded on the transformant selection medium,
And transformants are selected from the growth of the colonies for 3 to 5 days.

IX [Measurement of Cell Proliferation] 100 ml of YPD medium (hereinafter referred to as YPDE) supplemented with ergosterol to a final concentration of 4 μg / ml
Yeast cells pre-cultured in the same amount of YPD medium and YPD
Inoculate 10 μl of each into E medium, and aerobically culture at 30 ° C. with shaking.

Aliquots of 1 ml are taken every 4 hours, diluted with sterile water, and the absorbance (OD) at 550 nm is measured.
However, the measurement wavelength of the absorbance need not be limited to 550 nm.

X [Measurement of Squalene] YPD medium or YPD medium 10 containing sterols
The cells cultured in 0 ml are collected, and the collected cells are suspended by adding a chloroform-methanol solution, followed by adding glass beads and homogenizing. Next, the solvent and the cells are centrifuged, and the extraction is repeated with a chloroform-methanol solution. The whole extract is evaporated under a stream of nitrogen, chloroform is added to the residue, and squalene is measured by gas chromatography or thin-layer chromatography.

[0047]

Embodiment 1

(1) Preparation of homologous recombination yeast Saccharomyces cerevisiae SHY3 (ATCC44
771) (hereinafter referred to as SHY3), and the chromosomal DNA was separated and randomly decomposed by DNaseI to a size smaller than 1 Kbp. These small fragments of DNA were blunted using T4 DNA polymerase so that the ends matched the insertion site of the vector, and the chromosomal integration vector p
Randomly inserted into the PvuII site of IA02, co
li (Escherichia coli) HB101 to obtain about 25,000 transformants. These were cultured and the plasmid was separated to prepare a gene bank.

These gene bank DNAs were transformed into SHY3, and a minimal agar medium without leucine (Bacto) was used.
yeast nitrogen base with
out amino acid, glucose, histidine, adenine, agar, pH 5.5), and the transformant was transformed using the expression of the indicator gene on the vector.
That is, a strain in which homologous recombination occurred in the chromosome was obtained. About 15,000 strains of this transformant were screened for sterols and analyzed for squalene in parallel. As a result, two ergosterol-requiring squalene-producing strains were isolated.

Thus, based on the evaluation described below, the Saccharomyces cerevisiae strain E13 (hereinafter referred to as E1 strain) was used.
3 strains) and Saccharomyces cerevisiae E16
(Hereinafter referred to as E16 strains)
-12600 and FERMP-12601.

(2) Confirmation of requirement for sterols In order to further clarify the observation of the requirement for sterols of the cells grown on the transformant selection medium (minimal agar medium without leucine), a YPD medium was used. Aerobic liquid culture was performed on YPDE and YPDE (YPD medium containing ergosterol) medium, and the requirement of sterols was examined from the viability thereof, that is, the cell concentration. (Fig. 2)

The cell concentration is determined by measuring the absorbance of the culture solution.
The absorbance was measured at a wavelength of 550 nm, and the culture was appropriately diluted and measured.

The E13 strain and the E16 strain were found to recover more viable in the presence of ergosterol than in the absence of ergosterol, thus confirming the requirement for sterols.

(3) Properties of Uptake of Sterols The E13 and E16 strains recovered viability by the addition of ergosterol, even though they did not reach SHY3. However, ergosterol was taken up into the cells due to a sterol-deficient mutation. In order to confirm whether the strains had recovered, both mutant strains and the SHY3 strain were cultured in a YPD medium supplemented with cholesterol that does not biosynthesize by yeast (hereinafter referred to as YPDC medium), and squalene and sterols were analyzed. . (Fig. 3)

Cholesterol and squalene were not detected in SHY3 cells, but cholesterol and ergosterol were detected in strains E13 and E16, and uptake of sterols into cells and accumulation of squalene were confirmed.

The E13 strain and the E16 strain are not completely ergosterol-deficient biosynthesis strains and have almost the same accumulation of squalene, but are characterized by different growth rates. Then, squalene is accumulated under aerobic conditions at 30 ° C. in a YPD medium, which is a usual nutrient medium, but accumulation of squalene is not detected in the SHY3 strain under the same culture conditions.

In general, it is known that yeast accumulates squalene in cells in semi-anaerobic culture, but does not accumulate squalene in aerobic culture.

(4) Detection of Incorporation of DNA into Chromosome by Southern Hybridization Method The chromosomal DNA of SHY3, E13 strain and E16 strain was digested with restriction enzymes XbaI, EcoRI and BglII and a well-known Southern hybridization method [Journ
al of Molecular Biology, 9
8, pp. 503 to 517 (1975)], a well-known plasmid DNA pBR322 occupying about 3.7 Kbp of the chromosomal integration vector pIA02 [Gen
e, vol. 2, p. 95 (1977)], and digested with AluI to obtain a random primer Label.
ing Kit, and (Takara Shuzo Co., Ltd.) [α- ³² P] dCT
P was used to perform radiolabelling, and hybridization was performed using it as a probe. (FIG. 4)

In SHY3, no hybridized band of the probe was observed at all, but in the E13 strain and the E16 strain, the hybridized band was clearly observed.
Therefore, it was confirmed that homologous recombination occurred in the chromosomes of the E13 strain and the E16 strain, and the plasmid DAN of the gene bank was integrated.

[0060]

Example 2 For a mutant yeast strain E13 (FERM P-12600) that produces squalene, a medium obtained by adding ergosterol or cholesterol to a YPD medium (1% yeast extract, 2% peptone, 2% glucose) as a normal nutrient medium And squalene was produced. (Fig. 5)

The addition of ergosterol showed better accumulation of squalene than that of addition of cholesterol. The amount of squalene produced per gram of dry cell weight was about 5 milligrams. E16 strain (FER
(MP-12601) also exhibited excellent squalene productivity.

[0062]

As described above, the Saccharomyces cerevisiae E13 strain and the Saccharomyces cerevisiae E16 strain of the present invention are known Saccharomyces cerevisiae strains that do not accumulate squalene.
It is a novel transformant produced by genetic manipulation of S. cerevisiae SHY3 strain, and can accumulate squalene in aerobic culture on a normal nutrient medium. Furthermore, the use of the present yeast or a gene that has undergone homologous recombination isolated from the present yeast enables the breeding of another yeast, and the production amount and price associated with the current capture and production from natural organisms Can be compensated for, which is extremely useful when performing commercial fermentation production of squalene.

Further, according to the present invention, by using a chromosome integration type vector, for example, pIA02, even if the target gene is unknown, the target plasmid can be efficiently produced. The invention is extremely useful from an industrial or commercial point of view.

[Brief description of the drawings]

FIG. 1 shows the construction of the vector pIA02 and its construction.

FIG. 2 shows the difference in cell growth in aerobic culture on YPD medium in the presence and absence of ergosterol.

FIG. 3 shows a gas chromatograph of a simple lipid content in a cholesterol-supplemented YPD medium cultured cells of strains SHY3, E13 and E16.

FIG. 5 shows the amount of squalene accumulated in the aerobic culture of YPD medium in the presence of ergosterol (A) and cholesterol (B).

[Explanation of symbols]

 A squalene B cholesterol C ergosterol

FIG. 4: SHY3 strain, E13 using pBR322 DNA in vector pIA02 as probe
2 shows Southern hybridization analysis of the strain E16.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ identification code FI C12R 1: 865) (58) Investigated field (Int.Cl. ⁶ , DB name) C12N 1/16-1/19 C12N 15 / 00-15/90 BIOSIS (DIALOG) WPI (DIALOG)

Claims (6)

(57) [Claims] 1. A host yeast having a host-vector system and a yeast-Escherichia coli shuttle vector are subjected to genetic manipulation to cause homologous recombination to cause a mutation in the sterol metabolic pathway, thereby requiring sterols. A method for producing yeast that produces squalene. 2. The method according to claim 1, wherein the mutation in the sterol metabolism pathway is disruption, alteration and / or removal of a squalene epoxidase enzyme gene. 3. The host-vector yeast according to claim 1, wherein the yeast is Saccharomyces cerevisiae SHY3 (ATC).
C 44971), the yeast-E. Coli shuttle vector is pIA02, and the shuttle vector pIA02 is obtained by replacing the shuttle vector YEp13 with Sa1I.
, Self-ligating the larger fragment, comprising a replication region in Escherichia coli, an ampicillin resistance gene and a yeast LEU2 gene, and having a PvuII cleavage site. A method for producing yeast producing squalene having ergosterol requirement. 4. A chromosome-integrated shuttle vector having a restriction enzyme map shown in the following figure, pIA02. 5. A yeast for producing squalene having a requirement for sterols, produced by the production method according to any one of claims 1 to 3. 6. The yeast according to claim 5, wherein the yeast is Saccharomyces cerevisiae strain E13 (FERM P-1260).
0) or Saccharomyces cerevisiae strain E16 (FE
RM P-12601), a yeast that produces squalene having ergosterol requirement.