JP2012024084A - New yeast, and method of producing δ5,7-sterol and hydrocortisone using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of efficiently producing Δ5,7-sterol of cholesta-5,7,24-trien-3β-ol or the like and hydrocortisone.SOLUTION: The Δ5,7-sterol and the hydrocortisone are produced using a yeast modified so that the expression of an acyl-CoA: sterolacyltransferase gene and a steryl ester hydrolase gene may be reinforced.

Description

  The present invention relates to a yeast for producing Δ5,7-sterol and hydrocortisone useful as a pharmaceutical intermediate and the like, and a method for producing Δ5,7-sterol and hydrocortisone using the same.

  Sterols are substances essential for life activities contained in animals, plants and fungi, and are contained in the cell membrane in the form of free sterols, and some are stored as ester forms with fatty acids. Among them, C5,7-diene sterol is a sterol having carbon-carbon double bonds at the 5-position and 7-position of the cholesterol skeleton, and among them, cholesta-5,7,24-trien-3β-ol (also known as 7-dehydrodesmosterol (hereinafter referred to as TEOL) is a very useful substance as an intermediate of pharmaceuticals, such as used as a raw material for synthesizing chenodeoxycholic acid and ursodeoxycholic acid, which are pharmaceuticals.

  In addition, 11β, 17α, 21-trihydroxy-4-pregnene-3,20-dione (hydrocortisone), together with its precursors pregnenolone, progesterone, and 7-dehydropregnenolone, is industrially useful as a pharmaceutical and pharmaceutical intermediate. Compound. As an existing production method of pregnenolone, a method of synthesizing by a plurality of organic synthesis reactions using a naturally occurring sterol compound such as cholesterol, diosgenin, and stigmasterol as a raw material has been reported (Non-patent Document 1).

  Conventionally, C5,7-diene sterols purified from animal tissue extracts have been used. On the other hand, as the biosynthesis pathway of ergosterol is clarified in fungi such as yeast (Non-patent Document 2), in recent years, studies have been conducted to accumulate specific sterols by modifying the biosynthesis pathway. Yes.

  In fungi such as yeast and mold, several studies have been reported in which the sterol composition is changed by overexpression of ergosterol biosynthetic enzyme deficient strain or ergosterol biosynthetic enzyme gene. For example, in the yeast Saccharomyces cerevisiae, the sterol C-24 methyltransferase-encoding gene (ERG6) is deleted so that only sterols having no methyl group at the side chain position 24 accumulate (Non-patent Document 3). ), Or a gene encoding sterol C-22 desaturase (ERG5) in yeast and modified by metabolic engineering so that Arabidopsis Δ7-sterol reductase is highly expressed. Ergosta-5,24-Dienol, Ergosta-5 -An example in which enenol and ergosta-5,22-dienol accumulate is reported (Non-patent Document 4).

  In Patent Document 1, the activities of both sterol C-22 desaturase and sterol C-24 methyltransferase are reduced, and sterol Δ7-reductase and / or sterol Δ5-desaturase are highly expressed. A method of culturing fungi (yeast, mold) modified by metabolic engineering and collecting sterols from the culture is disclosed.

On the other hand, acyl CoA: sterol acyltransferase is an enzyme that catalyzes esterification of sterol compounds, and steryl ester hydrolase is an enzyme that catalyzes hydrolysis of steryl esters. A yeast with enhanced expression of ARE1, which is a protein having acyl CoA: sterol acyltransferase activity, is disclosed in Non-Patent Document 5, and a fusion protein in which protein A is added to the C-terminus of YEH1 having steryl ester hydrolase activity. Non-patent document 6 discloses a yeast into which is introduced. However, these documents do not describe the effects of enhanced expression of ARE1 and YEH1 on Δ5,7-sterol production such as C5,7-diene sterol and ergosterol, and both ARE1 and YEH1. No yeast with enhanced expression has been known so far.

JP 2004-141125 A

J. Org. Chem. 44, 1582-1584 (1979) Appl. Microbiol. Biotechnol. 63, 635-646 (2004) Biochem. Biophys. Res. Commun. 181, 509-517 (1988) J. Biol. Chem. 271 (18), 10866-10873 (1996) Science 272, 1353-1356 (1996) Biochim. Biophys. Acta 1791, 118-124 (2009)

  The present invention provides a modified yeast that efficiently produces Δ5,7-sterol and hydrocortisone such as C5,7-diene sterol and ergosterol, and C5,7-diene sterol and ergo using the same. It is an object of the present invention to provide a method for efficiently producing Δ5,7-sterol such as sterol and hydrocortisone.

The present inventors have intensively studied to solve the above problems.
As described above, most of the sterol compounds are accumulated in the cells as ester bodies. Therefore, it is generally considered that it is important to promote esterification of a sterol compound in order to efficiently accumulate a desired sterol compound. For those skilled in the art, in order to efficiently produce Δ5,7-sterol. Acyl CoA that esterifies sterols: It is thought that sterol ester hydrolase that enhances only the sterol acyltransferase activity and conversely promotes hydrolysis of the ester is reduced or not modified. However, when the present inventors tried to enhance not only the acyl CoA: sterol acyltransferase gene but also the expression of steryl ester hydrolase gene at the same time, surprisingly, the expression of both of these genes was enhanced. It has been found that Δ5,7-sterol can be produced very efficiently. Furthermore, the inventors have found that hydrocortisone can also be produced by modifying the yeast, and the present invention has been completed.

That is, the present invention is as follows.
[1] Acyl CoA: yeast modified to enhance the expression of sterol acyltransferase gene and steryl ester hydrolase gene.
[2] The yeast according to [1], further modified so that expression of the sterol C-22 desaturase gene (ERG5) and the sterol C-24 methyltransferase gene (ERG6) is reduced.
[3] The yeast according to [1], further modified so that expression of a sterol C-22 desaturase gene (ERG5) is reduced.
[4] Further, Δ7-reducing activity, activity of cleaving the bond between positions 20 and 22 of the sterol side chain, sterol 3-position oxidative isomerization activity, steroid 17α hydroxylation activity, steroid 21-position hydroxylation activity, and steroid The yeast according to [3], which has been modified to have at least one activity selected from 11β-position hydroxylation activity.
[5] Further, Δ7-reducing activity, activity of cleaving the bond between positions 20 and 22 of the sterol side chain, sterol 3-position oxidative isomerization activity, steroid 17α hydroxylation activity, steroid 21-position hydroxylation activity, and steroid The yeast according to [3], which has been modified to have 11β-position hydroxylation activity.
[6] The yeast according to any one of [1] to [5], which is Saccharomyces cerevisiae.
[7] The yeast according to [6], wherein the parent strain is Saccharomyces cerevisiae YPH499 strain, Saccharomyces cerevisiae FY1679-06c strain, Saccharomyces cerevisiae KA311A strain or Saccharomyces cerevisiae X2181-1B strain.
[8] A method for producing Δ5,7-sterol, wherein the yeast according to any one of [1] to [3] is cultured to produce and accumulate Δ5,7-sterol and to recover Δ5,7-sterol.
[9] The method according to [8], wherein the yeast to be cultured is the yeast according to [1] or [2], and Δ5,7-sterol is a C5,7-diene sterol.
[10] The method according to [9], wherein the C5,7-diene sterol is cholesta-5,7,24-trien-3β-ol or 7-dehydrocholesterol.
[11] The method according to [8], wherein the yeast to be cultured is the yeast according to [1] or [3], and Δ5,7-sterol is ergosta-5,7,24-trienol.
[12] The method according to [8], wherein the yeast to be cultured is the yeast according to [1], and Δ5,7-sterol is ergosterol.
[13] A method for producing hydrocortisone, comprising culturing the yeast according to [5] to produce and accumulate hydrocortisone, and recovering hydrocortisone.

  By using the yeast of the present invention, Δ5,7-sterol and hydrocortisone can be produced. If Δ5,7-sterol and hydrocortisone are produced by the method of the present invention, and a drug is synthesized using these, it is considered that the production process of the drug can be improved.

(A) It is a figure which shows the analysis result of sterols by gas chromatography. “C7,24” represents cholesta-7,24-dien-3β-ol, “ZYMO” represents zymosterol, and “others” represents other sterols. (B), (c) It is a figure which shows the result of having analyzed the transcription | transfer amount of ARE1 and YEH1 by quantitative PCR. The transferred amount is shown as a ratio to ACT1. (B) shows the expression level ratio between ARE1 and ACT1, and (c) shows the expression level ratio between YEH1 and ACT1. In the figure, Δe5Δe6 is an erg5, erg6 double disruption strain (SX1313) in which neither ARE1 nor YEH1 is enhanced, Δe5Δe6ARE1 is an erg5, erg6 double disruption strain (SX1316), Δe5Δe6ARE1YEH1 in which only ARE1 is introduced, And erg5 and erg6 disruption strains (SX1317) into which YEH1 has been introduced. All are strains produced using YPH499 as a parent strain. (A) It is a figure which shows the analysis result of sterols by gas chromatography. (B), (c) It is a figure which shows the result of having analyzed the transcription | transfer amount of ARE1 and YEH1 by quantitative PCR. The transferred amount is shown as a ratio to ACT1. (B) shows the expression level ratio between ARE1 and ACT1, and (c) shows the expression level ratio between YEH1 and ACT1. In the figure, Δe5Δe6 is an erg5, erg6 double disruption strain (SX1312) in which neither ARE1 nor YEH1 is enhanced, Δe5Δe6ARE1 is an erg5, erg6 double disruption strain (SX1319), or Δe5Δe6ARE1YEH1 in which only ARE1 is introduced. And erg5 and erg6 disruption strains (SX1320) introduced with YEH1. Three clones were evaluated for SX1319 and SX1320. All are strains produced using FY1679-06c as a parent strain. (A) It is a figure which shows the analysis result of sterols by gas chromatography. (B), (c) It is a figure which shows the result of having analyzed the transcription | transfer amount of ARE1 and YEH1 by quantitative PCR. The transferred amount is shown as a ratio to ACT1. (B) shows the expression level ratio between ARE1 and ACT1, and (c) shows the expression level ratio between YEH1 and ACT1. In the figure, Δe5Δe6 is an erg5, erg6 double disruption strain (SX1307) in which neither ARE1 nor YEH1 is enhanced, Δe5Δe6ARE1 is an erg5, erg6 double disruption strain (SX1321), Δe5Δe6ARE1YEH1 in which only ARE1 is introduced, And erg5 and erg6 disruption strains (SX1322) into which YEH1 has been introduced. All are strains produced using KA311A as a parent strain. (A) It is a figure which shows the analysis result of sterols by gas chromatography. (B), (c) It is a figure which shows the result of having analyzed the transcription | transfer amount of ARE1 and YEH1 by quantitative PCR. The transferred amount is shown as a ratio to ACT1. (B) shows the expression level ratio between ARE1 and ACT1, and (c) shows the expression level ratio between YEH1 and ACT1. In the figure, Δe5Δe6 is erg5, erg6 double disruption strain (SX1353) that does not enhance both ARE1 and YEH1, ∆e5Δe6ARE1 is erg5, erg6 double disruption strain (SX1350), and Δe5Δe6ARE1YEH1 is ARE1. And erg5 and erg6 disruption strains (SX1351) introduced with YEH1. All are strains produced using X2181-1B as a parent strain. (A) It is a figure which shows the analysis result of sterols by gas chromatography. (B), (c) It is a figure which shows the result of having analyzed the transcription | transfer amount of ARE1 and YEH1 by quantitative PCR. The transferred amount is shown as a ratio to ACT1. (B) shows the expression level ratio between ARE1 and ACT1, and (c) shows the expression level ratio between YEH1 and ACT1. In the figure,-is an erg5 and erg6 double disruption strain (SX1319) into which only ARE1 has been introduced, and the other is an erg5 and erg6 double disruption strain into which ARE1 and YEH1 have been introduced. YEH1 locus is a combination of YEH1 and YEH1. A strain introduced into locus (SX1320), URA3 locus represents a strain introduced with YEH1 into locus of ura3 (SX1332), and LEU2 locus represents a strain introduced with YEH1 into locus of leu2 (SX1325). All are strains produced using FY1679-06c as a parent strain. (A) It is a figure which shows the analysis result of sterols by gas chromatography. (B), (c) It is a figure which shows the result of having analyzed the transcription | transfer amount of ARE1 and YEH1 by quantitative PCR. The transferred amount is shown as a ratio to ACT1. (B) shows the expression level ratio between ARE1 and ACT1, and (c) shows the expression level ratio between YEH1 and ACT1. In the figure,-is an erg5, erg6 double disruption strain (SX1335) into which neither ARE1 and YEH1 have been introduced, the other is an erg5, erg6 double disruption strain into which ARE1 and YEH1 have been introduced, and ARE1 locus is , ARE1 is introduced into the ARE1 locus (SX1320), and URA3 locus is the ARE1 introduced into the ura3 locus (SX1333). All are strains produced using FY1679-06c as a parent strain. (A) It is a figure which shows the analysis result of sterols by gas chromatography. (B), (c) It is a figure which shows the result of having analyzed the transcription | transfer amount of ARE2 and YEH1 by quantitative PCR. The transferred amount is shown as a ratio to ACT1. (B) shows the expression level ratio between ARE2 and ACT1, and (c) shows the expression level ratio between YEH1 and ACT1. In the figure, Δe5Δe6 is an erg5, erg6 double disruption strain (SX1335) in which neither ARE2 nor YEH1 is enhanced, Δe5Δe6ARE2 is an erg5, erg6 double disruption strain (SX1347), Δe5Δe6ARE2YEH1 in which only ARE2 is introduced, And erg5 and erg6 disruption strains (SX1346) introduced with YEH1. All are strains produced using FY1679-06c as a parent strain. (A) It is a figure which shows the analysis result of sterols by gas chromatography. “ERGO” represents ergosterol, “ZYMO” represents zymosterol, and “other” represents other sterols. (B), (c) It is a figure which shows the result of having analyzed the transcription | transfer amount of ARE1 and YEH1 by quantitative PCR. The transferred amount is shown as a ratio to ACT1. (B) shows the expression level ratio between ARE1 and ACT1, and (c) shows the expression level ratio between YEH1 and ACT1. In the figure,-indicates a strain in which neither ARE1 nor YEH1 is enhanced (SX1348), ARE1 indicates a strain in which only ARE1 is introduced (SX1338), and ARE1YEH1 indicates a strain in which ARE1 and YEH1 are introduced (SX1337). All are strains produced using FY1679-06c as a parent strain.

Hereinafter, embodiments of the present invention will be described in detail.

(1) Yeast of the Present Invention The yeast of the present invention is a yeast modified so that expression of acyl CoA: sterol acyltransferase gene and expression of steryl ester hydrolase gene are enhanced.

  In the present invention, Δ5,7-sterol means a sterol having carbon-carbon double bonds at the 5th and 7th positions of the sterol skeleton, preferably ergosterol and C5,7-diene sterol. C5,7-diene sterol means a sterol having carbon-carbon double bonds at the 5-position and 7-position of the cholesterol skeleton, but TEOL (cholesta-5,7,24-trien-3β-ol), 7-dehydrocholesterol is preferred and TEOL is particularly preferred. When hydrocortisone is the target product, ergosta-5,7,24-trienol is preferred. Δ5,7-sterol and C5,7-diene sterol also include esters of Δ5,7-sterol and C5,7-diene sterol, respectively.

  In the present invention, “acyl CoA: sterol acyltransferase gene” means a gene encoding a protein having acyl CoA: sterol acyltransferase activity, and “steryl ester hydrolase gene” means steryl ester hydrolysis. It means a gene encoding a protein having enzyme activity.

The phrase “acyl CoA: sterol acyltransferase gene expression is enhanced” means that the expression of the acyl CoA: sterol acyltransferase gene is enhanced as compared with the unmodified strain or the parent strain. The enhancement of the expression of the acyl CoA: sterol acyltransferase gene is preferably enhanced by 5 times or more, more preferably enhanced by 10 times or more per unit cell weight as compared with the unmodified strain or the parent strain. .
Acyl CoA: sterol acyltransferase is an enzyme that catalyzes an esterification reaction of sterol, and the expression of an acyl CoA: sterol acyltransferase gene can be measured by a quantitative PCR method or the like.

“The expression of the steryl ester hydrolase gene was enhanced” means that the expression of the steryl ester hydrolase gene was enhanced as compared with the unmodified strain or the parent strain. The enhancement of steryl ester hydrolase gene expression is preferably 5 times or more, more preferably 10 times or more per unit cell weight, as compared to the unmodified strain or the parent strain.
The expression of steryl ester hydrolase gene can be measured by a quantitative PCR method or the like.

The yeast used in the present invention can be obtained by using a yeast as shown below as a parent strain and modifying the parent strain.
The type of yeast used as the parent strain is not particularly limited, but Saccharomyces, Devariomyces, Schizosaccharomyces, Candida, Yarrowia, Rhodotorula, Lipomyces, Kluyveromyces, Rhodosporidium, Trichoderma or And yeast belonging to the genus Torlopsis or Pichia, such as Saccharomyces cerevisiae, Saccharomyces bayanus, Debaryomyces nilssonii, mys han , Schizosaccharomyces pombe, Candida glabrata, Candida tropicalis, Candida utilis , Candida boidinii, Yarrowia lipolytica, Rhodotorula glutinis, Lipomyces lipoferus, Kluyveromyces lactis, Rodispoli (Rhodosporidium toruloides), Trichoderma reesei, Torulopsis colliculosa, Pichia farinosa.

Of these, Saccharomyces is preferable because metabolic engineering techniques are easy to use. Furthermore, the genus Saccharomyces is preferably an auxotrophic genotype such as his3, leu2, trp1, ura3, and examples thereof include the following strains.
Saccharomyces cerevisiae YPH499 strain (MATa ura3 lys2 ade2 trp1 his3 leu2) (ATCC204679: available from American Type Culture Collection or STRATAGENE), Saccharomyces cerevisiae FY1679-06c strain (available from MATα ura3 leu2 trp1 his3) (available from Euroscarf) Saccharomyces cerevisiae strain X2181-1B (MATalpha his2 gal1 trp1 ade1
MAL SUC) (ATCC204822: available from American Type Culture Collection). Alternatively, the Saccharomyces cerevisiae KA311A strain (MATa his3 leu2 trp1 ura3) (Mol. Cell Biol. 13, 307-3083 (1993)) can also be used. The deposit number is FERM P-19053.

  Increased expression of acyl CoA: sterol acyltransferase gene and steryl ester hydrolase gene expression may be achieved by genetic recombination methods, for example, increasing the copy number of the gene encoding the enzyme or replacing the promoter of this gene By increasing the expression level of these genes.

  Examples of a gene encoding a protein having acyl CoA: sterol acyltransferase activity include the ARE1 gene and the ARE2 gene. The ARE1 gene and the ARE2 gene are known in many microorganisms and are not particularly limited as long as they encode a protein having an acyl CoA: sterol acyltransferase activity. For example, Saccharomyces cerevisiae having the nucleotide sequence of SEQ ID NO: 43 or 45, respectively. A gene derived from cerevisiae can be mentioned. The ARE1 gene and ARE2 gene hybridize under stringent conditions with DNA having a complementary sequence of the nucleotide sequence of SEQ ID NO: 43 or 45 as long as it encodes a protein having acyl CoA: sterol acyltransferase activity. It may be DNA or a homologue such as DNA having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 99% or more with these base sequences. Here, as stringent conditions, it corresponds to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, which is a normal washing condition for Southern hybridization. The conditions for hybridizing at a salt concentration are included.

  In addition, the ARE1 gene and the ARE2 gene have 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 99% or more identity with the amino acid sequence of SEQ ID NO: 44 or 46, respectively. It may be a DNA encoding a protein having CoA: sterol acyltransferase activity.

Furthermore, the ARE1 gene and the ARE2 gene have a sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 44 or 46, and have acyl CoA: sterol acyltransferase activity. It may be DNA encoding a protein. Here, one or several means preferably means 1 to 20, more preferably 1 to 10, particularly preferably 1 to 5.

  Moreover, ARE1 gene and ARE2 gene derived from yeast other than Saccharomyces cerevisiae, or other microorganisms or animals and plants can also be used. The ARE1 and ARE2 genes derived from microorganisms or animals and plants are genes whose base sequences have already been determined, or genes encoding proteins having acyl CoA: sterol acyltransferase activity based on homology, etc. More isolated and determined nucleotide sequences can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can be used. These can be obtained by amplifying a region containing the promoter and the ORF portion by a hybridization method or a PCR method.

  As a gene encoding a protein having steryl ester hydrolase activity, YEH1 gene can be mentioned. The YEH1 gene is known in many microorganisms and is not particularly limited as long as it encodes a protein having steryl ester hydrolase activity. For example, a gene derived from Saccharomyces cerevisiae having the base sequence of SEQ ID NO: 47 is mentioned. Can do. In addition, as long as the YEH1 gene encodes a protein having steryl ester hydrolase activity, DNA that hybridizes under stringent conditions with DNA having a complementary sequence of the nucleotide sequence of SEQ ID NO: 47, or these bases It may be a homologue such as DNA having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 99% or more with the sequence. Here, the stringent conditions are as described above.

Each YEH1 gene has 80% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more identity with the amino acid sequence of SEQ ID NO: 48, and steryl ester hydrolase activity. It may be a DNA encoding a protein having
Furthermore, the YEH1 gene is a DNA encoding a protein having a sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 48 and having steryl ester hydrolase activity. There may be. Here, one or several means preferably means 1 to 20, more preferably 1 to 10, particularly preferably 1 to 5.

  In addition, YEH1 gene derived from yeast other than Saccharomyces cerevisiae, other microorganisms or animals and plants can also be used. The YEH1 gene derived from a microorganism or animal or plant is a gene whose base sequence has already been determined, or a gene encoding a protein having steryl ester hydrolase activity based on homology or the like, isolated from a chromosome of a microorganism, animal or plant, etc. Those having a determined base sequence can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can be used. These can be obtained by amplifying a region containing the promoter and the ORF portion by a hybridization method or a PCR method.

  In order to enhance the expression of the acyl CoA: sterol acyltransferase gene and the expression of the steryl ester hydrolase gene, for example, the DNA as described above is incorporated into a plasmid capable of functioning in the host yeast so as to be expressed in the host yeast. What is necessary is just to introduce.

The plasmid vector into which the above gene can be incorporated is not particularly limited as long as it contains at least a gene responsible for the replication growth function in the host yeast. Specific examples of plasmids that can be used for introducing genes into yeast include pAUR vectors (manufactured by Takara Bio Inc.) derived from 2 μm plasmids and pESC vectors (manufactured by STRATAGENE). These vectors are commercially available and can be easily obtained.

  In addition, DNA cleavage, ligation, and other methods such as chromosomal DNA preparation, PCR, plasmid DNA preparation, transformation, setting of oligonucleotides used as primers, etc. adopt ordinary methods well known to those skilled in the art. can do. These methods are described in Sambrook, J., Fritsch, EF, and Maniatis, T., “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press, (1989), etc.

  By transforming yeast such as Saccharomyces cerevisiae with a recombinant vector obtained by inserting the ARE1 (or ARE2 gene) and YEH1 gene into the appropriate site of a plasmid vector that can replicate in yeast as described above. Yeast with increased expression of these genes is obtained.

Transformation can be performed, for example, by electroporation (Fromm ME, et al., (1986) Nature 319 (6056): 791-793), spheroplast method, lithium acetate method (Ito, H. et al, (1983 ) J. Bacteriol. 153 (1), 163-168).
Further, the expression of acyl CoA: sterol acyltransferase gene and steryl ester hydrolase gene is enhanced by making multiple copies of ARE1 (or ARE2 gene) and YEH1 gene on the host genome by a known homologous recombination method. It can also be done. For example, a method using a vector in which multiple copies of a genome integration plasmid are integrated into the genome (Bio / Technol. 9, 1382-1385 (1991)) can be mentioned. The place where the expression unit containing each gene on the genome is inserted may be any place as long as the expression of the sterol biosynthesis-related gene or the growth-related gene is not inhibited. Specifically, a DNA in which the ARE1 gene is linked so as to be under the control of an appropriate promoter is inserted into the ARE1 locus on the host genome so that there are two copies of the ARE1 gene in the host genome. Furthermore, the DNA is modified so that the YEH1 gene in the host genome becomes 2 copies by inserting the DNA ligated so that the YEH1 gene is also under the control of the same promoter into the YEH1 locus on the host genome. A method or the like is preferably used.

Further, the expression of acyl CoA: sterol acyltransferase gene and steryl ester hydrolase gene can be enhanced by replacing or modifying the promoters of ARE1 (or ARE2 gene) and YEH1 gene on the host genome.
Replacing the promoter for expressing the ARE1 (or ARE2 gene) and YEH1 gene, or the ARE1 (or ARE2 gene) and YEH1 gene on the chromosome by introduction with the above recombinant plasmid or homologous recombination on the chromosome The promoter used for this purpose is not particularly limited as long as it can function in the host yeast. For example, the promoter of 3-phosphoglycerate kinase 1 gene (hereinafter sometimes referred to as “PGK1 promoter”) or alcohol Examples include constitutive promoters such as dehydrogenase genes, or inducible promoters such as GAL1 and GAL10 (Mol. Cell Biol. 4 (8), 1440-1448 (1984)) (for example, manufactured by STRATAGENE).

It should be noted that the yeast of the present invention is designed to enhance or decrease the expression of other enzyme genes in addition to the enhanced expression of acyl CoA: sterol acyltransferase gene and steryl ester hydrolase gene as compared to the unmodified strain. It may be modified.
The modification of other enzymes is appropriately selected depending on the type of target Δ5,7-sterol.

For example, when producing TEOL, it is preferable to modify so that the expression of the sterol C-22 desaturase gene (ERG5) and the sterol C-24 methyltransferase gene (ERG6) decreases. These modifications are described in JP-A-2004-141125.
In addition, when hydrocortisone is the target product with ergosta-5,7,24-trienol as Δ5,7-sterol as an intermediate, the expression of only the sterol C-22 desaturase gene (ERG5) seems to decrease. It is preferable to modify it.

  Sterol C-22 desaturase is an enzyme having the ability to introduce a double bond at position 22 of the side chain of various sterols. Specifically, for example, cholesta-5,7,24-trienol is converted to cholesta-5. 7,22,24-tetraenol, ergosta-5,7-dienol in ergosta-5,7,22-trienol, ergosta-5,24-dienol in ergosta-5,22,24-trienol, ergosta It refers to an enzyme having the ability to convert 5,7,24-trienol to ergosta-5,7,22,24-tetraenol.

The sterol C-24 methyltransferase is an enzyme having the ability to transfer a methyl group from S-adenosylmethionine to position 24 of various sterols, and further to transfer a methyl group to the methyl group added by the transfer. Specifically, for example, it refers to an enzyme having the ability to convert zymosterol to fecosterol.
Some of the genes encoding these enzymes have already been isolated and nucleotide sequences have been determined. For example, a gene derived from Saccharomyces cerevisiae has been reported as a sterol C-22 desaturase (Biochem. Biophys. Acta). 1299, 313-324 (1996)), a gene derived from Saccharomyces cerevisiae has been reported as a sterol C-24 methyltransferase (Gene 169, 105-109 (1996)).

  Examples of methods for reducing the activity of both sterol C-22 desaturase and sterol C-24 methyltransferase include ethylmethanesulfonic acid (EMS) and N-methyl-N′-nitro-N-nitrosoguanidine (NTG). Method of mating mutant mutants of both enzyme genes obtained by treatment with chemical substances or UV, etc., method of suppressing transcription of both genes by introduction of antisense gene, and suppression of transcripts of both genes by RNAi technology And a method of suppressing enzyme activity with specific inhibitors of both enzymes can be used.

  In yeast, in gene fragments encoding sterol C-22 desaturase and sterol C-24 methyltransferase, auxotrophic gene DNA such as HIS3, LEU2, TRP1, URA3 or geneticin, aureobasidin A, etc. A method in which a gene fragment into which a resistance gene for an antibiotic or antifungal drug is inserted, or a plasmid in which the gene fragment is further inserted into a vector, is prepared and the gene in the host chromosome is disrupted by homologous recombination is preferably used. It is done.

  For example, in Saccharomyces cerevisiae, a DNA fragment containing a gene encoding sterol C-22 desaturase (ERG5) and a DNA fragment containing a gene encoding sterol C-24 methyltransferase (ERG6) are subjected to restriction enzyme cleavage or polymerase chain reaction. An intermediate vector is prepared by obtaining it by the method (hereinafter sometimes abbreviated as “PCR”) and incorporating it into the vector. This intermediate vector is cleaved into two at any restriction enzyme site inside the ERG5 gene and ERG6 gene, and any one of yeast auxotrophic gene cDNAs such as HIS3, LEU2, TRP1, and URA3 is A gene disruption vector or a DNA fragment for gene disruption obtained by PCR amplification is prepared by selecting and inserting the intermediate vectors so as not to overlap each other. Subsequently, the gene for gene disruption or the DNA fragment for gene disruption is introduced into yeast to induce homologous recombination to destroy the target gene.

As a specific example of the gene disruption DNA fragment or gene disruption vector thus constructed, a DNA fragment (erg5 :: TRP1) in which the internal 371 bp of the ERG5 gene has been deleted and the TRP1 gene has been inserted into the deleted portion, or Plasmid pSK-erg5 :: TRP1, ERG6 gene internal plasmid 476 bp having the DNA fragment deleted, and DNA fragment (erg6 :: HIS3) in which the HIS3 gene is inserted into the deleted portion or the DNA fragment pUC-erg6 :: HIS3 (see JP 2004-141125 A) and the like.

Confirmation of gene disruption in yeast is based on the fact that the gene part inserted with a specific auxotrophic gene is integrated into the yeast genome by homologous recombination. It can be known by the loss of demand. Furthermore, PCR was performed using chromosomal DNA extracted from the yeast strain subjected to gene disruption as a template using PCR primers set at the 5 ′ and 3 ′ ends of the ERG5 and ERG6 genes, respectively, and the length of the PCR fragment was inserted. By confirming that the length of the auxotrophic gene is increased, the gene disruption can be confirmed.
Specific examples of the gene-disrupted strain thus obtained include the Saccharomyces cerevisiae KAΔ5-5 strain in which the ERG5 gene is disrupted with the erg5 :: TRP1 fragment of the gene disruption vector pSK-erg5 :: TRP1, and the Δ5-5 strain Examples include KAΔ5Δ6-1 strain, KAΔ5Δ6-4 strain, and KAΔ5Δ6-5 strain in which the ERG6 gene is disrupted with the erg6 :: HIS3 fragment of pUC-erg6 :: HIS3.
Furthermore, as yeast of this invention, the yeast as described in the following (3) is also contained.

(2) Δ5,7-sterol production method The yeast of the present invention described above is adjusted in a medium containing a carbon source, nitrogen source, inorganic salt, amino acid, vitamin, etc., under aerobic conditions, temperature, pH, etc. However, if culturing is carried out, Δ5,7-sterol accumulates in the yeast cells and is collected.

As the carbon source, for example, carbohydrates such as glucose, glycerol, fructose, sucrose, maltose, mannose, starch, starch hydrolyzate, molasses, and various organic acids such as acetic acid, pyruvic acid, and mevalonic acid can be used. Further, depending on the assimilation ability of the yeast, hydrocarbons, alcohols and the like are also used. In particular, molasses is preferably used.
As the nitrogen source, for example, ammonia, various inorganic salts such as ammonium chloride and ammonium carbonate, and organic ammonium salts, or various substances such as peptone, meat extract, yeast extract, and casein hydrolyzate can be used.
As the inorganic salt, for example, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, ammonium chloride, magnesium sulfate, sodium chloride and the like are used. Vitamins and amino acids vary depending on the type of yeast used, but are added as necessary. Moreover, when the strain to be used shows auxotrophy, the required substance is added.

  The culture is performed under appropriate conditions depending on the strain to be used, but is usually performed under aerobic conditions such as shaking culture or aeration and agitation culture. When Saccharomyces cerevisiae is used as the production strain, the culture temperature is preferably 20 to 40 ° C., and the pH of the medium is preferably maintained near weak acidity. The culture period is usually 1 to 15 days, and Δ5,7-sterol accumulates in the cells. In addition, by performing fed-batch culture or continuous culture in which the above-described carbon source, nitrogen source, inorganic salt, vitamin, and amino acid are added continuously or intermittently during the culture period, cell growth and Δ5, It is possible to further extend the accumulation of 7-sterols.

Extraction of Δ5,7-sterol after completion of the culture can be generally performed by a method of extracting sterols from a biological tissue. Specifically, for example, the cells are crushed together with sea sand in acetone and extracted with ethyl acetate, or boiled in 50% ethanol containing 2M NaOH for 2 hours and then saponified, and then heptane or chloroform / methanol. / Water (volume ratio 86: 16: 1) extracted with a mixed solution, subjected to thin layer chromatography, scraped off the sterol fraction, dried under a nitrogen stream, and dissolved in a methanol / chloroform mixed solution to obtain total sterol from the cells. Can be extracted.

  Next, the sterol type is identified by subjecting the sterol fraction separated and extracted by thin layer chromatography to gas chromatography or high performance liquid chromatography using a combination of a suitable solvent and column known per se, and standardizing the retention time of the peak that appears. This can be done by comparing with the retention time of the sample. In addition, the proportion of each identified sterol in the total sterol can be calculated from the area of each peak. Furthermore, confirmation that these major sterol peaks are the target compound can be carried out by examining the molecular weight and cleavage pattern using mass spectrometry. Further, sterols extracted more efficiently can be identified by using GC-MS or HPLC-MS.

The obtained Δ5,7-sterol can be an intermediate for various drugs.
For example, TEOL is useful as a raw material for the synthesis of chenodeoxycholic acid and ursodeoxycholic acid. JP 2007-210888 A and JP 2006-056877 A disclose a method for synthesizing ursodeoxycholic acid from TEOL.
Specifically, using TEOL as a raw material, the following four steps are performed: (I) oxidation of 3-position hydroxyl group and isomerization of 5-position double bond to 4-position (II) side chain oxidative cleavage Step (III) Introducing an oxygen functional group at the 7-position (IV) 5β-3 through a step of reductive saturation of the 4-position double bond Ursodeoxycholic acid can be produced by producing 1,7-dioxocholanic acid and reducing it.
The reduction reaction of 5β-3,7-dioxocholanic acid to ursodeoxycholic acid can be performed by metal reduction or catalytic hydrogenation. Further, the conversion from 5β-3,7-dioxocholanic acid to ursodeoxycholic acid can be carried out in consideration of the descriptions in JP-A-60-228500 and JP-A-5-32692.

(3) Method for producing hydrocortisone Hydrocortisone is produced by culturing the yeast having the following activity in the same manner as in (2) above. The yeast having activity may have its own activity, a substance having the activity may be added to the culture solution, or the yeast cell having the activity or its You may add a processed material in a culture solution.

  Activities necessary for the production of hydrocortisone include Δ7-reduction activity, activity of cleaving the bond between positions 20 and 22 of the sterol side chain, sterol 3-position oxidative isomerization activity, steroid 17α hydroxylation activity, steroid 21 Hydroxylation activity and steroid 11β-position hydroxylation activity. The yeast of the present invention includes those having at least one of the above activities. As the yeast of the present invention for the purpose of producing hydrocortisone, those that produce the above-mentioned protein are preferable, and those in which all of the genes for expression and coenzymes necessary for the activity have been introduced are introduced. Are also preferred. In order to produce a protein having the above-described activity, a method using a transformed yeast obtained by transforming the yeast by inserting a DNA encoding the protein into the expression vector is preferably used.

As a method for obtaining an activity for producing a protein having an activity of cleaving the bond between the 20-position and the 22-position of the sterol side chain, (i) For example, an enzyme protein (CYP204A1 or CYPSS204A) described in WO2010 / 079594 is used. Enzyme genes having the same activity as encoded by genes and other genes, ferredoxin protein having electron transfer activity to the enzyme protein, and ferredoxin reductase protein having electron transfer activity to the ferredoxin protein A mixture of genes encoding, or (ii) a gene encoding the enzyme protein, a ferredoxin protein having electron transfer activity to the enzyme protein, and a ferredoxin reductase protein fusion protein having electron transfer activity to the ferredoxin protein For example, a method of gene introduction into the yeast so that it can be expressed. It is.

  Here, the ferredoxin protein having electron transfer activity and the ferredoxin reductase protein having electron transfer activity to the ferredoxin protein may be one kind of protein having both activities. For example, an artificially produced fusion protein can be used by linking the P450 reductase domain of the P450-BM3 gene of Bacillus megaterium to an enzyme protein (P450 2C11) (Helvig, C. and Capdevila, JH, Biochemistry, (2000) 39, 5196-5205).

Ferredoxin reductase is selected from among the following, for example.
Spinach-derived ferredoxin reductase,
Putidaredoxin reductase from Pseudomonas putida,
Animal-derived adrenodoxin reductase,
Ferredoxin reductase from Novosphingobium subterraneum,
Ferredoxin reductase from Novosphingobium aromaticivorans,
Flavodoxin reductase or ferredoxin reductase from Escherichia coli,
Ferredoxin reductase from Saccharomyces cerevisiae,
Or other ferredoxin reductase.

Ferredoxin is selected, for example, from:
Ferredoxin from spinach,
Putidaredoxin from Pseudomonas putida,
Animal-derived adrenodoxin,
Ferredoxin from Novosphingobium subterraneum,
Ferredoxin from Novosphingobium aromaticivorans,
Flavodoxin and ferredoxin from Escherichia coli,
Ferredoxin from Saccharomyces cerevisiae,
Or other ferredoxin-type protein.

Steroid 17α-position hydroxylase protein, steroid 21-position hydroxylase protein, steroid 11β-position hydroxylase protein, and sterol 3-position oxidase protein are described in, for example, Molecular and Cellular Endocrinology 1990, 73, 73-80. Things are used.
The sterol Δ7-reductase protein is derived from Arabidopsis thaliana described in Journal of Biological Chemistry 1996, 271, 10866-10873, or Mortierella described in Applied and Environmental Microbiology 2007, 73, 1736-1741. ) The one derived from the genus mold is used.
Specifically, the sterol Δ7-reductase protein is preferably an Arabidopsis derived NADPH-sterol Δ7-reductase Mortierella alpina-derived sterol Δ7-reductase (MoDELTA7SR).
The steroid 17α hydroxylase protein is preferably, for example, bovine, mouse, rat, or human-derived cytochrome P450c17.
The steroid 21 hydroxylase protein is preferably bovine, mouse, rat, or human cytochrome P450c21.
As the steroid 11β-position hydroxylase protein, bovine, mouse, rat, or human-derived cytochrome P450c11, Curvularia lunata-derived P-450lun, or the like is preferable.
Furthermore, the sterol 3-position oxidase protein is preferably bovine, mouse, rat, or human-derived 3β-hydroxysteroid dehydrogenase (3βHSD) or Streptomyces genus-derived cholesterol oxidase.

  Here, yeasts preferably used include yeasts of the genus Saccharomyces, yeasts of the genus Pichia, yeasts of the genus Schizosaccharomyces, yeasts of the genus Yarrowia, genus Aspergillus, and Mortierella. ) Genus. More preferred is Saccharomyces yeast. Specifically, it is Saccharomyces cerevisiae (Saccharomyces cerevisiae), more specifically, Saccharomyces cerevisiae FY1679-28c strain (JP2004-528827 A1), and similarly, FY1679-18b strain (JP2004-528827) A1), KA311A strain (FERM: P-19053), YPH499 strain (ATCC # 204679), YPH500 strain (ATCC # 204680) and the like. DNA encoding each enzyme protein is pESC-LEU (Stratagene) It is preferable to be inserted into an expression vector such as a yeast chromosome.

In addition, these yeasts preferably have any or all of the following properties (1) to (5).
(1) A gene encoding a sterol-22 desaturase protein or a DNA sequence of a region controlling its expression is not desaturated between positions 22 and 23 of an endogenous sterol.
(2) NADP-cytochrome P450 reductase protein expression is enhanced by introducing one or more copies of the gene encoding this protein into the host cell together with its expression control region.
(3) The expression of sterol ester hydrolase is enhanced by introducing one or more copies of the gene encoding this protein together with its expression control region into the host cell.
(4) steroid 17α hydroxylase, steroid 21-hydroxylase, ferredoxin-type protein that transfers electrons to steroid 11β-hydroxylase, and genes encoding ferredoxin reductase proteins that have electron transfer activity to the protein. By introducing it into a host cell together with its expression control region, the expression of the protein is enhanced.
(5) The methyl group does not exist at the 24th position of the endogenous sterol by modifying the DNA sequence of the gene encoding the sterol-24 methyltransferase protein or the region controlling its expression.

  The method for fractionating hydrocortisone from the reaction mixture is not particularly limited, and techniques for separation or purification known to those skilled in the art can be used. For example, it can be carried out by solvent extraction, crystallization, resin adsorption, column chromatography, etc., but is not limited thereto.

  Moreover, various derivatives can be manufactured by a well-known method from the hydrocortisone manufactured above. Furthermore, the hydrocortisone and derivatives thereof thus produced can be obtained by screening their pharmaceutical activity and safety by pharmacological or physiological tests in vitro or in vivo by methods known per se. Can be a therapeutic agent. In this case, a method for producing a pharmaceutical composition containing the compound obtained in the above step as an active ingredient is also included in the present invention.

  Specifically, hydrocortisone itself can be used alone, but can also be used as a pharmaceutical composition by blending with a pharmaceutically acceptable carrier. The pharmaceutical compound thus obtained can itself be used alone, but can also be used as a pharmaceutical composition by blending with a pharmaceutically acceptable carrier. The ratio of the active ingredient to the carrier at this time can vary between 0.01 and 90% by weight.

  Pharmaceutical compositions containing Δ5,7-sterol and hydrocortisone obtained in the present invention are tablets, capsules, soft capsules, powders, granules, fine granules, syrups, if necessary. It may be administered orally as a dosage form such as an emulsion, suspension or liquid, or as an injection such as a sterile solution or suspension with a pharmaceutically acceptable liquid, for example, intravenous administration , Intramuscular administration, topical administration, and subcutaneous administration. In addition, mucosal administration such as suppositories, sublingual tablets, nasal and pulmonary preparations, and administration as external preparations such as ointments and patches are also possible. When an oral or parenteral composition is prepared, it can be mixed by mixing in an organic or inorganic solid or liquid carrier or diluent in a unit volume form usually used. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.

  Examples of excipients used in producing solid preparations for oral administration and mucosal administration include lactose, sucrose, starch, talc, cellulose, dextrin, kaolin, calcium carbonate and the like. Liquid preparations for oral administration, i.e. syrups, suspensions, solutions, etc., contain generally used inert diluents such as water, vegetable oils and the like. In addition to the inert diluent, the preparation can also contain adjuvants such as wetting agents, suspending aids, sweetening agents, flavoring agents, coloring agents, preservatives, stabilizers and the like. Examples of the solvent or suspending agent used in the manufacture of injections, mucosal administration agents and the like include water, propylene glycol, polyethylene glycol, benzyl alcohol, ethyl oleate, lecithin and the like. Examples of bases used for suppositories include cocoa butter, emulsified cocoa butter, laurin butter, witepsol and the like. For the preparation of external preparations, alcohol, fatty acid esters, propylene glycol, etc. are used as a solubilizing agent or solubilizing agent, carboxyvinyl polymer, polysaccharides, etc. are used as adhesives, surfactants, etc. are used as emulsifiers. The

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited at all by these Examples.

Example 1
Saccharomyces cerevisiae YPH499, FY1679-06c strain, KA311A strain, X2181-1B strain erg5 and erg6 gene double disruption and ARE1 and YEH1 enhanced strain preparation (1) Preparation of yeast chromosomal DNA Saccharomyces cerevisiae S288C strain (from ATCC) Obtained ATCC20458) chromosomal DNA was prepared in the same manner as in Japanese Patent Application Laid-Open No. 2004-141125.

(2) Preparation of erg5 gene disruption strains SX1311, SX1306 and SX1334 by introduction of DNA fragment erg5 :: TRP1 for gene disruption Plasmid pSK-erg5 :: TRP1 for disrupting gene ERG5 encoding Saccharomyces cerevisiae-derived sterol C-22 desaturase (See JP-A No. 2004-141125) was cleaved with restriction enzyme Sac I (Takara Bio, unless otherwise specified) and Kpn I. Saccharomyces cerevisiae YPH499 strain (MATa ura3 lys2 ade2 trp1 his3 leu2) (obtained from STRATAGENE) and FY1679-06c strain (MATα ura3 leu2 trp1 his3f) (Euroscal)
X2181-1B (MATalpha his2 gal1 trp1 ade1 MAL SUC) (obtained from ATCC) and lithium acetate method (Ito, H. et al, (1983) J. Bacteriol. 153 (1), 163-168) Or by electroporation (Fromm ME, et al., (1986) Nature 319 (6056): 791-793), and YPH499 strains are each 20 mg / l adenine, L-histidine, uracil, 30 mg / l SD containing l L-lysine, 100 mg / l L-leucine, FY1679-06c strain each containing 20 mg / l L-histidine, uracil, 100 mg / l L-leucine, and X2181-1B strain containing 20 mg / l adenine. Seed on agar medium (0.67% Yeast Nitrogen Base w / o Amino Acids (Difco), 2% glucose, 2% agar) And cultured at 30 ° C. for 3 to 5 days.

Tryptophan non-requiring colonies grown on an agar medium were treated with YPD liquid medium (1%
Yeast extract, 2% polypeptone, 2% glucose) was inoculated into 2 ml, and cultured with shaking at 30 ° C. for 16 hours. Chromosomal DNA was extracted from this culture solution by the method described in (1) above. Using this as a template, PCR was performed with a combination of forward primer TRP1-outF (SEQ ID NO: 1) and reverse primer ERG5-D31 (SEQ ID NO: 2), and the presence or absence of homologous recombination on the ERG5 locus was examined. PCR uses TaKaRa ExTaq DNA Polymerase (Takara Bio Inc.) (1) 94 ° C., 2 minutes, (2) 94 ° C., 1 minute, (3) 53 ° C., 1 minute, (4) 72 ° C., 3 minutes The reaction from (2) to (4) was repeated 30 times, and then the reaction was performed at 4 ° C. under cooling conditions. As a comparative control, PCR was performed in the same manner using the chromosomal DNAs of the KAΔ5-5 strain, YPH499 strain, FY1679-06c strain, and X2181-1B strain described in JP-A-2004-141125 as templates.

  As a result of analyzing the PCR product by agarose gel electrophoresis, the transformants named SX1311 (derived from YPH499 strain), SX1306 (derived from FY1679-06c strain), and SX1334 (derived from X2181-1B strain) were about 1. A 93 kbp DNA fragment was detected. Therefore, it was confirmed that the ERG5 gene was disrupted by homologous recombination in the SX1311, SX1306, and SX1334 strains.

(3) DNA fragment erg6 :: HIS3 for gene disruption into SX1311, SX1306 strain, and DNA fragment erg6 :: HIS2 for gene disruption into SX1334 strain, erg5, erg6 double gene disruption strains SX1313, SX1312, Preparation of SX1340 strain (i) Gene fragment DNA erg6 :: Preparation of erg5 and erg6 double disruption strains SX1313 and SX1312 by introduction of HIS3 Plasmid for destroying gene ERG6 encoding sterol C-24 methyltransferase derived from Saccharomyces cerevisiae pUC-erg6 :: HIS3 (see Japanese Patent Application Laid-Open No. 2004-141125) was cleaved with restriction enzymes ScaI and EcoRI. This DNA fragment was transformed into the SX1311 and SX1306 strains prepared in (2) above by the lithium acetate method or electroporation method in the same manner as in (2) above. The SX1311 strains were each 20 mg / l adenine, uracil, 30 mg. / L L-lysine, 100 mg / l L-leucine, SX1306 strain is 20 mg / l uracil and 100 mg / l L-leucine, respectively. SD agar medium (0.67% Yeast Nitrogen Base w / o Amino Acids (Difco) Manufactured), 2% glucose, 2% agar) and cultured at 30 ° C. for 3-5 days.

Colonies that do not require histidine and tryptophan grown on an agar medium are inoculated into 2 ml of YPD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose) and cultured with shaking at 30 ° C. for 16 hours. It was. Chromosomal DNA was extracted from this culture solution by the method described in (1) above. Using this as a template, PCR was performed with a combination of forward primer ERG6-U52 (SEQ ID NO: 3) and reverse primer ERG6-D34 (SEQ ID NO: 4), and the presence or absence of homologous recombination on the ERG6 locus was examined. PCR was performed under the conditions described in (2) above. As a comparative control, PCR was performed in the same manner using the chromosomal DNA of KAΔ5Δ6-1 strain, YPH499 strain, FY1679-06c described in JP-A-2004-141125 as a template.

  As a result of analyzing the PCR product by agarose gel electrophoresis, a DNA fragment of about 1.78 kbp having the same size as the target size was detected in transformants named SX1313 (derived from YPH499 strain) and SX1312 (derived from FY1679-06c strain). Accordingly, it was confirmed that the ERG6 gene was disrupted by homologous recombination in the SX1313 and SX1312 strains.

(Ii) Preparation of ERG6 for disruption and HIS2 gene fragment for yeast expression A DNA fragment containing a gene encoding Ester 6 from Saccharomyces cerevisiae (ERG6) and a DNA fragment containing a HIS2 gene are as described in (1) above. Using the obtained chromosomal DNA of Saccharomyces cerevisiae S288C strain as a template, it was synthesized and isolated by PCR. Details of the method are shown below.

For the synthesis of the ERG6 gene fragment by PCR, the forward primer ERG6-5L (SEQ ID NO: 5) and the reverse primer ERG6-3L (SEQ ID NO: 6) were used. This primer combination includes the protein coding region of ERG6 and can amplify from 238 bases before the start codon to 258 bases after the stop codon. Furthermore, the restriction enzyme PstI recognition sequence can be synthesized on the 5 ′ side and the EcoRI recognition sequence can be synthesized on the 3 ′ side.
For the synthesis of the HIS2 gene fragment by PCR, the forward primer HIS2-F (SEQ ID NO: 7) and the reverse primer HIS2-R (SEQ ID NO: 8) were used. This primer combination includes the HIS2 protein coding region and can amplify from 430 bases before the start codon to 290 bases after the stop codon. Further, the restriction enzyme SmaI can be synthesized on the 5 ′ side and the EcoRI recognition sequence on the 3 ′ side.
PCR was performed using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) (1) 98 ° C., 2 minutes, (2) 98 ° C., 10 seconds, (3) 55 ° C., 5 seconds, (4) 72 ° C. The reaction from (2) to (4) was repeated 30 times for 2 minutes, (5) after reacting at 72 ° C. for 2 minutes, the reaction was performed at 4 ° C. under cooling conditions. Next, the PCR product was subjected to agarose gel electrophoresis, and an ERG6 gene fragment of about 1.65 kbp and a HIS2 gene fragment of about 1.73 kbp were confirmed.

(Iii) Preparation of erg6 disruption plasmid pUC-erg6 :: HIS2 The pUC19 (manufactured by Takara Bio Inc.) was cleaved with restriction enzymes PstI and EcoRI, and the ERG6 gene fragment PCR-synthesized in (ii) above was prepared with PstI and EcoRI. The digested DNA was ligated with TaKaRa DNA Ligation Kit Ver. 2 (manufactured by Takara Bio Inc.). This was transformed into Escherichia coli JM109, and a plasmid was extracted from colonies grown on LB agar medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 2% agar) containing 100 mg / l ampicillin. did. A plasmid in which about 0.64 kbp and 3.66 kbp fragments were confirmed by agarose gel electrophoresis and cleaved with the restriction enzyme HincII was named pUC-ERG6L.
Next, using pGEM-T Easy Vector Systems (manufactured by Promega), the HIS2 gene fragment PCR-synthesized in (ii) above was linked to the cloning site of pGEM-T Easy (manufactured by Promega) by direct cloning. This was transformed into Escherichia coli JM109, and a plasmid was extracted from colonies grown on LB agar medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 2% agar) containing 100 mg / l ampicillin. did. By agarose gel electrophoresis, restriction enzyme Sm
A plasmid in which about 3.0 kbp and 1.73 kbp fragments were confirmed by digestion with aI and EcoRI was named pGEM-T-HIS2 as a target plasmid.

Further, the above pUC-ERG6L was cleaved with the restriction enzyme BspT104I and cloned.
DNA plasmid blunting treatment using DNA Polymerase I (Takara Bio Inc.), dephosphorylation treatment using Alkaline Phosphatase (E. coli C75) (Takara Bio Inc.), and plasmid pGEM-T-HIS2 was digested with restriction enzymes SmaI and XbaI and subjected to DNA end blunting treatment using DNA Polymerase I (manufactured by Takara Bio Inc.). TaKaRa DNA Ligation Kit Ver. 2 (manufactured by Takara Bio Inc.). This was transformed into Escherichia coli JM109, and a plasmid was extracted from colonies grown on LB agar medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 2% agar) containing 100 mg / l ampicillin. did. A plasmid in which about 1.71 kbp, 1.67 kbp, and 1.89 kbp fragments were confirmed by digestion with the restriction enzyme ScaI by agarose gel electrophoresis was named pUC-erg6 :: HIS2.

(Iv) Preparation of erg5, erg6 double gene disruption strain SX1340 Plasmid pUC-erg6 :: HIS2 for ERG6 disruption prepared in (iii) above was digested with restriction enzymes EcoRI and PstI. This DNA fragment was transformed into the SX1334 strain prepared in the above (2) by the lithium acetate method or the electroporation method in the same manner as in the above (2), and an SD agar medium (0.67% Yeast containing 20 mg / l adenine). Nitrogen Base w / o Amino Acids (manufactured by Difco), 2% glucose, 2% agar) and cultured at 30 ° C. for 3 to 5 days.

Colonies that do not require histidine and tryptophan grown on an agar medium are inoculated into 2 ml of YPD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose) and cultured with shaking at 30 ° C. for 16 hours. It was. Chromosomal DNA was extracted from this culture solution by the method described in (1) above. Using this as a template, PCR was performed with a combination of forward primer ERG6-U52 (SEQ ID NO: 3) and reverse primer ERG6-D34 (SEQ ID NO: 4), and the presence or absence of homologous recombination on the ERG6 locus was examined. PCR was performed under the conditions described in (i) above. As a comparative control, PCR was performed in the same manner using the chromosomal DNA of the KAΔ5Δ6-1 strain and the X2181-1B strain described in JP-A-2004-141125 as a template.
As a result of analyzing the PCR product by agarose gel electrophoresis, a DNA fragment of about 2.76 kbp having the same size as the target size was detected in the transformant named SX1340. Therefore, it was confirmed that the ERG6 gene was disrupted by homologous recombination in the SX1340 strain.

(4) Preparation of ARE1 gene introduction strain into erg5, erg6 double disruption strain (i) Preparation of plasmid pPG-LEU for yeast expression 3-phosphoglycerate kinase for constitutive expression of foreign genes in yeast cells The promoter region of 1 gene (PGK1) is a primordial primer PPGK1-5A (SEQ ID NO: 9), reverse primer PPGK1-3A (SEQ ID NO: 9) using the chromosomal DNA of Saccharomyces cerevisiae KA311A strain (Accession No .: FERM P-19053) as a template. It was synthesized by PCR using 10). With this primer combination, it can be synthesized in a form including a restriction enzyme BamHI on the 5 ′ side and a recognition sequence for the restriction enzyme EcoR I on the 3 ′ side. PCR uses TaKaRa ExTaq DNA Polymerase (Takara Bio Inc.) (1) 94 ° C., 2 minutes, (2) 94 ° C., 1 minute, (3) 55 ° C., 1 minute, (4) 72 ° C., 1 minute The reaction from (2) to (4) was repeated 30 times, and (5) the reaction was carried out at 72 ° C. for 4 minutes, followed by reaction at 4 ° C. under cooling conditions. Next, the PCR product was subjected to agarose gel electrophoresis, and a DNA fragment of about 0.6 kbp PGK1 promoter was confirmed.
This PCR product was ligated to pCR2.1 (manufactured by Invitrogen). The ligated product was used to transform Escherichia coli JM109, and plasmids were extracted from the resulting colonies.
The obtained plasmid was cleaved with BamH I and EcoR I, and a DNA fragment (PGK1 promoter) of about 0.6 kbp was extracted from the agarose gel with GENE CLEAN II KIT (Funakoshi).

On the other hand, the yeast expression vector pADNSΔE (Japanese Patent Application Laid-Open No. 2004-141125) was also cut with BamHI and EcoRI, and a DNA fragment of about 6.58 kbp was extracted from the agarose gel.
The above two DNA fragments were added to TaKaRa DNA Ligation Kit Ver. 2 (manufactured by Takara Bio Inc.), transformed into Escherichia coli JM109, and a plasmid was extracted from the obtained colonies. A plasmid whose size was confirmed to be about 7.2 kbp by agarose gel electrophoresis was designated as a target plasmid and named pPG-LEU. pPG-LEU has a PGK1 promoter as a promoter and an ADH1 terminator derived from the alcohol dehydrogenase 1 gene (ADH1) as a terminator.

(Ii) Preparation of yeast recombination plasmid pPG-LEU (-2μ) The plasmid pPG-LEU prepared in (i) above was cleaved with restriction enzymes Hpa I and Eco47III, and a DNA fragment of about 5.4 kbp was obtained from the agarose gel. 2μm by extracting
DNA was removed, the resulting DNA fragment was self-ligated, transformed into Escherichia coli JM109, and a plasmid was extracted from the resulting colony. The plasmid which confirmed about 5.4 kbp by agarose gel electrophoresis was made into the target plasmid, and it was named pPG-LEU (-2 micro | micron | mu).

(Iii) Preparation of ARE1, URA3, and ADE1 gene fragments for yeast expression A DNA fragment containing Saccharomyces cerevisiae-derived acyl CoA: sterol acyltransferase-encoding gene (ARE1), and a DNA fragment containing URA3 and ADE1 The Saccharomyces cerevisiae S288C strain chromosomal DNA obtained in (1) was synthesized and isolated by PCR. Details of the method are shown below.
For synthesis of the ARE1 gene fragment by PCR, the forward primer ARE1-Sma
I (F) (SEQ ID NO: 11) and reverse primer ARE1-EcoR I (R) (SEQ ID NO: 12) were used. This primer combination can be used to amplify the ARE1 protein coding region from the start codon to the end codon. Further, the restriction enzyme Sma I recognition sequence can be synthesized on the 5 ′ side, and the EcoR I recognition sequence can be synthesized on the 3 ′ side. For the synthesis of the URA3 gene fragment by PCR, the forward primer URA3-2F (SEQ ID NO: 13) and the reverse primer URA3-1R (SEQ ID NO: 14) were used. This primer combination includes the protein coding region of URA3 and can amplify from 254 bases before the start codon to 178 bases after the stop codon. The synthesized gene fragment is synthesized in such a manner that a recognition sequence for Hind III is included 223 bases before the start codon and 136 bases after the start codon. For the synthesis of the ADE1 gene fragment by PCR, the forward primer ADE1-F (SEQ ID NO: 15) and the reverse primer ADE1-R (SEQ ID NO: 16) were used. This primer combination includes the ADE1 protein coding region and can amplify from 346 bases before the start codon to 312 bases after the stop codon. Further, the synthesized gene fragment is synthesized in such a way that an EcoRV recognition sequence is included 106 bases after the termination codon. PCR was performed using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) (1) 98 ° C., 2 minutes, (2) 98 ° C., 10 seconds, (3) 55 ° C., 5 seconds, (4) 72 ° C. The reaction from (2) to (4) was repeated 30 times for 2 minutes, (5) the reaction was performed at 72 ° C. for 7 minutes, and then the reaction was performed at 4 ° C. under cooling conditions. Next, the PCR product was subjected to agarose gel electrophoresis, and an ARE1 gene fragment of about 1.83 kbp, an URA3 gene fragment of about 1.23 kbp, and an ADE1 gene fragment of about 1.57 kbp were confirmed.

(5) Preparation of ARE1 expression plasmids pPGU-ARE1 (-2μ) and pPGADE1-ARE1 (-2μ) Plasmid pPG-LEU (-2μ) prepared in (2) above was digested with restriction enzymes SmaI and EcoRI A PCR-synthesized ARE1 gene fragment cleaved with SmaI and EcoRI was prepared using TaKaRa DNA Ligation Kit Ver. 2 (manufactured by Takara Bio Inc.). This was transformed into Escherichia coli JM109, and a plasmid was extracted from colonies grown on LB agar medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 2% agar) containing 100 mg / l ampicillin. did. A plasmid in which about 5.4 kbp and 1.83 kbp fragments were confirmed by agarose gel electrophoresis and digested with restriction enzymes SmaI and EcoRI was named pPGL-ARE1 (−2 μ).

Next, pUC18 (manufactured by Takara Bio Inc.) was cut with HindIII, and Alkaline
A product obtained by dephosphorylation using Phosphatase (E. coli C75) (manufactured by Takara Bio Inc.) and a product obtained by cleaving the PCR-synthesized URA3 gene fragment with HindIII were prepared using TaKaRa DNA Ligation Kit Ver. 2 (manufactured by Takara Bio Inc.). This was transformed into Escherichia coli JM109, and a plasmid was extracted from colonies grown on LB agar medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 2% agar) containing 100 mg / l ampicillin. did. A plasmid in which fragments of about 2.68 kbp and 1.23 kbp were confirmed by digestion with restriction enzyme Hind III by agarose gel electrophoresis was named pUC-URA3 as a target plasmid.

  Further, the ADE1 gene fragment PCR-synthesized in (iii) above was ligated to the cloning site of pCR-Blunt (manufactured by Invitrogen) by direct cloning. This was transformed into Escherichia coli JM109, and a plasmid was extracted from colonies grown on LB agar medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 2% agar) containing 100 mg / l ampicillin. did. A plasmid that had been confirmed to have fragments of about 3.47 kbp and 1.62 kbp by digestion with restriction enzymes SpeI and PstI by agarose gel electrophoresis was named pCR-B-ADE1 as a target plasmid.

  Subsequently, pUC19 (manufactured by Takara Bio Inc.) was cleaved with XbaI and SmaI, subjected to DNA end blunting using cloned DNA Polymerase I (manufactured by Takara Bio Inc.), and then Alkaline Phosphatase (E. coli C75) ( PCR-B-ADE1 was cleaved with SpeI and EcoRV, and DNA end smoothing treatment was performed using cloned DNA Polymerase I (manufactured by Takara Bio Inc.). Were subjected to TaKaRa DNA Ligation Kit Ver. 2 (manufactured by Takara Bio Inc.). This was transformed into Escherichia coli JM109, and a plasmid was extracted from colonies grown on LB agar medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 2% agar) containing 100 mg / l ampicillin. did. A plasmid in which about 1.01 kbp, 0.39 kbp and 2.68 kbp fragments could be confirmed by digestion with restriction enzyme EcoRI by agarose gel electrophoresis was named pUC19-ADE1.

Furthermore, the above-described pPGL-ARE1 (-2 μ) was cleaved with SspI and subjected to dephosphorylation using Alkaline Phosphatase (E. coli C75) (manufactured by Takara Bio Inc.), and pUC-URA3 was treated with HindIII. Cleaved and subjected to DNA end blunting treatment using cloned DNA Polymerase I (Takara Bio Inc.), pUC19-ADE1 was cleaved with PstI and KpnI, and cloned.
d DNA Polymerase I (manufactured by Takara Bio Inc.) and those subjected to DNA end blunting treatment were respectively used in TaKaRa DNA Ligation Kit Ver. 2 (manufactured by Takara Bio Inc.). This was transformed into Escherichia coli JM109, and a plasmid was extracted from colonies grown on LB agar medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 2% agar) containing 100 mg / l ampicillin. did. By agarose gel electrophoresis, fragments of about 3.56 kbp and 3.07 kbp were confirmed by cleavage with restriction enzymes EcoR I and Pst I, and about 3.74 kbp and 1.29 kbp by cutting with restriction enzyme BspT104I. Those in which a fragment of .85 kbp could be confirmed were named pPGU-ARE1 (−2 μ) and pPGADE1-ARE1 (−2 μ), respectively, as target plasmids.

(6) Production of ARE1 gene-introduced strains SX1314, SX1318, SX1315, SX1350 strains into erg5, erg6 double disruptive strains SX1313, SX1312, KAΔ5-4, SX1340 erg5, erg6 double disruptive strains SX1313, SX1312, For the purpose of further introducing the ARE1 gene into the ARE1 locus on the genome of KAΔ5-4 described in 141125 (hereinafter referred to as SX1307), the following steps were performed. Saccharomyces cerevisiae-derived sterol acyl CoA: A plasmid pPGU-ARE1 (-2 μ) for expression of the gene ARE1 encoding sterol acyltransferase was cleaved with a restriction enzyme Xho I (in the ARE1 gene). This DNA fragment was transformed into the SX1313, SX1312, and SX1307 strains prepared in (3) above by the lithium acetate method or electroporation method as in (2) above, and SD agar containing 100 mg / l L-leucine. Medium (0.67% Yeast Nitrogen Base w / o Amino
Acids (manufactured by Difco), 2% glucose, 2% agar) and cultured at 30 ° C. for 3 to 5 days. In addition, 20 mg / l adenine and 30 mg / l L-lysine were added only to the SD agar medium in which the SX1313 strain was transformed.

  Uracil, histidine and tryptophan non-requiring colonies grown on an agar medium were inoculated into 2 ml of YPD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose) and cultured at 30 ° C. for 16 hours with shaking. Went. Chromosomal DNA was extracted from this culture solution by the method described in (1) above. Using this as a template, PCR was performed with a combination of the forward primer PPGK1-F (SEQ ID NO: 17) and the reverse primer ARE1-check (SEQ ID NO: 18), and the presence or absence of homologous recombination on the ARE1 locus was examined. PCR was performed under the conditions described in (2) above.

  As a result of analyzing the PCR product by agarose gel electrophoresis, as a result, transformants named SX1314 (derived from YPH499 strain), SX1318 (derived from FY1679-06c strain), and SX1315 (derived from KA311A strain) have a size of about 2.6 kbp which is the same as the target size. A DNA fragment was detected. Accordingly, the ARE1 gene was introduced into the ARE1 locus of the genomes of the SX1314, SX1318, and SX1315 strains by the homologous recombination, and it was confirmed that there were 2 copies in total.

Furthermore, the following steps were performed in the same manner as described above for the purpose of further introducing the ARE1 gene into the ARE1 locus on the genome of the erg5, erg6 double disruption strain SX1340. Saccharomyces cerevisiae-derived sterol acyl CoA: A plasmid pPGADE1-ARE1 (-2 μ) for expression of the gene ARE1 encoding sterol acyltransferase was cleaved with a restriction enzyme Xho I (in the ARE1 gene). In order to prepare a control strain into which ARE1 had not been introduced, pUC19-ADE1 prepared in (5) above was cleaved with the restriction enzyme HpaI. These DNA fragments were transformed into the SX1340 strain prepared in (3) above by the lithium acetate method or electroporation method in the same manner as in (2) above.
Agar medium (0.67% Yeast Nitrogen Base w / o Amino
Acids (manufactured by Difco), 2% glucose, 2% agar) and cultured at 30 ° C. for 3 to 5 days.

  Adenine, histidine, and tryptophan non-requiring colonies grown on an agar medium are inoculated into 2 ml of YPD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose) and cultured at 30 ° C. for 16 hours with shaking. Went. Chromosomal DNA was extracted from this culture solution by the method described in (1) above. Using this as a template, PCR was performed with a combination of the forward primer PPGK1-F (SEQ ID NO: 17) and the reverse primer ARE1-check (SEQ ID NO: 18), and the presence or absence of homologous recombination on the ARE1 locus was examined. PCR was performed under the conditions described in (2) above.

As a result of analyzing the PCR product by agarose gel electrophoresis, a DNA fragment of about 2.6 kbp having the same size as the target size was detected in the transformant named SX1350 (derived from X2181-1B strain). Therefore, the ARE1 gene was introduced into the ARE1 locus of the genome of the SX1350 strain by the homologous recombination, and it was confirmed that a total of 2 copies exist.
Moreover, about the strain | stump | stock which transformed using the DNA fragment which cut | disconnected pUC19-ADE1 by HpaI, PCR was performed by the combination of forward primer ADE1-1F (sequence number 19) and reverse primer M13 forward (sequence number 20). The presence or absence of homologous recombination on the ADE1 locus was examined. PCR was performed under the conditions described in (2) above. As a result of analyzing the PCR product by agarose gel electrophoresis, a DNA fragment of about 1.45 kbp having the same size as the target size was detected in the transformant named SX1353. Therefore, it was confirmed that ADE1 gene was complemented by homologous recombination in SX1353 strain.

(7) Preparation of YEH1 gene introduction strain into erg5, erg6 double disruption strain introduced with ARE1 gene (i) Preparation of YEH1, GAL1 gene fragment for yeast expression Gene encoding Saccharomyces cerevisiae steryl ester hydrolase A DNA fragment containing (YEH1) and a DNA fragment containing GAL1 were synthesized and isolated by PCR using the chromosomal DNA of Saccharomyces cerevisiae S288C obtained in (1) above as a template. Details of the method are shown below.
For the synthesis of the YEH1 gene fragment by PCR, the forward primer YLL-OF (SEQ ID NO: 21) and the reverse primer YLL-OR (SEQ ID NO: 22) were used. This primer combination can be used to amplify from the start codon to the end codon, which is the protein coding region of YEH1. Alternatively, the restriction enzyme EcoR I recognition sequence can be synthesized on both the 5 ′ side and the 3 ′ side.

  The forward primer GAL1-51 (SEQ ID NO: 23) and the reverse primer GAL1-31 (SEQ ID NO: 24) were used for the synthesis of the GAL1 gene fragment by PCR. This primer combination includes the GAL1 protein coding region and can amplify from 499 bases before the start codon to 230 bases after the stop codon. Further, it can be synthesized in such a way that a recognition sequence for the restriction enzyme SmaI is included on both the 5 'side and the 3' side.

  PCR was performed using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) (1) 98 ° C., 2 minutes, (2) 98 ° C., 10 seconds, (3) 55 ° C., 5 seconds, (4) 72 ° C. The reaction from (2) to (4) was repeated 30 times for 2 minutes, (5) the reaction was performed at 72 ° C. for 7 minutes, and then the reaction was performed at 4 ° C. under cooling conditions. Next, the PCR product was subjected to agarose gel electrophoresis, and a YEH1 gene fragment of about 1.72 kbp and a GAL1 gene fragment of about 2.33 kbp were confirmed.

(Ii) Preparation of YEH1 expression plasmids pPGL-YEH1 (-2μ) and pPGGAL-YEH1 (-2μ) The plasmid pPG-LEU (-2μ) prepared in (4) (ii) above was digested with the restriction enzyme EcoR I. The one obtained by dephosphorylation using Alkaline Phosphatase (E. coli C75) (manufactured by Takara Bio Inc.) and the one obtained by cleaving the PCR-synthesized YEH1 gene fragment with EcoR I were prepared using TaKaRa DNA Ligation Kit Ver. 2 (manufactured by Takara Bio Inc.). This was transformed into Escherichia coli JM109, and a plasmid was extracted from colonies grown on LB agar medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 2% agar) containing 100 mg / l ampicillin. did. A plasmid in which about 5.4 kbp and 1.72 kbp fragments were confirmed by agarose gel electrophoresis and digested with the restriction enzyme EcoRI was named pPGL-YEH1 (−2 μ).

Next, pUC19 (manufactured by Takara Bio Inc.) was digested with SmaI, dephosphorylated using Alkaline Phosphatase (E. coli C75) (manufactured by Takara Bio Inc.), and the PCR-synthesized GAL1 gene fragment. What was cut | disconnected with SmaI and TAKARA DNA Ligation Kit Ver. 2 (manufactured by Takara Bio Inc.). This was transformed into Escherichia coli JM109, and a plasmid was extracted from colonies grown on LB agar medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 2% agar) containing 100 mg / l ampicillin. did. A plasmid in which about 0.48 kbp, 0.72 kbp, 1.45 kbp, and 2.36 kbp fragments were confirmed by agarose gel electrophoresis and digested with the restriction enzyme PvuII was named pUC19-GAL1.
Subsequently, the pPGL-YEH1 (−2 μ) was cleaved with SspI and subjected to dephosphorylation using Alkaline Phosphatase (E. coli C75) (manufactured by Takara Bio Inc.), and the pUC19-GAL1 was treated with SmaI. Cleaved with TAKARA DNA Ligation Kit Ver. 2 (manufactured by Takara Bio Inc.). This was transformed into Escherichia coli JM109, and a plasmid was extracted from colonies grown on LB agar medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 2% agar) containing 100 mg / l ampicillin. did. By agarose gel electrophoresis, fragments of about 3.30 kbp and 2.66 kbp were confirmed by cleavage with restriction enzyme EcoRI, and fragments of about 1.28 kbp and 4.68 kbp were confirmed by cleavage with restriction enzyme ScaI. Was designated as pPG-GAL1 (-2μ) as the target plasmid. Furthermore, the above-described pPGL-YEH1 (-2 μ) was cleaved with BamHI and subjected to dephosphorylation using Alkaline Phosphatase (E. coli C75) (manufactured by Takara Bio Inc.), and the above-described pPG-GAL1 (-2 μm). ) Was digested with BamHI and TAKARA DNA Ligation Kit Ver. 2 (manufactured by Takara Bio Inc.). This was transformed into Escherichia coli JM109, and a plasmid was extracted from colonies grown on LB agar medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 2% agar) containing 100 mg / l ampicillin. did. A fragment of about 0.37 kbp, 0.57 kbp, 0.19 kbp, 3.73 kbp, and 2.82 kbp, which was confirmed by agarose gel electrophoresis and digested with the restriction enzyme HindIII, was used as a target plasmid. 2μ).

(Iii) Preparation of YEH1 gene-introduced strains SX1317, SX1320, SX1322, and SX1351 strains into erg5 and erg6 double-disrupted strains SX1314, SX1318, SX1315, and SX1350 into which ARE1 gene was introduced erg5 and erg6 double-disrupted strains into which ARE1 gene was introduced For the purpose of further introducing the YEH1 gene into the YEH1 locus on the genomes of SX1314, SX1318, and SX1315, the following steps were performed. The plasmid pPGL-YEH1 (-2μ) for expression of the gene YEH1 encoding the Saccharomyces cerevisiae-derived steryl ester hydrolase was cleaved with the restriction enzyme NdeI (in the YEH1 gene). Further, in order to prepare a control strain into which YEH1 had not been introduced, the yeast recombination plasmid pPG-LEU (-2 μ) prepared in (4) (ii) was cleaved with the restriction enzyme Bsp1407 I. These DNA fragments were transformed into the SX1314, SX1318, and SX1315 strains prepared in (6) above by the lithium acetate method or the electroporation method as in (2) above, and the SD agar medium (0.67% Yeast Nitrogen) was transformed. Base w / o Amino Acids (manufactured by Difco), 2% glucose, 2% agar) and cultured at 30 ° C. for 3 to 5 days. In addition, 20 mg / l adenine and 30 mg / l L-lysine were added only to the SD agar medium which transformed SX1314 strain.

  The leucine and uracil, histidine, and tryptophan non-requiring colonies grown on the agar medium are inoculated into 2 ml of YPD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose), and shaken at 30 ° C. for 16 hours. Culture was performed. Chromosomal DNA was extracted from this culture solution by the method described in (1) above. Using this as a template, PCR was performed with a combination of forward primer PPGK1-F (SEQ ID NO: 17) and reverse primer YEH1-RR (SEQ ID NO: 25), and the presence or absence of homologous recombination on the YEH1 locus was examined. PCR was performed under the conditions described in (2) above.

  As a result of analyzing the PCR product by agarose gel electrophoresis, the transformants named SX1317 (derived from the YPH499 strain), SX1320 (derived from the FY1679-06c strain), and SX1322 (derived from the KA311A strain) have a size of about 2.0 kbp which is the same as the target size. A DNA fragment was detected. Therefore, the YEH1 gene was introduced into the genomes of the SX1317, SX1320, and SX1322 strains by the homologous recombination, and it was confirmed that two copies existed.

  In addition, for a strain transformed with a DNA fragment obtained by cutting plasmid pPG-LEU (-2μ) with Bsp1407I, forward primer PPGK1-R1 (SEQ ID NO: 26) and reverse primer LEU2-R2 (SEQ ID NO: 27). PCR was performed with the combination of the above, and the presence or absence of homologous recombination on the leu2 locus was examined. PCR was performed under the conditions described in (2) above.

  As a result of analyzing the PCR product by agarose gel electrophoresis, the transformants named SX1316 (derived from YPH499 strain), SX1319 (derived from FY1679-06c strain), and SX1321 (derived from KA311A strain) were about 2.0 kbp which is the same size as the target size. A DNA fragment was detected. Therefore, it was confirmed that SX1316, SX1319, and SX1321 strains were complemented with the LEU2 gene by homologous recombination.

  Further, for the purpose of further introducing the YEH1 gene into the YEH1 locus on the genome of the erg5 and erg6 double disruption strain SX1350 into which the ARE1 gene was introduced, the following steps were performed in the same manner as described above. The plasmid pPGGAL1-YEH1 (-2 μ) for expression of the gene YEH1 encoding the Saccharomyces cerevisiae-derived steryl ester hydrolase was cleaved with the restriction enzyme Bsp1407 I (in the YEH1 gene). This DNA fragment was transformed into the SX1350 strain prepared in the above (6) by the lithium acetate method or the electroporation method in the same manner as in the above (2), and SG agar medium (0.67% Yeast Nitrogen Base w / o Amino) was obtained. Acids (manufactured by Difco), 2% galactose, 2% agar) and cultured at 30 ° C. for 3 to 5 days.

Adenine, histidine, and tryptophan non-requiring colonies grown on an agar medium using galactose as a carbon source were inoculated into 2 ml of YPD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose), and 16 ml at 30 ° C. Shaking culture was performed for a time. Chromosomal DNA was extracted from this culture solution by the method described in (1) above. Using this as a template, PCR was performed with a combination of forward primer PPGK1-F (SEQ ID NO: 17) and reverse primer YEH1-RR (SEQ ID NO: 25), and the presence or absence of homologous recombination on the YEH1 locus was examined. PCR was performed under the conditions described in (2) above.

  As a result of analyzing the PCR product by agarose gel electrophoresis, a DNA fragment of about 2.0 kbp having the same size as the target size was detected in the transformant named SX1351 (derived from X2181-1B strain). Accordingly, the YEH1 gene was introduced into the ARE1 locus of the genome of the SX1351 strain by the homologous recombination, and it was confirmed that there were 2 copies in total.

(Example 2)
Expression of erg5, erg6 double disruption strain SX1312 Production of ARE1 and YEH1 enhanced strains with different locus (1) Preparation of ARE1 gene-introduced strain SX1326 into erg5, erg6 double disruption strain SX1312 For the purpose of further introducing the ARE1 gene into the above ARE1 locus, the following steps were performed. Saccharomyces cerevisiae-derived sterol acyl CoA: A plasmid pPGL-ARE1 (-2 μ) for expression of the gene ARE1 encoding sterol acyltransferase was cleaved with a restriction enzyme Xho I (in the ARE1 gene). This DNA fragment was transformed into the SX1312 strain prepared in Example 1 (3) by the lithium acetate method or electroporation method as in Example 1 (2), and an SD agar medium containing 20 mg / l uracil. (0.67% Yeast Nitrogen Base w / o Amino Acids (manufactured by Difco), 2% glucose, 2% agar), and cultured at 30 ° C. for 3 to 5 days.

Colonies that do not require leucine, histidine, and tryptophan grown on an agar medium are inoculated into 2 ml of YPD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose), and cultured at 30 ° C. for 16 hours with shaking. Went. Chromosomal DNA was extracted from this culture solution by the method described in Example 1 (1) above. Using this as a template, PCR was performed with a combination of the forward primer PPGK1-F (SEQ ID NO: 17) and the reverse primer ARE1-check (SEQ ID NO: 18), and the presence or absence of homologous recombination on the ARE1 locus was examined. PCR was performed under the conditions described in Example 1 (2) above.
As a result of analyzing the PCR product by agarose gel electrophoresis, a DNA fragment of about 2.6 kbp having the same size as the target size was detected in the transformant named SX1326. Therefore, the ARE1 gene was introduced into the ARE1 locus of the genome of the SX1326 strain by the homologous recombination, and it was confirmed that a total of 2 copies were present.

(2) Preparation of YEH1 gene-introduced strains SX1325 and SX1332 having different expression locus into SX1318 and SX1326 strains into which ARE1 gene has been introduced (i) Preparation of YEH1 expression plasmid pPGU-YEH1 (-2μ) Example 1 (5) The plasmid pPGU-ARE1 (−2 μ) prepared in (1) was cleaved with the restriction enzyme BamHI and dephosphorylated using Alkaline Phosphatase (E. coli C75) (manufactured by Takara Bio Inc.), and pPGL-YEH1 ( -2μ) was digested with BamHI and TaKaRa DNA Ligation Kit Ver. 2 (manufactured by Takara Bio Inc.). This was transformed into Escherichia coli JM109, and a plasmid was extracted from colonies grown on LB agar medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 2% agar) containing 100 mg / l ampicillin. did. A plasmid in which fragments of about 4.88 kbp and 1.64 kbp were confirmed by agarose gel electrophoresis and digested with the restriction enzyme SmaI was named pPGU-YEH1 (−2 μ).

(Ii) Preparation of YEH1 gene-introduced strains SX1325 and SX1332 having different expression locus in erg5 and erg6 double-disrupted strains SX1318 and 1326 introduced with ARE1 gene leu2 gene of erg5 and erg6 double-disrupted strain SX1318 introduced with ARE1 gene The following steps were performed for the purpose of further introducing the YEH1 gene into the locus and the ura3 locus on the SX1326 genome. A plasmid pPGL-YEH1 (-2 μ) for expression of the gene YEH1 encoding the steryl ester hydrolase derived from Saccharomyces cerevisiae was cleaved with the restriction enzyme BstEII (in the leu2 gene), and pPGU-YEH1 (-2 μ) was cleaved with the restriction enzyme StuI ( cleaved at ura3 gene). These DNA fragments were transformed into the SX1318 and SX1326 strains by the lithium acetate method or the electroporation method in the same manner as in Example 1 (2) above, and the SD agar medium (0.67% Yeast Nitrogen Base w / o Amino Acids). (Difco), 2% glucose, 2% agar) and cultured at 30 ° C. for 3-5 days.

  The leucine and uracil, histidine, and tryptophan non-requiring colonies grown on the agar medium are inoculated into 2 ml of YPD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose), and shaken at 30 ° C. for 16 hours. Culture was performed. Chromosomal DNA was extracted from this culture solution by the method described in (1) above. Using this as a template, a combination of forward primer LEU2-R2 (SEQ ID NO: 27) and reverse primer YLL-Seq3R (SEQ ID NO: 28), forward primer URA3-RR (SEQ ID NO: 29) and reverse primer YEH1-seqR1 (SEQ ID NO: 30) PCR was performed to examine the presence or absence of homologous recombination on the leu2 locus and the ura3 locus. PCR was performed under the conditions described in Example 1 (2) above.

  As a result of analyzing the PCR product by agarose gel electrophoresis, the transformants named SX1325 (leu2 locus) and SX1332 (ura3 locus) had DNA fragments of about 2.8 kbp and about 2.6 kbp, which were the same size as the target size, respectively. was detected. Therefore, the YEH1 gene was introduced into the genome of the SX1325 and SX1332 strains by the homologous recombination, and it was confirmed that two copies existed.

(3) Preparation of ARE1 gene-introduced strain SX1327 with different expression locus in erg5 and erg6 double-disrupted strain SX1312 For the purpose of further introducing the ARE1 gene into the ura3 locus on the genome of erg5 and erg6 double-disrupted strain SX1312 The following steps were performed. Saccharomyces cerevisiae-derived sterol acyl CoA: a plasmid pPGU-ARE1 (-2 μ) for expression of the gene ARE1 encoding sterol acyltransferase was cleaved with the restriction enzyme StuI (in the ura3 gene). This DNA fragment was transformed into the SX1312 strain prepared in Example 1 (3) by the lithium acetate method or electroporation method as in Example 1 (2), and SD containing 100 mg / l L-leucine. The cells were seeded on an agar medium (0.67% Yeast Nitrogen Base w / o Amino Acids (Difco), 2% glucose, 2% agar) and cultured at 30 ° C. for 3 to 5 days.
Uracil, histidine and tryptophan non-requiring colonies grown on an agar medium were inoculated into 2 ml of YPD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose) and cultured at 30 ° C. for 16 hours with shaking. Went. Chromosomal DNA was extracted from this culture solution by the method described in Example 1 (1) above. Using this as a template, PCR was performed with a combination of forward primer URA3-RR (SEQ ID NO: 29) and reverse primer ARE1-RR (SEQ ID NO: 31), and the presence or absence of homologous recombination on the ARE1 locus was examined. PCR was performed under the conditions described in Example 1 (2) above.

As a result of analyzing the PCR product by agarose gel electrophoresis, a DNA fragment of about 2.5 kbp having the same size as the target size was detected in the transformant named SX1326. Therefore, the ARE1 gene was introduced into the ura3 locus of the SX1327 strain genome by the above homologous recombination, and it was confirmed that there were 2 copies in total.

(4) Preparation of YEH1 gene-introduced strain SX1333 into SX1327 strain into which ARE1 gene was introduced For the purpose of further introducing YEH1 gene into YEH1 locus on the genome of erg5 and erg6 double-disrupted strain SX1327 into which ARE1 gene was introduced The following steps were performed. The plasmid pPGL-YEH1 (-2 μ) for expression of the gene YEH1 encoding the Saccharomyces cerevisiae-derived steryl ester hydrolase was cleaved with the restriction enzyme NdeI (in the YEH1 gene). This DNA fragment was transformed into the SX1327 strain by the lithium acetate method or the electroporation method in the same manner as in Example 1 (2) above, and the SD agar medium (0.67% Yeast Nitrogen Base w / o Amino Acids (Difco) Manufactured), 2% glucose, 2% agar) and cultured at 30 ° C. for 3-5 days.
The leucine and uracil, histidine, and tryptophan non-requiring colonies grown on the agar medium are inoculated into 2 ml of YPD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose), and shaken at 30 ° C. for 16 hours. Culture was performed. Chromosomal DNA was extracted from this culture solution by the method described in Example 1 (1) above. Using this as a template, PCR was carried out using a combination of forward primer PPGK1-F (SEQ ID NO: 17) and reverse primer YEH1-RR (SEQ ID NO: 25), and the presence or absence of homologous recombination on the YEH1 locus was examined. PCR was performed under the conditions described in Example 1 (2) above.

  As a result of analyzing the PCR product by agarose gel electrophoresis, a DNA fragment of about 2.0 kbp having the same size as the target size was detected in the transformant named SX1333 (YEH1 locus). Therefore, the YEH1 gene was introduced by homologous recombination into the YEH1 locus of the SX1333 genome, and it was confirmed that two copies existed.

(5) Preparation of erg5, erg6 double disruption strain SX1335 strain not introducing ARE1, YEH1 gene In order to prepare a control strain not introducing ARE1, YEH1, yeast group prepared in Example 1 (5) above The replacement plasmid pUC-URA3 was cleaved with the restriction enzyme StuI. These DNA fragments were transformed into the erg5 and erg6 double disruption strain SX1312 prepared in Example 1 (3) by the lithium acetate method or the electroporation method in the same manner as in Example 1 (2). l Seed on SD agar medium (0.67% Yeast Nitrogen Base w / o Amino Acids (manufactured by Difco), 2% glucose, 2% agar) containing L-leucine and cultured at 30 ° C. for 3 to 5 days.
Uracil, histidine and tryptophan non-requiring colonies grown on an agar medium were inoculated into 2 ml of YPD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose) and cultured at 30 ° C. for 16 hours with shaking. Went. Chromosomal DNA was extracted from this culture solution by the method described in Example 1 (1) above. Using this as a template, PCR was performed with a combination of forward primer M13 forward (SEQ ID NO: 20) and reverse primer URA3-RR (SEQ ID NO: 29), and the presence or absence of homologous recombination on the ura3 locus was examined. PCR was performed under the conditions described in Example 1 (2) above.

  As a result of analyzing the PCR product by agarose gel electrophoresis, a DNA fragment of about 1.2 kbp having the same size as the target size was detected in the transformant named SX1329. Therefore, it was confirmed that the URA1 gene was complemented in the SX1329 strain by homologous recombination.

Next, the yeast recombination plasmid pPG-LEU (-2μ) was cleaved with the restriction enzyme Bsp1407I. This DNA fragment was transformed into the SX1329 strain prepared above by the lithium acetate method or the electroporation method in the same manner as in Example 1 (2), and the SD agar medium (0.67% Yeast Nitrogen Base w / o Amino) was transformed. Acids (manufactured by Difco), 2% glucose, 2% agar) and cultured at 30 ° C. for 3 to 5 days.

  The leucine and uracil, histidine, and tryptophan non-requiring colonies grown on the agar medium are inoculated into 2 ml of YPD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose), and shaken at 30 ° C. for 16 hours. Culture was performed. Chromosomal DNA was extracted from this culture solution by the method described in Example 1 (1) above. Using this as a template, PCR was performed with a combination of the forward primer PPGK1-R1 (SEQ ID NO: 26) and the reverse primer LEU2-R2 (SEQ ID NO: 27), and the presence or absence of homologous recombination on the leu2 locus was examined. PCR was performed under the conditions described in Example 1 (2) above.

  As a result of analyzing the PCR product by agarose gel electrophoresis, a DNA fragment of about 2.1 kbp having the same size as the target size was detected in the transformant named SX1335. Therefore, it was confirmed that the SX1335 strain was complemented with the LEU2 gene by homologous recombination.

(Example 3)
Production of ARE2 and YEH1-enhanced strains of erg5, erg6 double disruption strain SX1312 (1) Production of ARE2 gene introduction strain SX1342 in erg5, erg6 double disruption strain SX1312 (i) Preparation of ARE2 gene fragment for yeast expression Saccharomyces A DNA fragment containing the cerevisiae-derived acyl CoA: sterol acyltransferase-encoding gene (ARE2) was synthesized and isolated by PCR using the chromosomal DNA of Saccharomyces cerevisiae S288C obtained in Example 1 (1) as a template. did. Details of the method are shown below.
For the synthesis of the ARE2 gene fragment by PCR, the forward primer ARE2-SmaI (F) (SEQ ID NO: 32) and the reverse primer ARE2-EcoRI (R) (SEQ ID NO: 33) were used. This primer combination can be used to amplify the ARE2 protein coding region from the start codon to the end codon. Further, the restriction enzyme SmaI can be synthesized on the 5 ′ side and the EcoRI recognition sequence on the 3 ′ side. PCR was performed using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) (1) 98 ° C., 2 minutes, (2) 98 ° C., 10 seconds, (3) 55 ° C., 5 seconds, (4) 72 ° C. The reaction from (2) to (4) was repeated 30 times for 2 minutes, (5) the reaction was performed at 72 ° C. for 7 minutes, and then the reaction was performed at 4 ° C. under cooling conditions. Next, the PCR product was subjected to agarose gel electrophoresis, and an ARE2 gene fragment of about 1.93 kbp was confirmed.

(Ii) Preparation of ARE2 expression plasmid pPGL-ARE2 (-2μ) Plasmid pPG-LEU (-2μ) prepared in Example 1 (4) above was digested with restriction enzymes SmaI and EcoRI, and PCR-synthesized ARE2 A gene fragment cleaved with SmaI and EcoRI was prepared using TaKaRa DNA Ligation Kit Ver. 2 (manufactured by Takara Bio Inc.). This was transformed into Escherichia coli JM109, and a plasmid was extracted from colonies grown on LB agar medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 2% agar) containing 100 mg / l ampicillin. did. A plasmid in which about 5.4 kbp and 1.93 kbp fragments were confirmed by digestion with restriction enzymes SmaI and EcoRI by agarose gel electrophoresis was named pPGL-ARE2 (−2 μ).

(Iii) Preparation of ARE2 gene introduction strain SX1342 into erg5, erg6 double disruption strain SX1312 For the purpose of introducing the ARE2 gene into the ARE2 locus on the genome of erg5, erg6 double disruption strain SX1312, the following The process was performed. Saccharomyces cerevisiae-derived sterol acyl CoA: a plasmid pPGL-ARE2 (-2 μ) for expression of the gene ARE2 encoding sterol acyltransferase was cleaved with the restriction enzyme SalI (in the ARE2 gene). This DNA fragment was transformed into the SX1312 strain prepared in Example 1 (3) by the lithium acetate method or electroporation method as in Example 1 (2), and an SD agar medium containing 20 mg / l uracil. (0.67% Yeast Nitrogen Base w / o Amino Acids (manufactured by Difco), 2% glucose, 2% agar), and cultured at 30 ° C. for 3 to 5 days.
Colonies that do not require leucine, histidine, and tryptophan grown on an agar medium are inoculated into 2 ml of YPD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose), and cultured at 30 ° C. for 16 hours with shaking. Went. Chromosomal DNA was extracted from this culture solution by the method described in Example 1 (1) above. Using this as a template, PCR was performed with a combination of forward primer PPGK1-F (SEQ ID NO: 17) and reverse primer ARE2-R1 (SEQ ID NO: 34), and the presence or absence of homologous recombination on the ARE2 locus was examined. PCR was performed under the conditions described in Example 1 (2) above.

  As a result of analyzing the PCR product by agarose gel electrophoresis, a DNA fragment of about 2.6 kbp having the same size as the target size was detected in the transformant named SX1342. Therefore, the ARE2 gene was introduced into the ARE2 locus of the SX1342 genome by the homologous recombination, and it was confirmed that a total of 2 copies were present.

(2) Production of YEH1 gene-introduced strain SX1346 into SX1342 strain into which ARE2 gene has been introduced For the purpose of further introducing YEH1 gene into the YEH1 locus on the genome of erg5 and erg6 double-disrupted strain SX1342 into which ARE2 gene has been introduced The following steps were performed. Plasmid pPGU-YEH1 (-2μ) for expression of gene YEH1 encoding Saccharomyces cerevisiae-derived steryl ester hydrolase was cleaved with restriction enzyme Bsp1407I (in YEH1 gene). Moreover, in order to produce a control strain into which YEH1 had not been introduced, the yeast recombination plasmid pUC-URA3 produced in Example 1 (5) above was cleaved with the restriction enzyme StuI. These DNA fragments were transformed into the SX1342 strain prepared in (1) above by the lithium acetate method or the electroporation method in the same manner as in Example 1 (2) above, and the SD agar medium (0.67% Yeast Nitrogen Base) was transformed. W / o Amino Acids (manufactured by Difco), 2% glucose, 2% agar) and cultured at 30 ° C. for 3 to 5 days.

The leucine and uracil, histidine, and tryptophan non-requiring colonies grown on the agar medium are inoculated into 2 ml of YPD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose), and shaken at 30 ° C. for 16 hours. Culture was performed. Chromosomal DNA was extracted from this culture solution by the method described in Example 1 (1) above. Using this as a template, PCR was performed with a combination of forward primer PPGK1-F (SEQ ID NO: 17) and reverse primer YEH1-RR (SEQ ID NO: 25), and the presence or absence of homologous recombination on the YEH1 locus was examined. PCR was performed under the conditions described in Example 1 (2) above.
As a result of analyzing the PCR product by agarose gel electrophoresis, a DNA fragment of about 2.0 kbp having the same size as the target size was detected in the transformant named SX1346. Therefore, the YEH1 gene was introduced into the genome of the SX1346 strain by the above homologous recombination, and it was confirmed that two copies existed.

For a strain transformed with a DNA fragment obtained by cutting plasmid pUC-URA3 with StuI, forward primer M13 forward (SEQ ID NO: 20)
And reverse primer URA3-RR (SEQ ID NO: 29) were used for PCR, and the presence or absence of homologous recombination at the ura3 locus was examined. PCR was performed under the conditions described in Example 1 (2) above.

  As a result of analyzing the PCR product by agarose gel electrophoresis, a DNA fragment of about 1.2 kbp having the same size as the target size was detected in the transformant named SX1347. Therefore, it was confirmed that the URA3 gene was complemented in the SX1347 strain by homologous recombination.

Example 4
Saccharomyces cerevisiae FY1679-06c ARE1 and YEH1 enhanced strains (1) ARE1 gene-enhanced strain SX1330 to FY1679-06c strain Saccharomyces cerevisiae FY1679-06c strain ARE1 The following steps were carried out for the purpose of introduction. Saccharomyces cerevisiae-derived sterol acyl CoA: A plasmid pPGU-ARE1 (-2 μ) for expression of the gene ARE1 encoding sterol acyltransferase was cleaved with a restriction enzyme Xho I (in the ARE1 gene). This DNA fragment was transformed into FY1679-06c strain by the lithium acetate method or the electroporation method in the same manner as in Example 1 (2), and 20 mg / l L-histidine, tryptophan, and 100 mg / l L-leucine were obtained. The cells were seeded on an SD agar medium (0.67% Yeast Nitrogen Base w / o Amino Acids (manufactured by Difco), 2% glucose, 2% agar) and cultured at 30 ° C. for 3 to 5 days.

  The uracil non-requiring colonies grown on the agar medium were inoculated into 2 ml of YPD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose), and cultured with shaking at 30 ° C. for 16 hours. Chromosomal DNA was extracted from this culture solution by the method described in Example 1 (1) above. Using this as a template, PCR was performed with a combination of the forward primer PPGK1-F (SEQ ID NO: 17) and the reverse primer ARE1-check (SEQ ID NO: 18), and the presence or absence of homologous recombination on the ARE1 locus was examined. PCR was performed under the conditions described in Example 1 (2) above.

  As a result of analyzing the PCR product by agarose gel electrophoresis, a DNA fragment of about 2.6 kbp having the same size as the target size was detected in the transformant named SX1330. Therefore, the ARE1 gene was introduced into the ARE1 locus of the genome of the SX1330 strain by the homologous recombination, and it was confirmed that a total of 2 copies were present.

(2) Preparation of YEH1 gene-introduced strain SX1337 into SX1330 strain into which ARE1 gene was introduced The following steps were carried out for the purpose of further introducing YEH1 gene into the YEH1 locus on the genome of SX1330 strain into which ARE1 gene was introduced. It was. The plasmid pPGL-YEH1 (-2μ) for expression of the gene YEH1 encoding the Saccharomyces cerevisiae-derived steryl ester hydrolase was cleaved with the restriction enzyme NdeI (in the YEH1 gene). In order to prepare a control strain into which YEH1 had not been introduced, the yeast recombination plasmid pPG-LEU (-2 μ) prepared in Example 1 (4) (ii) was cleaved with the restriction enzyme Bsp1407 I. These DNA fragments were transformed into the SX1330 strain prepared in the above (1) by the lithium acetate method or the electroporation method in the same manner as in Example 1 (2), and each contained 20 mg / l L-histidine and tryptophan. The cells were seeded on an SD agar medium (0.67% Yeast Nitrogen Base w / o Amino Acids (Difco), 2% glucose, 2% agar) and cultured at 30 ° C. for 3 to 5 days.

The leucine and uracil non-requiring colonies grown on the agar medium are inoculated into 2 ml of YPD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose) and cultured with shaking at 30 ° C. for 16 hours. It was. Chromosomal DNA was extracted from this culture solution by the method described in Example 1 (1) above. Using this as a template, PCR was performed with a combination of forward primer PPGK1-F (SEQ ID NO: 17) and reverse primer YEH1-RR (SEQ ID NO: 25), and the presence or absence of homologous recombination on the YEH1 locus was examined. PCR was performed under the conditions described in Example 1 (2) above.

  As a result of analyzing the PCR product by agarose gel electrophoresis, a DNA fragment of about 2.0 kbp having the same size as the target size was detected in the transformant named SX1337. Therefore, the YEH1 gene was introduced into the genome of the SX1337 strain by the homologous recombination, and it was confirmed that two copies existed.

  In addition, for a strain transformed with a DNA fragment obtained by cutting plasmid pPG-LEU (-2μ) with Bsp1407I, forward primer PPGK1-R1 (SEQ ID NO: 26) and reverse primer LEU2-R2 (SEQ ID NO: 27). PCR was performed with the combination of the above, and the presence or absence of homologous recombination on the leu2 locus was examined. PCR was performed under the conditions described in Example 1 (2) above.

  As a result of analyzing the PCR product by agarose gel electrophoresis, a DNA fragment of about 2.1 kbp having the same size as the target size was detected in the transformant named SX1338. Therefore, it was confirmed that the SX1338 strain was complemented with the LEU2 gene by homologous recombination.

(3) Preparation of control strain SX1348 without ARE1 and YEH1 genes introduced In order to prepare a control strain without ARE1 and YEH1 introduced, plasmid pUC-URA3 for yeast recombination prepared in Example 1 (5) above was used. Was digested with the restriction enzyme StuI. This DNA fragment was transformed into FY1679-06c strain by the lithium acetate method or the electroporation method in the same manner as in Example 1 (2), and 20 mg / l L-histidine, tryptophan, and 100 mg / l L-leucine were obtained. SD agar medium containing (0.67% Yeast
Nitrogen Base w / o Amino Acids (manufactured by Difco), 2% glucose, 2% agar) and cultured at 30 ° C. for 3 to 5 days.

  The uracil non-requiring colonies grown on the agar medium were inoculated into 2 ml of YPD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose), and cultured with shaking at 30 ° C. for 16 hours. Chromosomal DNA was extracted from this culture solution by the method described in Example 1 (1) above. Using this as a template, PCR was performed with a combination of forward primer M13 forward (SEQ ID NO: 20) and reverse primer URA3-RR (SEQ ID NO: 29), and the presence or absence of homologous recombination on the ura3 locus was examined. PCR was performed under the conditions described in Example 1 (2) above.

As a result of analyzing the PCR product by agarose gel electrophoresis, a DNA fragment of about 1.2 kbp having the same size as the target size was detected in the transformant named SX1343. Therefore, it was confirmed that the URA1 gene was complemented in the SX1343 strain by homologous recombination.
Next, the yeast recombination plasmid pPG-LEU (-2μ) was cleaved with the restriction enzyme Bsp1407I. This DNA fragment was transformed into the SX1343 strain prepared above by the lithium acetate method or the electroporation method in the same manner as in Example 1 (2), and an SD agar medium containing 20 mg / l L-histidine and tryptophan ( 0.67% Yeast Nitrogen Base w / o Amino Acids (manufactured by Difco), 2% glucose, 2% agar) and cultured at 30 ° C. for 3 to 5 days.

The leucine and uracil non-requiring colonies grown on the agar medium are inoculated into 2 ml of YPD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose) and cultured with shaking at 30 ° C. for 16 hours. It was. Chromosomal DNA was extracted from this culture solution by the method described in Example 1 (1) above. Using this as a template, PCR was performed with a combination of the forward primer PPGK1-R1 (SEQ ID NO: 26) and the reverse primer LEU2-R2 (SEQ ID NO: 27), and the presence or absence of homologous recombination on the leu2 locus was examined. PCR was performed under the conditions described in Example 1 (2) above.
As a result of analyzing the PCR product by agarose gel electrophoresis, a DNA fragment of about 2.1 kbp having the same size as the target size was detected in the transformant named SX1348. Therefore, it was confirmed that the LEU2 gene was complementary to the SX1348 strain by homologous recombination.

(Example 5)
Analysis of sterols of various strains with enhanced ARE1 and YEH1 genes (1) Culture of various yeast strains and analysis of sterols 2 ml of various strains prepared in Examples 1 to 4 using 0.5% glucose as a carbon source 1 platinum ear inoculated into a YPAD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose, 40 mg / l adenine), and cultured at 30 ° C. for 18 hours to form an inoculum. From this yeast culture solution, an amount of culture solution having an initial OD660 = 0.2 was recovered, and centrifuged at 4 ° C., 10,000 rpm for 3 minutes to remove the culture supernatant. 0.1 ml of 0.85% physiological saline was added to the cell pellet, suspended, and 5 ml of YPAD liquid medium (1% yeast extract, 2% polypeptone, 2% glucose, 40 mg / 40%) using 2% glucose as a carbon source. l adenine) and cultured at 30 ° C. for 72 hours. Centrifugation was performed with 0.5 ml of yeast culture solution at 4 ° C., 14,000 rpm for 3 minutes, and the cells were collected. 0.4 ml of ethanol and 0.02 ml of 40% potassium hydroxide aqueous solution were added to the cell pellet, and the mixture was shaken at 80 ° C. for 90 minutes to saponify the sterol ester accumulated in the cell. This liquid was subjected to a vacuum concentrator at 35 ° C. and concentrated to dryness.
The obtained concentrated dried product is suspended by adding 0.1 ml of ethanol and 0.05 ml of sterilized water, further adding 0.8 ml of heptane and mixing vigorously, and then centrifuging at 14,000 rpm for 1 minute at room temperature. Separated into a layer and an oil layer. The upper heptane layer was taken in another tube, and 0.1 ml of ethanol and 0.8 ml of heptane were added again to the remaining aqueous layer, and the extraction operation was repeated. 0.04 ml of sterilized water was added to about 1.6 ml of the extracted heptane solution, mixed vigorously and then centrifuged again at 14,000 rpm for 1 minute at room temperature to separate into an aqueous layer and an oil layer. About 1.6 ml of a heptane layer was collected and concentrated in a vacuum concentrator at 35 ° C. to dryness. The obtained concentrated dried product was redissolved in 0.2 ml toluene, and sterols were analyzed by gas chromatography using testosterone as an internal standard. The results are shown in FIGS.

(2) Analysis of sterols of erg5 and erg6 double disruption strains with enhanced ARE1 and YEH1 genes As shown in FIG. 1 (a), erg5 and erg6 double disruption strains with only ARE1 produced using YPH499 as a parent strain In contrast to (SX1316), in the erg5 and erg6 double disruption strain (SX1317) in which ARE1 and YEH1 were enhanced, it was found that the ZYMO accumulation amount decreased and the TEOL accumulation amount increased. From this, it was clarified that by enhancing YEH1 under the enhancement of ARE1, ZYMO, which is a precursor of TEOL, is reduced, and accumulation of TEOL is improved.
On the other hand, as shown in FIG. 2 (a), erg5 and erg6 double disruption strains (SX1319) that enhanced only ARE1 produced using FY1679-06c as a parent strain were further enhanced against erg5 and erg6 double disruptions that enhanced YEH1. Also in the strain (SX1320), the ZYMO accumulation amount decreased and the TEOL accumulation amount increased. From these facts, it was revealed that by enhancing YEH1 under the enhancement of ARE1, ZYMO, which is a precursor of TEOL, is reduced and TEOL accumulation is improved even when FY1670-06c is used as a parent strain.

Next, as shown in FIG. 3 (a), erg5 and erg6 double disruption strains (SX1321) that only enhanced ARE1 produced using KA311A as a parent strain were further compared to erg5 and erg6 double disruption strains that further enhanced YEH1. Even in (SX1322), the ZYMO accumulation amount decreased and the TEOL accumulation amount increased. From these facts, it was revealed that by enhancing YEH1 under ARE1 enhancement, ZYMO, which is a TEOL precursor, is reduced and TEOL accumulation is improved even when KA311A is used as a parent strain.
Furthermore, as shown in FIG. 4 (a), erg5 and erg6 double disruption strains (SX1350) that enhanced only ARE1 produced using X2181-1B as a parent strain, and erg5 and erg6 double disruptions that further enhanced YEH1. Also in the strain (SX1351), the ZYMO accumulation amount decreased and the TEOL accumulation amount increased. From these facts, it was revealed that by enhancing YEH1 under the enhancement of ARE1, ZYMO, which is a TEOL precursor, is reduced and TEOL accumulation is improved even when X2181-1B is used as a parent strain.

(3) Analysis of sterols of erg5 and erg6 double disruption strains in which ARE1 and YEH1 having different expression loci were enhanced As shown in FIG. 5 (a), In the enhanced erg5, erg6 double disruption, and ARE1-enhanced strains, the strain in which YEH1 was expressed in any of YEH1, URA3, and LEU2 locus showed equivalent TEOL accumulation. From this, it has been clarified that, regardless of the locus of YEH1 expression enhancement under ARE1 enhancement, ZYMO, which is a precursor of TEOL, is reduced, and TEOL accumulation is improved.
On the other hand, as shown in FIG. 6 (a), in the erg5, erg6 double disruption, and YEH1-enhanced strains in which ARE1 having different expression locus was enhanced and FY1679-06c was produced as a parent strain, ARE1 was expressed in either ARE1 or URA3 locus. The expressed strain also showed equivalent TEOL accumulation. From this, it was clarified that the expression of ARE1 under any Ycus1 enhancement causes the ZOLMO precursor TEOL to be reduced and the TEOL accumulation to be improved.

(4) Analysis of sterols of erg5 and erg6 double disruption strains with enhanced ARE2 and YEH1, as shown in FIG. 7 (a), erg5 and erg6 double disruption with only ARE2 prepared using FY1679-06c as a parent strain The erg5 and erg6 double disruption strain (SX1346) further enhanced YEH1 compared to the strain (SX1347) also decreased the ZYMO accumulation amount and increased the TEOL accumulation amount. From these facts, it is clear that by enhancing YEH1 under ARE2 enhancement, ZYMO which is a precursor of TEOL is reduced and TEOL accumulation is improved, even when ARE2 is used as a sterol acyl CoA: sterol acyl transferase. became.

(5) Analysis of sterols of strains in which ARE1 and YEH1 were enhanced As shown in FIG. 8 (a), YEH1 was further enhanced with respect to a strain (SX1338) in which only ARE1 produced using FY1679-06c as a parent strain was enhanced. In the same strain (SX1337), the ZYMO accumulation amount decreased and the ERGO accumulation amount increased. From these facts, it is clear that by enhancing YEH1 under ARE1 enhancement, ZYMO, which is a precursor of TEOL, is reduced and TEOL accumulation is improved, even when a strain without erg5erg6 double disruption is used. became.

(Example 6)
Analysis of transcription amounts of various strains with enhanced ARE1 and YEH1 genes (1) Preparation of total RNA from yeast For the various strains prepared in Example 5, RNeasy plus Mini Kit and RNase-Free DNase Set (both manufactured by QIAGEN) ) Was used to prepare total RNA as follows.
After culturing the microbial cells in the same manner as in Example 5, a yeast microbial cell solution was prepared using the collected culture solution so as to have 2 × 10 ⁷ cells (OD660 = 1 was about 1.5 × 10 ^7). The cell number obtained by centrifuging at 4 ° C., 14,000 rpm for 1 minute is suspended in 0.85% physiological saline and centrifuged again at 4 ° C., 10,000 rpm for 2 minutes. Separation was performed. The obtained cells are suspended in 0.6 ml of Buffer RLT attached to the kit and 6 μl of 2-mercaptoethanol, and this suspension is transferred to a tube of MagNA Lyser Green Beads (Roche), MagNA Lyser (Roche). ) At room temperature at 6,000 rpm for 1 minute. This cell disruption solution was placed in a genomic DNA-removed mini spin column, and total RNA was prepared according to the instruction manual attached to the RNeasy plus Mini Kit. As an option, 0.08 ml of DNase mix solution was added after column adsorption of total RNA, and allowed to stand at room temperature for 15 minutes to decompose chromosomal DNA. Finally, RNase-free water kept at 50 ° C. was added, allowed to stand at room temperature for 1 minute, and total RNA was eluted by centrifugation.

(2) Reverse Transcription Reaction Using the total RNA prepared in (1) above as a template, SuperScript III First-Strand Synthesis System for RT-PCR (manufactured by Invitrogen) is used to perform the reverse transcription reaction according to the attached instruction manual. To synthesize single-stranded cDNA. In the reverse transcription reaction, 300 ng of total RNA and 50 μM Oligo (dT) ₂₀ were used as primers.

(3) Quantitative PCR
Quantitative PCR reaction is performed according to the attached instruction manual using SYBR premix Ex Taq (Perfect Real Time) (manufactured by Takara Bio Inc.). (1) 95 ° C., 10 seconds, (2) 95 ° C. The reaction of (3) 60 ° C., 20 seconds, (2) to (3) was repeated 40 times for 5 seconds. The temperature change rate was 20 ° C./sec. Melting curve analysis was performed at (1) 95 ° C., 0 seconds, (2) 65 ° C., 15 seconds, (3) 95 ° C., 0 seconds, and the temperature change rate was 20 ° C./second. As PCR primers, ARE1-F (SEQ ID NO: 35) and ARE1-R (SEQ ID NO: 36) are used for quantitative PCR of ARE1, and ARE2-F (SEQ ID NO: 37) and ARE2-R (SEQ ID NO: 38) are used for quantitative PCR of ARE2. ), YEH1-F (SEQ ID NO: 39) and YEH1-R (SEQ ID NO: 40) were used for quantitative PCR of YEH1. In addition, ACT1 encoding actin was used as an expression control, and ACT1-F (SEQ ID NO: 41) and ACT1-R (SEQ ID NO: 42) were used as primers.
For quantitative analysis, Light-Cycler manufactured by Roche Diagnostics, and Light-Cycler Software Ver. 3.5, Fit
Analysis was performed using the Point method. The results are shown in FIGS. 1 and 2 (b) and (c).

(4) Analysis of transcription amount of erg5 and erg6 double disruption strains with enhanced ARE1 and YEH1 genes As shown in FIGS. 1 (b) and (c), strains having Yerg499 erg5 and erg6 double disruption strains as parent strains As a result of the transcription analysis in SX1316, the amount of ARE1 transferred was increased in SX1316 in which only ARE1 was enhanced, confirming that expression was enhanced. Moreover, since the transcription amounts of ARE1 and YEH1 of SX1317 in which both ARE1 and YEH1 were enhanced were also increased compared to SX1313, it was confirmed that the expression of both genes was enhanced.
Similarly, as a result of transcriptional analysis in strains in which the erg5 and erg6 double disruption strains of FY1679-06c are parent strains (FIGS. 2 (b) and 2 (c)), SX1319, which only enhanced ARE1, was compared to SX1312. It was confirmed that the amount of transcription of ARE1 and YEH1 was increased in SX1320 in which the amount of transcription increased and both ARE1 and YEH1 were enhanced.

Subsequently, as a result of transcription analysis in the KA311A erg5, erg6 double disruption strain as a parent strain (FIGS. 3 (b) and 3 (c)), SX1321 in which only ARE1 is enhanced with respect to SX1307 is the transcription amount of ARE1. It was confirmed that SX1322 in which both ARE1 and YEH1 were enhanced increased the transcription amounts of ARE1 and YEH1.
Furthermore, as a result of transcription analysis in a strain in which the erg5 and erg6 double disruption strains of X2181-1B are parent strains (FIGS. 4 (b) and (c)), SX1350 in which only ARE1 is enhanced relative to SX1353 is transcription of ARE1. It was confirmed that the amount of transcription of ARE1 and YEH1 was increased in SX1351 in which the amount was increased and both ARE1 and YEH1 were enhanced.

(5) Analysis of transcription amount of erg5 and erg6 double disruption strains with enhanced ARE1 and YEH1 with different expression loci Strengthening YEH1 with different expression locus as the parent strain of erg5 and erg6 double disruption and ARE1 enhancement strains of FY1679-06c Results of transcription analysis in the strains (Figs. 5 (b) and (c)), SX1319, SX1320 (YEH1 locus), SX1332 (URA3 locus), and SX1325 (LEU2 locus) all strains of YEH1 transcription Was confirmed to increase.

  Furthermore, as a result of transcriptional analysis in strains in which ARE5 and erg6 double disruption strains of FY1679-06c were parental strains and ARE1 having different expression locus was enhanced together with YEH1 (FIGS. 6 (b) and (c)), SX1335, It was confirmed that both SX1320 (ARE1 locus) and SX1333 (URA3 locus) strains increased the transcription amounts of ARE1 and YEH1.

(6) Analysis of transcription amount of erg5 and erg6 double disruption strains with enhanced ARE2 and YEH1 Results of transcription analysis in strains of FY1679-06c using erg5 and erg6 double disruption strains as parent strains (FIG. 7 (b), ( c)) Compared with SX1335, it was confirmed that SX1347 enhanced only ARE2 increased the transcription amount of ARE2, and SX1346 enhanced both ARE2 and YEH1 increased the transcription amounts of ARE2 and YEH1. .

(7) Analysis of transcription amount of strains with enhanced ARE1 and YEH1 Results of transcription analysis in strains with FY1679-06c as the parent strain (FIGS. 8 (b) and (c)), only ARE1 was enhanced relative to SX1348 It was confirmed that SX1338 increased the transcription amount of ARE1, and SX1337 which enhanced both ARE1 and YEH1 increased the transcription amounts of ARE1 and YEH1.

  Table 1 shows numerical data of FIGS. 1A, 1B, and 1C. The numerical value is shown as (A) mg of sterol accumulation per 1 g of dry cell weight. (B) Expression ratios of ARE1 and ACT1, and YEH1 and ACT1 are shown as a transcription amount ratio to ACT1, respectively.

  Table 2 shows numerical data of FIGS. 2 (a), (b), and (c). The numerical value is shown as (A) mg of sterol accumulation per 1 g of dry cell weight. (B) Expression ratios of ARE1 and ACT1, and YEH1 and ACT1 are shown as a transcription amount ratio to ACT1, respectively.

Table 3 shows numerical data in FIGS. 3A, 3B, and 3C. The numerical value is shown as (A) mg of sterol accumulation per 1 g of dry cell weight. (B) Expression ratios of ARE1 and ACT1 and YEH1 and ACT1 are shown as the transcription amount ratio to ACT1, respectively.

  Table 4 shows numerical data of FIGS. 4A, 4B, and 4C. The numerical value is shown as (A) mg of sterol accumulation per 1 g of dry cell weight. (B) Expression ratios of ARE1 and ACT1, and YEH1 and ACT1 are shown as a transcription amount ratio to ACT1, respectively.

  Table 5 shows numerical data of FIGS. 5A, 5B, and 5C. The numerical value is shown as (A) mg of sterol accumulation per 1 g of dry cell weight. (B) Expression ratios of ARE1 and ACT1, and YEH1 and ACT1 are shown as a transcription amount ratio to ACT1, respectively.

  Table 6 shows numerical data of FIGS. 6 (a), (b), and (c). The numerical value is shown as (A) mg of sterol accumulation per 1 g of dry cell weight. (B) Expression ratios of ARE1 and ACT1, and YEH1 and ACT1 are shown as a transcription amount ratio to ACT1, respectively.

  Table 7 shows numerical data of FIGS. 7 (a), (b), and (c). The numerical value is shown as (A) mg of sterol accumulation per 1 g of dry cell weight. (B) Expression ratios of ARE2 and ACT1, and YEH1 and ACT1 are shown as the transcription ratio for ACT1, respectively.

  Table 8 shows numerical data of FIGS. 8A, 8B, and 8C. The numerical value is shown as (A) mg of sterol accumulation per 1 g of dry cell weight. (B) Expression ratios of ARE1 and ACT1, and YEH1 and ACT1 are shown as a transcription amount ratio to ACT1, respectively.

  The Δ5,7-sterol produced by the present invention is useful in the pharmaceutical field, cosmetic field, food field and the like.

Claims (13)

Acyl CoA: yeast modified to enhance expression of sterol acyltransferase gene and steryl ester hydrolase gene. Furthermore, the yeast of Claim 1 modified so that the expression of a sterol C-22 desaturase gene (ERG5) and a sterol C-24 methyltransferase gene (ERG6) may be reduced. Furthermore, the yeast of Claim 1 modified | changed so that the expression of a sterol C-22 desaturase gene (ERG5) might be reduced. Furthermore, Δ7-reducing activity, activity of cleaving the bond between positions 20 and 22 of the sterol side chain, sterol 3-position oxidative isomerization activity, steroid 17α hydroxylation activity, steroid 21-position hydroxylation activity, and steroid 11β-position water The yeast according to claim 3, which has been modified to have at least one activity selected from oxidative activity. Further, Δ7-reducing activity, activity of cleaving the bond between positions 20 and 22 of the sterol side chain, sterol 3-position oxidative isomerization activity, steroid 17α hydroxylation activity, steroid 21-position hydroxylation activity, and steroid 11β-position water The yeast according to claim 3, which has been modified to have an oxidative activity. The yeast according to any one of claims 1 to 5, which is Saccharomyces cerevisiae. The yeast according to claim 6, wherein the parent strain is Saccharomyces cerevisiae YPH499 strain, Saccharomyces cerevisiae FY1679-06c strain, Saccharomyces cerevisiae KA311A strain or Saccharomyces cerevisiae X2181-1B strain. A method for producing Δ5,7-sterol, wherein the yeast according to any one of claims 1 to 3 is cultured to produce and accumulate Δ5,7-sterol and to recover Δ5,7-sterol. The method of Claim 8 whose yeast to culture | cultivate is the yeast of Claim 1 or 2, and (DELTA) 5,7-sterol is C5,7-diene sterol. 10. The method of claim 9, wherein the C5,7-diene sterol is cholesta-5,7,24-trien-3 [beta] -ol or 7-dehydrocholesterol. The method according to claim 8, wherein the yeast to be cultured is the yeast according to claim 1 or 3, and Δ5,7-sterol is ergosta-5,7,24-trienol. The method according to claim 8, wherein the yeast to be cultured is the yeast according to claim 1, and Δ5,7-sterol is ergosterol. A method for producing hydrocortisone, comprising culturing the yeast according to claim 5 to produce and accumulate hydrocortisone, and recover hydrocortisone.

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