Method for producing squalene using yeast

Abstract

Disclosed are: yeast which is capable of highly producing squalene, the yeast being genetically modified in such a manner that the expression or activity of at least one enzyme in the ergosterol biosynthesis pathway can be increased or decreased and being also genetically modified in such a manner that the expression of a protein involved in the synthesis of a cell wall can be increased or decreased; and a method for producing squalene, the method including a step for culturing the yeast in a culture medium.

Description

Method for producing squalene using yeast

[0001] This disclosure relates to yeast and a method for producing squalene using the yeast.

[0002] Squalene is a 30-carbon hydrocarbon widely present in eukaryotes, including humans. Squalene and squalane, derived from squalene, have moisturizing properties and are used as natural oils in skincare and cosmetics. Because they are biologically derived and have few side effects, they are also used as adjuvants to enhance the immune activity of vaccines. Furthermore, due to their physical properties, they can be repurposed as lubricants such as engine oil and fuels.

[0003] When considering the industrial production of squalene, the limited availability of natural resources is a major problem. Currently, it is extracted from shark liver and olive oil, but shark catches are limited due to conservation concerns, and while olive oil is in high demand as a food product, olive production is also subject to the unpredictable influence of weather conditions.

[0004] Given this situation, production methods using genetically modified microorganisms such as yeast are being employed. This involves genetically modifying the yeast to produce large quantities of squalene, collecting the yeast after growth, and extracting the squalene from within by destroying the cells. Such methods for producing squalene using yeast are reported in Patent Documents 1 to 3.

[0005] Patent Document 1 reports a method for producing squalene using ergosterol-metabolizing mutant yeast that accumulates squalene, characterized by culturing the yeast in a culture medium containing sterols and yeast extract and urea as nitrogen sources, and then collecting squalene from the culture.

[0006] Patent Document 2 reports a process for preparing a squalene-containing product, the process comprising: (a) obtaining purified squalene from yeast that produces squalene in high yield during cultivation; and (b) combining the squalene from step (a) with a non-squalene component to form the squalene-containing product.

[0007] Patent Document 3 reports a method for producing squalene using genetically modified yeast, comprising increasing or decreasing the activity or expression of one or more enzymes in the isoprenoid biosynthesis pathway, wherein the enzyme activity or expression is increased or decreased by one or more design mutations, the one or more design mutations are located at a predetermined position within the enzyme, and the genetically modified yeast produces an increased amount of isoprenoids compared to natural yeast.

[0008] However, there is room for improvement in terms of cost when it comes to production using microorganisms. First, there are costs associated with collecting the cells (cell harvesting). One method of cell harvesting is centrifugation, but this requires the cost of introducing a large centrifuge and electricity. Another method involves using flocculating yeast, but the squalene produced may hinder efficient flocculation, making it not a versatile method. Next, there are costs associated with disrupting the cells. Physical shearing involves the cost of introducing equipment and power, while enzyme addition involves the cost of purchasing enzymes. Finally, there are also the costs of processing the cell residue after extraction.

[0009] Japanese Patent Publication No. 4-311393, Japanese Patent Publication No. 2015-61546, Japanese Patent Publication No. 2017-140037

[0010] This disclosure aims to provide a method for producing squalene using yeast.

[0011] To reduce the cost of extracting squalene from yeast, it is conceivable to create yeast that secretes squalene outside the cell rather than accumulating it internally.

[0012] As a result of diligent research to achieve the above objective, the inventors have discovered that, in order to produce high levels of squalene, it is possible to secrete squalene outside the cell rather than storing it inside the cell, not only by modifying the genes of the yeast squalene metabolic system, but also by modifying the genes involved in cell wall synthesis. To date, there have been no reports that inhibiting cell wall synthesis in yeast can promote the secretion of intracellular lipids.

[0013] This disclosure is a further refinement of these findings and includes, for example, the following subjects:

[0014] Item 1. A yeast that produces a large amount of squalene, wherein the gene has been modified to increase or decrease the expression or activity of at least one enzyme in the ergosterol biosynthesis pathway, and further, the gene has been modified to increase or decrease the expression of a protein involved in cell wall synthesis. Item 2. The yeast described in Item 1, which has the effect of promoting the secretion of squalene into the extracellular space. Item 3. The enzymes mentioned above are acetyl-CoA acetyltransferase (ERG10), 3-hydroxy-3-methylglutaryl-CoA synthase (ERG13), HMG1, HMG2, mevalonate kinase (ERG12), phosphomevalonate kinase (ERG8), mevalonate pyrophosphate decarboxylase (ERG19), isopentenyl diphosphate δ-isomerase (Idi1), kinase farnesyl diphosphate synthase (ERG20), squalene synthase (ERG9), squalene epoxidase (ERG1), lanosterol synthase (ERG7), and lanosterol 14-α-demethylase. The yeast according to claim 1 or 2, wherein at least one selected from the group consisting of (ERG11), C-14 sterol reductase (ERG24), C-4 methylsterol oxidase (ERG25), C-3 sterol dehydrogenase (ERG26), 3-ketosterol reductase (ERG27), sterol 24-C-methyltransferase (ERG6), C-8 sterol isomerase (ERG2), C-5 sterol desaturase (ERG3), C-22 desaturase (ERG5), C-24 sterol reductase (ERG4), acetyl-CoA carboxylase, and ATP citrate lyase. Claim 4. The yeast according to any one of claims 1 to 3, wherein cell wall synthesis is inhibited by increased or decreased expression of proteins involved in cell wall synthesis. Claim 5. The yeast according to any one of claims 1 to 4, wherein the protein involved in the synthesis of the cell wall is at least one selected from the group consisting of proteins encoded by FKS1, FKS2, DAN1, PAU24, PAU2, HES1, SET4, PBI1, TIR1, TIR2, TIR3, TIR4, YSR3, HSP30, FHN1, PAU9, EMT1, FMP49, FDH2, and UPC2.6. The yeast according to any one of items 1 to 5, wherein the yeast belongs to at least one genus selected from the group consisting of Saccharomyces, Candida, Kluiveromyces, Pichia, Schizosaccharomyces, Torlaspora, and Jarrowvia. 7. A method for producing squalene, comprising the step of culturing the yeast according to any one of items 1 to 6 in a culture medium. 8. The method according to item 7, further comprising the step of recovering squalene from the culture medium.

[0015] This disclosure provides a yeast that secretes squalene outside the cell rather than accumulating it internally, and a method for producing squalene using this yeast. The yeast and method of this disclosure allow for the extraction of squalene without separating and disrupting cells, thus eliminating problems such as the cost of centrifugation for separating the yeast, the cost of reagents such as enzymes and physical disruption for disrupting the yeast, and the cost of residue disposal. Furthermore, the yeast and method of this disclosure allow for the reuse of the yeast because it is not necessary to kill it.

[0016] This is a conceptual diagram of transformation by pYK4. It shows the squalene biosynthesis pathway and the genetic modification sites. The graph shows the amount of squalene in each strain (top: intracellular squalene, bottom: cell wall squalene). The vertical axis represents squalene per dry weight. WT (KY767), UHSQ (KY1233), UHSQΔfks1 (KY1359), values ​​are mean ± SD. This is a conceptual diagram of the production of n=3 self-cloning yeast.

[0017] The embodiments included in this disclosure will be described in more detail below. This disclosure preferably includes, but is not limited to, yeast, methods for producing squalene using such yeast, and encompasses everything disclosed herein and recognizable to those skilled in the art.

[0018] In this disclosure, “gene” includes double-stranded DNA, single-stranded DNA (sense strand or antisense strand), and fragments thereof, unless otherwise specified. In this disclosure, “gene” refers to regulatory regions, coding regions, exons, and introns without distinction, unless otherwise specified.

[0019] The genes used in this disclosure include not only those with known nucleotide sequences registered in databases, but also their degenerate forms and variants. Preferably, the variants encode proteins with equivalent biological activity to the protein composed of the amino acid sequence encoded by the gene. Examples of proteins with equivalent biological activity include proteins derived from other organisms.

[0020] Examples of mutants include (1) genes encoding proteins whose amino acid sequences consist of amino acid sequences in which one or more amino acids, for example, 1 to 50, 1 to 25, 1 to 12, 1 to 9, or 1 to 5 amino acids are substituted, deleted, or added in the amino acid sequences registered in the aforementioned database, and (2) genes whose nucleotide sequences have 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the nucleotide sequences registered in the aforementioned database.

[0021] The identity of nucleotide sequences can be calculated using commercially available analysis tools or tools accessible via telecommunication lines (the Internet). The percentage of nucleotide sequence identity can be determined using a program commonly used in the field (e.g., BLAST, FASTA, etc.) with default settings.

[0022] "Genetically modified yeast" refers to yeast into which genes have been introduced from an external source, or yeast in which the gene sequence has been altered to differ from that of natural (wild-type) yeast.

[0023] The yeast disclosed herein is a squalene-producing yeast whose genes have been modified to increase or decrease the expression or activity of at least one enzyme in the ergosterol biosynthesis pathway, and which has been further modified to increase or decrease the expression of a protein involved in cell wall synthesis (hereinafter sometimes referred to as "the yeast disclosed herein").

[0024] Squalene is a carbon atom with an unsaturated bond. ３０ H５０ It refers to an acyclic triterpene hydrocarbon (molecular weight = 410.73), which is a polyunsaturated compound called 2,6,10,15,19,23 - hexamethyl - 2,6,10,14,18,22 - tetracos - hexane or squalene (CAS registration number 111 - 02 - 4). Squalene has the following structure.

[0025] In the present disclosure, the parent yeast to be modified by genes is not particularly limited, and any yeast can be used. As the parent yeast, natural (wild - type) yeast, mutant strains treated with drugs, ultraviolet rays, radiation, etc., and yeast with modified genes can be used.

[0026] Specific examples of the types of parent yeast include, for example, yeasts belonging to the genus Saccharomyces, Candida, Kluyveromyces, Pichia, Schizosaccharomyces, Torulaspora, Yarrowia, etc. These yeasts can be used alone or in combination of two or more.

[0027] Among such yeasts, yeasts belonging to the genus Saccharomyces and Yarrowia are preferably used. As the yeast belonging to the genus Saccharomyces, Saccharomyces cerevisiae is particularly preferably used, and as the yeast belonging to the genus Yarrowia, Yarrowia lipolytica is particularly preferably used.

[0028] The yeast disclosed herein is a squalene-producing yeast. Here, a squalene-producing yeast means a yeast that produces more squalene than a natural (wild-type) yeast of the same species. Here, high production refers to cases where the amount of squalene produced is, for example, 10 times or more, 50 times or more, 100 times or more, 200 times or more, 300 times or more, 400 times or more, 500 times or more, 600 times or more, 700 times or more, 800 times or more, 900 times or more, 1000 times or more, etc., compared to a natural (wild-type) yeast of the same species. Furthermore, for yeasts that produce a large amount of squalene, examples of squalene production (mass%) per unit weight of dry yeast include 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, and 40% or more.

[0029] The yeast described herein has been genetically modified to increase or decrease the expression or activity of at least one enzyme in the ergosterol biosynthesis pathway, with the aim of increasing squalene production. The enzyme to be genetically modified is not particularly limited as long as it can produce squalene in large quantities. For example, in the yeast ergosterol biosynthesis pathway, the expression or activity of an enzyme in the upstream pathway leading to squalene synthesis can be increased by genetic modification, thereby increasing the amount of squalene synthesized. Alternatively, in the yeast ergosterol biosynthesis pathway, the expression or activity of an enzyme in the downstream pathway leading to squalene can be decreased by genetic modification, thereby suppressing further metabolism of synthesized squalene and preventing a decrease in the amount of squalene.

[0030] Enzymes involved in the biosynthesis of squalene include, for example, acetyl-CoA acetyltransferase (ERG10), 3-hydroxy-3-methylglutaryl-CoA synthase (ERG13), HMG1 (3-hydroxy-3-methylglutaryl-CoA reductase), HMG2 (3-hydroxy-3-methylglutaryl-CoA reductase 2), ERG9 (squalene synthase), mevalonate kinase (ERG12), phosphomevalonate kinase (ERG8), mevalonate pyrophosphate decarboxylase (ERG19), isopentenyl-diphosphate δ-isomerase (Idi1), kinase farnesyl pyrophosphate synthase (ERG20), squalene synthase (ERG9), and the like.

[0031] Enzymes involved in converting squalene to ergosterol include, for example, squalene epoxidase (ERG1), lanosterol synthase (ERG7), lanosterol 14-α-demethylase (ERG11), C-14 sterol reductase (ERG24), C-4 methylsterol oxidase (ERG25), C-3 sterol dehydrogenase (ERG26), 3-ketosterol reductase (ERG27), sterol 24-C-methyltransferase (ERG6), C-8 sterol isomerase (ERG2), C-5 sterol desaturase (ERG3), C-22 desaturase (ERG5), C-24 sterol reductase (ERG4), and the like.

[0032] Other enzymes involved in the metabolism of squalene include, for example, acetyl-CoA carboxylase, ATP citrate lyase, and the like. Since acetyl-CoA is led to fatty acid biosynthesis by acetyl-CoA carboxylase, the gene may be modified so that the expression or activity of acetyl-CoA carboxylase is decreased so that more acetyl-CoA is used for the synthesis of squalene. Since acetyl-CoA is generated by the reaction of ATP citrate lyase, the gene may be modified so that the expression or activity of ATP citrate lyase is increased so that more acetyl-CoA is used for the synthesis of squalene.

[0033] The level of enzyme expression or activity can be increased or decreased by modifying the yeast gene using various known methods. Increases or decreases in enzyme expression include increases or decreases in the transcription of the enzyme-encoding mRNA, and increases or decreases in the translation of the enzyme mRNA.

[0034] The expression level of an enzyme can be reduced by deleting part or all of the region of the gene encoding the enzyme on the chromosome, or by modifying the promoter or expression regulatory sequences such as the Kozak sequence. The expression level can also be reduced by modifying non-coding regions other than the expression regulatory sequences. Furthermore, the entire gene encoding the enzyme on the chromosome may be deleted. Additionally, gene recombination can be used to introduce amino acid substitutions (missense mutations), stop codons (nonsense mutations), or frameshift mutations involving the addition or deletion of one or two bases into the enzyme-coding region on the chromosome. Finally, the expression level can also be reduced by substituting a less active promoter.

[0035] The level of enzyme activity can be reduced in yeast by deleting a portion of the coding region of the gene encoding the enzyme on a chromosome, or by inserting other nucleic acid sequences into these regions. When deleting a coding region, the deleted region can be the N-terminal, internal, or C-terminal region, as long as it reduces the activity of the enzyme produced. Similarly, when inserting other nucleic acid sequences into a coding region, the insertion site can be the N-terminal, internal, or C-terminal region, as long as it reduces the activity of the enzyme produced.

[0036] To modify the gene encoding the enzyme on the chromosome as described above, for example, a modified gene is created to reduce its expression or activity, and homologous recombination is induced between the modified gene and the gene on the parent yeast chromosome, thereby replacing the naturally occurring enzyme-coding gene on the chromosome with the modified gene. Gene disruption by gene substitution using such homologous recombination can be carried out by conventional methods.

[0037] The expression level of an enzyme can be increased, for example, by increasing the copy number of the gene encoding the enzyme already present in yeast. Increasing the copy number of the gene in yeast thus increases the enzyme's expression level. Alternatively, the expression level can be increased by substituting a stronger promoter. The activity level of an enzyme can be increased, for example, by substituting a portion of the coding region of the enzyme-coding gene on the chromosome to enhance stability. When substituting the coding region, the region to be increased can be the N-terminal region, the internal region, or the C-terminal region, as long as it increases the activity of the enzyme produced.

[0038] As described above, in order to increase the expression of endogenous genes, known promoters such as TEF1, TDH3, GPD1, PGK1, ENO1, GAL1, MET3, and PHO5 promoters can be used in a method of incorporating a promoter that can highly express the target enzyme onto a chromosome by gene substitution.

[0039] Gene introduction into yeast can be performed using plasmids or expression cassettes. A plasmid or expression cassette may contain one or more genes capable of expressing the enzyme used in this disclosure. Such plasmids may include, in addition to the enzyme-coding gene, a multi-cloning site, promoter, enhancer, terminator, selection marker cassette, etc. Linkers and restriction enzyme sites may also be added as needed during nucleic acid insertion. These operations can be performed using conventional genetic engineering techniques.

[0040] The method for introducing plasmids into yeast is not particularly limited and includes known methods such as lithium acetate, electroporation, calcium phosphate, lipofection, and DEAE dextran.

[0041] Furthermore, the yeast of this disclosure can be produced by homologous recombination, which incorporates the target gene onto the chromosome of the parent yeast. Those skilled in the art can produce the yeast of this disclosure by homologous recombination using known methods.

[0042] Self-cloning yeast can also be used as the yeast described herein. Self-cloning yeast refers to yeast composed of nucleotides substantially derived from the same species of organism. Even if nucleotides not derived from the same species of yeast are included, it is desirable that they are not derived from the same species of yeast at all, for example, only about 1 to 10 bases. When using self-cloning yeast in which a promoter that can express the target enzyme at high or low levels is substituted, a promoter derived from the same species of yeast should be used. Such self-cloning yeast can be produced by various known methods (e.g., S. Scherer and R.W. Davis (1979) Proc. Natl. Acad. Sci. USA, 76:4951), or by the methods described in the examples below. For example, after introducing the target sequence by homologous recombination, unwanted sequences not derived from the same species of yeast can be removed by homologous recombination using GIN11 (galactose-containing medium), URA3 (5-fluorotic acid-containing medium), etc. as removal selection markers.

[0043] The yeast of this disclosure has been genetically modified to further increase or decrease the expression of proteins involved in cell wall synthesis. It is desirable that the yeast of this disclosure inhibits cell wall synthesis by increasing or decreasing the expression of proteins involved in cell wall synthesis. Here, the enzyme to be genetically modified is not particularly limited as long as the genetic modification results in an increase or decrease in the expression of proteins involved in cell wall synthesis, thereby inhibiting cell wall synthesis. For example, in yeast that produces a large amount of squalene, proteins involved in cell wall synthesis whose expression is increased or decreased can be targeted. By conversely decreasing or increasing the expression of such proteins, it is possible to inhibit cell wall synthesis.

[0044] Proteins involved in cell wall synthesis include, for example, enzymes involved in cell wall synthesis reactions and cell wall proteins. Specific examples of proteins involved in cell wall synthesis include proteins encoded by the 1,3-β-D-glucan synthase gene (FKS1, FKS2), DAN1, PAU24, PAU2, HES1, SET4, PBI1, TIR1, TIR2, TIR3, TIR4, YSR3, HSP30, FHN1, PAU9, EMT1, FMP49, FDH2, UPC2, etc. These proteins can be used individually or in combination of two or more. Among these, 1,3-β-D-glucan synthase is particularly preferred. It is desirable to modify the gene to reduce the expression of 1,3-β-D-glucan synthase.

[0045] The expression or activity levels of proteins involved in cell wall synthesis can be increased or decreased by modifying yeast genes using various known methods. Increases or decreases in the expression of proteins involved in cell wall synthesis include increases or decreases in the transcription of protein-coding mRNA, and increases or decreases in the translation of protein mRNA. The explanation for increases or decreases in protein expression or activity levels is the same as above.

[0046] The yeast of this disclosure preferably has the effect of promoting the secretion of squalene outside the cell as a result of inhibiting cell wall synthesis by increasing or decreasing the expression of proteins involved in cell wall synthesis. Here, the cell wall includes the area outside the cell membrane (plasma membrane), i.e., the cell wall fraction. Because the yeast has the effect of promoting the secretion of squalene outside the cell in this way, it is possible to extract squalene without separating and disrupting the cells. As a result, problems such as the cost of centrifugation to separate the yeast, the cost of reagents such as enzymes and physical disruption to disrupt the yeast, and the cost of residue disposal can be eliminated. Furthermore, since it is not necessary to kill the yeast according to the yeast of this disclosure, the yeast can be reused.

[0047] The yeast of this disclosure has the effect of promoting the secretion of squalene outside the cell. Here, "promoted secretion of squalene outside the cell" means yeast that secretes more squalene outside the cell compared to yeast having the same properties except that cell wall synthesis is not inhibited. The increase in squalene secretion rate of the yeast of this disclosure compared to yeast having the same properties except that cell wall synthesis is not inhibited can be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, etc. Furthermore, the amount of squalene secreted per dry yeast weight (mass%) of the yeast of this disclosure can be, for example, 2% or more by mass, 2.5% or more by mass, 3.0% or more by mass, 3.5% or more by mass, 4.0% or more by mass, 4.5% or more by mass, etc.

[0048] Method for producing squalene The method for producing squalene according to the present disclosure includes the step of culturing the yeast according to the present disclosure in a culture medium (hereinafter sometimes referred to as the "method for producing squalene according to the present disclosure").

[0049] The manufacturing method of the present disclosure may further include a step of recovering squalene from the culture medium.

[0050] The culture medium used for culturing yeast is not particularly limited as long as it is a medium in which yeast can grow and produce squalene or a medium in which yeast can proliferate. The appropriate medium should be selected as appropriate, taking into consideration the type of yeast, etc. Such a medium may be a synthetic medium or a natural medium containing nutrients such as carbon sources, nitrogen sources, and inorganic salts. For example, carbon sources for the medium include glucose, sucrose, fructose, maltose, mannose, xylose, arabinose, galactose, lactose, cellobiose, xylobiose, trehalose, mannitol, sorbitol, xylitol, glycerin, dextrin, starch, oligosaccharides, molasses, malt extract, and organic acids (e.g., acetic acid, citric acid, lactic acid, fumaric acid, maleic acid, gluconic acid). Furthermore, examples of nitrogen sources include organic nitrogen sources such as various peptones, yeast extract, corn steep liquor, soybean flour, bran extract, meat extract, casein, protein hydrolysates, amino acids, and urea, as well as inorganic nitrogen sources such as nitrates and ammonium salts. Examples of inorganic salts include sodium salts, potassium salts, magnesium salts, iron salts, and other metal salts. In addition, examples of other nutrients include vitamins (e.g., biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid), amino acids, and nucleic acids. Carbon sources and nitrogen sources may be used individually or in combination of two or more.

[0051] Culturing can be carried out using either liquid culture or solid culture, and can be performed according to general microbial culture methods. Solid culture is preferred. Liquid culture can be carried out by aeration-stirring culture or shaking culture. There are also methods that allow stirring by aeration alone, such as airlift. Furthermore, liquid culture can be carried out using any of the following methods: batch culture, fed-batch culture, or continuous culture. Culture conditions (temperature, pH, culture time, etc.) can be set appropriately according to the growth characteristics of the yeast to be cultured. The culture temperature is usually 10 to 40°C, preferably 26 to 30°C, and the pH is usually pH 4 to 8, preferably pH 5 to 7. The culture time can vary depending on the culture method, type and amount of culture medium, culture temperature, etc., and should be the time required to produce the desired amount of squalene, for example, about 12 to 24 hours, preferably about 14 to 18 hours.

[0052] The yeast may be pre-cultured before the main culture. Pre-culture can be performed, for example, by inoculating a small amount of the yeast of this disclosure into a culture medium and culturing it for 12 to 72 hours. Then, 0.1 to 10%, preferably 1%, of the pre-culture solution can be added to the culture medium of the main culture, and the main culture can be started.

[0053] The culture medium (culture supernatant) obtained by culturing contains squalene secreted by the yeast of this disclosure. The squalene-containing culture supernatant can be subjected to further purification steps as needed. Examples of purification methods include solvent extraction, chromatography (e.g., affinity chromatography, ion exchange chromatography, hydroxyapatite column chromatography, hydrophobic interaction chromatography, gel filtration chromatography, etc.), membrane treatment (e.g., membrane filtration, ultrafiltration, microfiltration, reverse osmosis, etc.), activated carbon treatment, supercritical fluid extraction, distillation, and crystallization, which can be performed individually or in any combination of two or more in any order.

[0054] In the manufacturing method disclosed herein, it is not necessary to kill the yeast, and the culture medium (culture supernatant) obtained by cultivation can be reused. Therefore, squalene can be produced by obtaining the culture medium (culture supernatant), recovering the squalene, and then adding new culture medium as needed to continue cultivation.

[0055] Squalene obtained by the manufacturing method of this disclosure can be used, for example, in skincare and cosmetics, supplements for health foods, adjuvants to enhance the immune activity of live vaccines, lubricants such as engine oil, and fuels. Furthermore, confirmation and quantification of squalene obtained by the manufacturing method of this disclosure can be performed, for example, by using gas chromatography as shown in the examples.

[0056] In this specification, the term “comprising” includes “consisting essentially of” and “consisting of.” Furthermore, this disclosure encompasses all any combination of the constituent elements described herein.

[0057] Furthermore, the various characteristics (properties, structure, function, etc.) described for each embodiment of this disclosure described above may be combined in any way to identify the subject matter covered by this disclosure. In other words, this disclosure covers all subject matter consisting of any combination of the combinable characteristics described herein.

[0058] Examples are given below to further illustrate the present disclosure. However, the present disclosure is not limited in any way to these examples.

[0059] <Experimental Method> 1. Plasmid Preparation: To prepare a plasmid containing pYK1 ERG1, KY354 genomic DNA was used as a template. ERG1 was amplified by PCR using ERG1F primers and ERG1R primers. A PstI-treated fragment (2402 bp excluding the PstI sequence) was inserted, and pBluescriptII SK- treated with PstI was used as the vector for ligation. The plasmid obtained here was designated as pYK1 (5363 bp).

[0060] To create a plasmid containing a non-functional ERG1 in pYK2, pYK1 was treated with EcoRV, and the longest resulting fragment (3842 bp) was self-ligated. This mutates the ERG1 from the 108th amino acid onward, causing the 128th position to become a stop codon (this short ERG1 is designated as erg1Δ), preventing the production of a normally functioning protein. The plasmid obtained here was designated as pYK2 (3842 bp).

[0061] ・pYK3 P ＭＥＴ３ To create a plasmid containing a gene fused with erg1Δ downstream, P was amplified from the genomic DNA of strain KY354. ＭＥＴ３ Using a plasmid containing as a template, PCR was performed using primers PMET3_ERG1F, which has the BamHI sequence added, and primer PMET3_ERG1R, which has the HpaI sequence added, to identify the MET3 promoter site (P ＭＥＴ３ After amplifying the 490bp sequence from -1 to -490, a fragment treated with BamHI and HpaI (499bp excluding the BamHI and HpaI sequences) was inserted. Ligation was performed using the BamHI and HpaI-treated pYK2 fragment as the vector. The plasmid obtained here was designated as pYK3 (3754bp).

[0062] ・pYK4 P ＭＥＴ３To construct a yeast genome insertion plasmid (YIp vector) containing a gene fused with erg1Δ downstream, a fragment of pYK3 treated with SacI and SalI (868 bp excluding the SacI and SalI sequences) was inserted, and a fragment of the YIp vector pRS402 (ADE2 marker, ATCC87538, ACC. No. U93717) treated with SacI and SalI was used as the vector for ligation. The plasmid obtained here was designated as pYK4 (6321 bp).

[0063] ・ pMSK4 P ＴＥＦ１ To construct a plasmid containing a gene fused with HMG1 downstream, a plasmid (pKY265) was created by inserting the -349 to +3451 region of HMG1 into the BamHI region of YEplac181. This plasmid was used as a template and amplified by PCR using primers HMG1inFusionF, which had a SpeI cleavage sequence and vector sequence attached, and primer HMG1inFusionR2, which had a SalI cleavage sequence and vector sequence attached. The amplified fragment (3195 bp) was used as an insert, and a fragment (7145 bp) treated with p425TEF using SpeI and SalI was used as the vector. The insert and vector were fused using the in fusion method, and the resulting plasmid was designated pMSK4.

[0064] ・ pMSK6 P ＴＥＦ１ To construct a yeast genome insertion plasmid (YIp vector) containing a gene fused with HMG1 downstream, pMSK4 was used as a template and amplified by PCR using primers pTEF to pRS SpeIF and pTEF to pRS SalIR, which had SalI cleavage sequences and vector sequences added to them. The amplified fragment (3866 bp) was used as an insert, and the fragment treated with pRS405 (LEU2 marker, ATCC87538, ACC. No. U03445) with SpeI and SalI was used as the vector. These were joined together by in fusion, and the resulting plasmid was designated pMSK6.

[0065] ・To replace the vectors pYA1 and pYK4 with a vector having the HIS3 marker, a fragment (868 bp, not containing the SacI, SalI sequence) obtained by treating pYK3 with SacI and SalI was used as an insert, and a fragment obtained by treating pRS403 (HIS3 marker, ATCC87538, ACC. No. U03443) with SacI and SalI was used as a vector, and ligation was performed. The plasmid obtained here was designated as pYA1.

[0066] 2. Preparation of yeast strains

[0067] ・KY1018 KY1018 is obtained by replacing the promoter of HMG1 in KY354 (wild type) with P ＴＥＦ１ Specifically, pMSK6 was treated with PstI and transformed into KY354, and transformants in which LEU2 was inserted in a leucine-deficient medium were selected.

[0068] ・UHSQ (KY1233) UHSQ is obtained by replacing the promoter (P ＥＲＧ１ ) of KY1018 with P ＭＥＴ３ that can be repressed by methionine using pYK4 (Figure 1). pYK4 is a plasmid in which the MET3 promoter (P ＭＥＴ３ ) is fused upstream of the functionally lost ERG1 (erg1Δ) between the SacI - SalI sites of the pRS402 vector with ADE2 as a marker (P ＭＥＴ３ ::erg1Δ) is inserted. When this is cut with AgeI and introduced into yeast, homologous recombination occurs with the ERG1 of the recipient strain's genome. As a result, the ERG1 promoter (P ＥＲＧ１ ), erg1Δ, ADE2, P ＭＥＴ３ , and ERG1 will be arranged in this order. Eventually, normal ERG1 will be under the control of P ＭＥＴ３ and its expression can be repressed by methionine.

[0069] ・fks1Δ (KY1357) A URA3 fragment having 50 bp at the 5' side and 50 bp at the 3' side of the cds of FKS1 at both ends was amplified by PCR using pUG72 as a template and FKS1deltaF and FKS1deltaR, and introduced into KY767, and transformants in which LEU2 was inserted in a uracil-deficient medium were selected.

[0070] ・HSQfks1Δ (KY1358) To prepare HSQfks1Δ, the HMG1 promoter of fks1Δ (KY1357) was changed to P ＴＥＦ１ Specifically, pMSK6 was treated with PstI to transform it into fks1Δ, and transformants with LEU2 inserted in leucine-deficient medium were selected.

[0071] • UHSQfks1Δ (KY1359) For the fabrication of UHSQfks1Δ, P ＥＲＧ１ P ＭＥＴ３ Specifically, pYA1 was treated with AgeI and transformed into HSQfks1Δ, and transformants with HIS3 inserted in histidine-deficient medium were selected.

[0072] 3. Yeast cells intended for squalene content measurement and lipid extraction were inoculated into 2 mL of YPD medium and cultured with shaking at 28°C for 18 hours. Then, 2 mL of fresh liquid medium was added to the culture solution so that the absorbance at 600 nm (O.D. 600) of the culture solution was 1, and the culture was cultured with shaking at 28°C for 6 hours. After culturing, the absorbance was measured, and the culture was subcultured into two tubes of 20 mL of fresh liquid medium so that the O.D. 600 was 0.1, and the culture was cultured with shaking at 28°C for 18 hours.

[0073] The culture medium was transferred to a 50 mL tube and centrifuged at 3,000 rpm for 5 minutes at room temperature to collect the cells. The supernatant was removed, 1 mL of spheroplast buffer was added and the cells were suspended. The tube was then transferred to a lidded test tube (AGC Techno Glass Co., Ltd.) and centrifuged at 3,000 rpm for 5 minutes at room temperature. After removing the supernatant, 2 mL of spheroplast buffer was added to the collected cells and they were suspended. Further suspension was performed by adding 100 μL of Zymolyase 100T (1 mg / mL, Nacalai Tesque Co., Ltd.) and 20 μL of 1 M DTT, and the cells were allowed to stand at 37°C for 90 minutes. Subsequently, the cells were centrifuged at 3,000 rpm for 5 minutes at room temperature and the supernatant was saved as the cell wall fraction. Further centrifugation was performed by adding 1 mL of spheroplast buffer and centrifuging again. The supernatant was removed and the precipitate was saved as the intracellular fraction.

[0074] To the intracellular fraction, 4 mL of acetone (other extraction solvents such as hexane and ethanol can also be used) was added, suspended for 1 minute, allowed to stand at room temperature for 3 minutes, centrifuged at 3,000 rpm at room temperature for 3 minutes, and the supernatant was collected in a 30 mL round-bottom flask using a Pasteur pipette. This process was repeated three times. The cell wall fraction was adjusted to a total volume of 4 mL by adding purified water, 4 mL of chloroform and 8 mL of methanol were added, and suspended for 1 minute. Further 4 mL of purified water and 8 mL of chloroform were added, suspended for 1 minute, left to stand at room temperature for 10 minutes, then centrifuged at 3,000 rpm at room temperature for 3 minutes, and the lower layer was collected in a 30 mL round-bottom flask using a Pasteur pipette. The solvent in the round-bottom flask was evaporated using a rotary evaporator (AGC Techno Glass Co., Ltd.), and 500 μL of 100% ethanol was added to evaporate the water. 1 mL of acetone was added to the round-bottom flask to dissolve the lipids, and the mixture was stored in a vial (Mighty Vial). After acetone extraction, the cells were left to stand in a dryer (DG82, Yamato Scientific Co., Ltd.) set to 60°C for at least 24 hours, and then weighed to obtain the dry weight (DW).

[0075] • Gas chromatography analysis: 200 μL of the acetone-extracted sample was transferred to a 10 mL glass centrifuge tube, nitrogen gas was blown to evaporate the acetone, and 20 μL of acetone was added to redissolve the lipids and concentrate the sample 10-fold. 2 μL of the sample was taken with a glass syringe and injected into an InertCap-1 column (0.25-mm inner diameter × 60 m) (GL Sciences Co., Ltd.) attached to a GC-4000 Plus (GL Sciences Co., Ltd.). The gas chromatography instrument was set to an inlet temperature of 250°C and an FID detector temperature of 260°C. The temperature was increased from 50°C for 1 minute, to 220°C at 10°C / min, to 260°C at 2°C / min, and then held at 260°C for 90 minutes. Peak area was calculated using SICμ7 Data Station. To quantify the amount of squalene, squalene was prepared at concentrations of 1 μg / mL, 2 μg / mL, and 4 μg / mL. 2 μL of the sample was injected using a glass syringe, and a quantitative curve was constructed using the area of ​​the resulting peaks.

[0076] 4. RNA extraction and purification KY354 or KY1233 was inoculated into 2 mL of synthetic minimal medium containing 8 mM L-methionine and incubated overnight. The next day, the OD of the culture medium was measured. ６００ The solution was added to a new culture medium until the concentration was 0.5, and incubated at 28°C for 18 hours. The culture medium was transferred to a 50 mL tube, cooled on ice, and immediately centrifuged at 4°C at 5,000 rpm for 5 minutes. The precipitate was suspended in 1 mL of TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA), transferred to a 2 mL tube, centrifuged at 4°C at 5,000 rpm for 5 minutes, and the supernatant was removed. In a tube, 250 μL of acid-washed glass beads (0.5 mm), 350 μL of phenol:chloroform:isoamyl alcohol 25:24:1 solution (Nacalai Tesque Co., Ltd.), and 350 μL of CrossRNA solution (0.3 M NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.2% SDS) were added. The cells were disrupted by vortexing for 30 seconds and standing on ice for 30 seconds, repeated 10 times. The mixture was then centrifuged at 15,000 rpm at 4°C for 5 minutes, and 250 μL of the supernatant was transferred to a new tube. 250 μL of 70% ethanol was added to this supernatant, and RNA was purified using the RNeasy Mini Kit (QIAGEN) according to the instructions provided. Next, using the TURBO DNA-free Kit (Thermo Fisher Scientific), DNase treatment was performed according to the attached instructions, and then the RNA sample was purified again using the RNeasy Mini Kit (QIAGEN).

[0077] 5. RNA-Seq Analysis RNA-Seq analysis was performed using the prepared RNA samples and the RNA-Seq contract analysis service (for Saccharomyce cerevisiae) provided by Biotechnology Research Institute Co., Ltd. Changes in expression levels were evaluated by using the TPM values ​​for each provided gene as an indicator and calculating the values ​​for the KY1233-derived RNA sample relative to the values ​​for the KY354-derived RNA sample.

[0078] 6. Reagents, Culture Mediums, and YPD Medium YPD medium was prepared by dissolving 5 g of dried yeast extract (Nacalai Tesque Co., Ltd.) and 10 g of Bactone (Becton Dickinson) in 500 mL of pure water, autoclaving (121°C, 20 mins), and then adding 20 mL of 50% glucose solution. For agar medium, agar powder (Nacalai Tesque Co., Ltd.) was added at a rate of 20 g / L.

[0079] - A synthetic minimum liquid medium was prepared by dissolving 3.35 g of Yeast Nitrogene Base w / o Amino Acids (Formedium) in 500 mL of pure water, autoclaving it (121°C, 20 mins), and then adding 20 mL of 50% glucose solution and 5 mL of Stock Supplements. For agar media, purified agar powder (Nacalai Tesque Co., Ltd.) was added at a rate of 20 g / L before autoclaving.

[0080] Stock Supplements: A mixed aqueous solution of the following substances: 0.2% adenine sulfate, 0.2% uracil, 1% L-tryptophan, 1% L-histidine hydrochloride, 1% L-leucine, and 1% L-lysine hydrochloride. However, drop-out solutions without each additive were used to prepare the nutrient-requiring medium. When L-methionine was added to the medium, it was made to a concentration of 8 mM.

[0081] 7. List of primers used

[0082] 8. List of Yeast Strain Genotypes

[0083] <Results> To create a yeast strain (Saccharomyces cerevisiae) that produces large amounts of squalene, we performed genetic modification to enable the steady expression of HMG-CoA reductase and the suppression of squalene epoxidizing enzyme by adding methionine (UHSQ strain) (see Figure 2). Furthermore, we created a modified strain in which the β1,3-glucan synthase gene (FKS1) involved in cell wall synthesis of the UHSQ strain was disrupted (UHSQfks1Δ strain). By growing the created yeast strains on a solid medium containing methionine, we promoted the production and secretion of squalene, which was then harvested.

[0084] After treating the yeast strains with Zymolase, they were centrifuged to separate the supernatant (cell wall) and precipitate (intracellular). Lipids were extracted from each by acetone extraction and analyzed by chromatography. Figure 3 shows the results of squalene content measurement for each strain. The proportion of squalene detected from the cell wall fraction relative to the total amount was 14% for the UHSQ strain, while it was significantly higher at 41% for the UHSQΔfks1 strain.

[0085] Furthermore, in the ultra-high squalene-producing strain (UHSQ strain), some cell wall proteins (cell wall mannoproteins) are highly expressed. For example, the expression levels of DAN1, PAU24, PAU2, HES1, SET4, PBI1, TIR1, TIR2, TIR3, TIR4, YSR3, HSP30, FHN1, PAU9, EMT1, FMP49, FDH2, and UPC2 increased 4 to 60 times.

[0086] In ultra-high squalene-producing strains (UHSQ strains), squalene leaks into the cell wall rather than remaining inside the cell, but disrupting the FKS1 gene significantly increased this leakage. In strains with disrupted FKS1 genes, squalene was able to be secreted extracellularly rather than stored intracellularly. This ability to secrete squalene is thought to be due not only to modification of the squalene metabolic pathway genes, but also to the disruption of the β1,3-glucan synthase gene involved in cell wall synthesis. To date, there have been no reports suggesting that inhibiting cell wall synthesis can promote intracellular lipid secretion.

[0087] <Preparation of self-cloning yeast> The gene modification applied to the yeast described above is the HMG1 promoter (P ＨＭＧ１ ) TEF1 promoter (P ＴＥＦ１ Substitution to ) ERG1 promoter (P ＥＲＧ１ ) MET3 promoter (P ＭＥＴ３ The three sites are substitution to ) and FKS1 disruption. FKS1 disruption is performed using DNA that does not contain foreign gene sequences, P ＨＭＧ１ and P ＥＲＧ１ In this substitution, a plasmid-derived DNA fragment is inserted into the yeast genome. Therefore, promoter substitution is performed in a way that does not leave any foreign DNA fragments, and self-cloning yeast is created.

[0088] To achieve this, the YIp vector is used to introduce a foreign gene into the genome, and then utilizes the phenomenon in which DNA fragments derived from the vector are removed through homologous recombination, which naturally occurs between yeast genomic DNAs.

[0089] P ＨＭＧ１ P ＴＥＦ１ For substitution to P ＴＥＦ１ A plasmid containing HMG1 downstream was prepared using the same method as for pMSK4, and the P of this plasmid was prepared. ＴＥＦ１ Upstream P ＨＭＧ１ A sequence containing at least 30 base pairs of the upstream sequence (5'UP) is inserted (Figure 4). The plasmid thus constructed (pMSK4URA) is linearized using inverse PCR with a primer prepared at the PstI site in HMG1 or at any position in HMG1, and then introduced into yeast, where it is inserted into the HMG1 site on the genomic DNA by homologous recombination (Figure 4).

[0090] To remove plasmid-derived DNA fragments from the resulting yeast, a metabolic reaction mediated by URA3 is utilized. Since pMSK4URA possesses URA3 as a marker gene, yeast with this plasmid inserted cannot grow in a medium containing 5-fluoroorotic acid (5FOA). This is because URA3 products metabolize 5FOA into 5-fluorouracil, which inhibits yeast growth. However, if URA3 is lost through homologous recombination, 5-fluorouracil is not produced, allowing the yeast to grow in 5FOA medium. Homologous recombination can occur at any position between homologous DNA sequences, but if it occurs at the 5'UP region, the plasmid-derived sequence is lost along with URA3 (Figure 4).

[0091] In practice, the target yeast is cultured at 28°C for 3 days on a synthetic minimal agar medium containing 1 mg / mL of 5FOA and 50 μg / mL of uracil, and the resulting colonies are isolated. The isolated colonies are cultured, genomic DNA is extracted, the gene region near HMG1 is amplified by PCR, and the DNA sequence of the amplified product is determined to confirm the presence or absence of plasmid-derived sequences.

[0092] By a similar method, PＥＲＧ１ P ＭＥＴ３ Perform the substitution.

[0093] This application is based on Japanese Patent Application No. 2024-205276 (filing date: November 26, 2024), the contents of which are fully incorporated herein.

Claims

1. A yeast that produces a large amount of squalene, wherein the gene has been modified to increase or decrease the expression or activity of at least one enzyme in the ergosterol biosynthesis pathway, and further, the gene has been modified to increase or decrease the expression of a protein involved in cell wall synthesis.

2. The yeast according to claim 1, which has the effect of promoting the secretion of squalene outside the cell.

3. The enzymes include acetyl-CoA acetyltransferase (ERG10), 3-hydroxy-3-methylglutaryl-CoA synthase (ERG13), HMG1, HMG2, mevalonate kinase (ERG12), phosphomevalonate kinase (ERG8), mevalonate pyrophosphate decarboxylase (ERG19), isopentenyl diphosphate δ-isomerase (Idi1), kinase farnesyl diphosphate synthase (ERG20), squalene synthase (ERG9), squalene epoxidase (ERG1), lanosterol synthase (ERG7), and lanosterol 14-α-demethylase. The yeast according to claim 1, wherein the enzyme is at least one selected from the group consisting of (ERG11), C-14 sterol reductase (ERG24), C-4 methylsterol oxidase (ERG25), C-3 sterol dehydrogenase (ERG26), 3-ketosterol reductase (ERG27), sterol 24-C-methyltransferase (ERG6), C-8 sterol isomerase (ERG2), C-5 sterol desaturase (ERG3), C-22 desaturase (ERG5), C-24 sterol reductase (ERG4), acetyl-CoA carboxylase, and ATP citrate lyase.

4. The yeast according to claim 1, wherein cell wall synthesis is inhibited by an increase or decrease in the expression of proteins involved in cell wall synthesis.

5. The yeast according to claim 1, wherein the protein involved in the synthesis of the cell wall is at least one selected from the group consisting of proteins encoded by FKS1, FKS2, DAN1, PAU24, PAU2, HES1, SET4, PBI1, TIR1, TIR2, TIR3, TIR4, YSR3, HSP30, FHN1, PAU9, EMT1, FMP49, FDH2, and UPC2.

6. The yeast according to claim 1, wherein the yeast is a yeast belonging to at least one genus selected from the group consisting of the genera Saccharomyces, Candida, Kluiveromyces, Pichia, Schizosaccharomyces, Torraspora, and Jarrowvia.

7. A method for producing squalene, comprising the step of culturing the yeast described in any one of claims 1 to 6 in a culture medium.

8. The method according to claim 7, further comprising the step of recovering squalene from the culture medium.

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