Method for producing Labyrinthula microorganisms and sterol esters

Abstract

The object of this invention is to provide Labyrinthula microorganisms that have high sterol ester production capacity. [Solution] A Labyrinthull microorganism modified to have reduced or lost activity of sterol 24-C-methyltransferase (SMT1) compared to an unmodified strain, wherein the Labyrinthull microorganism is modified to express a modified diacylglycerol acyltransferase 2C (modified DGAT2C) gene, and the modified diacylglycerol acyltransferase 2C (modified DGAT2C) is modified to have a defect in presumed transmembrane regions 1 to 8 of the presumed transmembrane regions 1 to 12 in diacylglycerol acyltransferase 2C (DGAT2C).

Description

Technical Field

[0001] The present invention relates to a method for producing labyrinthulid microorganisms and sterol esters.

Background Art

[0002] Sterols are steroid derivatives found in various organisms such as animals, plants, and fungi. Sterols are produced, for example, by extraction from biological resources that produce those sterols. Among labyrinthulids, which are marine unicellular eukaryotes, there are known ones that produce sterols such as animal sterols, plant sterols, and fungal sterols (Non-Patent Document 1). Also, a method for producing sterols using a modified strain of labyrinthulid microorganisms has been reported.

[0003] For example, Patent Document 1 discloses a method for producing sterols, which includes culturing a labyrinthulid microorganism having sterol-producing ability in a medium and recovering sterols from the cells produced by the culture. The labyrinthulid microorganism is modified such that the activity of 24-dehydrocholesterol reductase is reduced as compared with the unmodified strain, and the sterol is at least one sterol selected from plant sterols and fungal sterols. Also, Patent Document 1 discloses a method for producing sterols, which includes culturing a labyrinthulid microorganism having sterol-producing ability in a medium and recovering sterols from the cells produced by the culture. The labyrinthulid microorganism is modified such that the activity of sterol 24-C-methyltransferase is reduced as compared with the unmodified strain, and the sterol is cholesterol.

[0004] Patent Document 2 also discloses a method for producing cholesterol, which includes culturing Labyrinthuria microorganisms capable of producing sterols in a culture medium and recovering sterols from the bacterial cells produced by the culture. In this method, the Labyrinthuria microorganisms are modified so that the activity of diacylglycerol acyltransferase (DGAT) is increased compared to the unmodified strain, and efforts are being made to enhance the sterol production capacity.

[0005] Sterols can also exist in various derivative forms, such as sterol esters. Sterol esters are compounds that have a structure in which free sterols and fatty acids are linked by an ester bond. The ester bond is formed between the hydroxyl group at the C3 position of the sterol and the carboxyl group of the fatty acid. For example, in higher animals such as humans, it is known that cholesterol esters are produced from cholesterol by the action of acyl-coenzyme A:cholesterol acyltransferase (ACAT). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2019-71860 [Patent Document 2] Japanese Patent Publication No. 2020-031572 [Non-patent literature]

[0007] [Non-Patent Document 1] Weete JD, et al., Lipids and ultrastructure of Thraustochytrium sp. ATCC 26185., Lipids. 1997 Aug;32(8):839-45. [Overview of the project] [Problems that the invention aims to solve]

[0008] However, a method for producing sterol esters using Labyrinthula microorganisms has not been established. Therefore, in order to solve the problems of the conventional technology, the present inventors conducted research with the aim of providing Labyrinthula microorganisms that have high sterol ester production capacity. [Means for solving the problem]

[0009] Examples of specific embodiments of the present invention are shown below.

[0010] [1] Labyrinthula microorganisms modified to have reduced or lost sterol 24-C-methyltransferase (SMT1) activity compared to unmodified strains, Labyrinthula microorganisms have been modified to express the modified diacylglycerol acyltransferase 2C (modified DGAT2C) gene. Modified diacylglycerol acyltransferase 2C (modified DGAT2C) is a Labyrinthula microorganism in which presumed transmembrane regions 1-8 of the presumed transmembrane regions 1-12 in diacylglycerol acyltransferase 2C (DGAT2C) have been modified to be missing. [2] Labyrinthula microorganisms as described in [1], wherein modified diacylglycerol acyltransferase 2C (modified DGAT2C) is a protein described in any of (a) to (c) below; (a) A protein consisting of the amino acid sequence shown in Sequence ID No. 1; (b) A protein having diacylglycerol acyltransferase activity, which includes substitution, addition, insertion and / or deletion of one or more amino acid residues in the amino acid sequence shown in Sequence ID No. 1; (c) A protein that has 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1 and has diacylglycerol acyltransferase activity. [3] Labyrinthuria microorganisms as described in [1] or [2], wherein the Labyrinthuria microorganism is a microorganism belonging to the genus Aurantiochytrium. [4] Labyrinthurid microorganisms described in [3], wherein the Labyrinthurid microorganism is a microorganism derived from Aurantiochytrium limacinum. [5] A Labyrinthula microorganism described in any of [1] to [4], wherein the growth ability of the Labyrinthula microorganism is 80% or more of the growth ability of the unmodified strain. [6] Labyrinthula microorganisms according to any of [1] to [5] that have the ability to produce sterol esters. [7] Labyrinthuli microorganisms described in [6], further having the ability to produce provitamin D3. [8] A Labyrinthula microorganism described in any of [1] to [7], wherein the microorganism with accession number: FERM P-22511 has been modified to express the modified diacylglycerol acyltransferase 2C (modified DGAT2C) gene. [9] A Labyrinthula microorganism described in any of [1] to [8], which is the microorganism with accession number: FERM P-22512. A method for producing sterol esters, comprising culturing a Labyrinthula microorganism described in any of [1] to [9] and recovering sterol esters from the microbial cells produced by the culturing.

[11] The method for producing a sterol ester according to

[10] , wherein the sterol ester comprises an acyl group derived from at least one selected from docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA). [Effects of the Invention]

[0011] According to the present invention, Labyrinthula microorganisms having high sterol ester production capacity can be obtained. [Brief explanation of the drawing]

[0012] [Figure 1]Figure 1 is a schematic diagram explaining the structure of the N-terminal deletion mutant of modified DGAT2C. [Figure 2] Figure 2 shows the results of Western blot analysis when non-modified (full-length) DGAT2C or modified DGAT2C is expressed. [Figure 3] Figure 3 is a graph showing the production amounts of cholesterol and Δ7 stigmasterol in the wild type strain, DHCR24-deficient strain, and SMT1-deficient strain. [Figure 4] Figure 4 is a graph showing the results of measuring the optical density (OD600) at a wavelength of 600 nm for the wild type strain, SMT1-deficient strain, and SMT1-deficient subculture strain. [Figure 5] Figure 5 is a graph showing the results of measuring the optical density (OD600) at a wavelength of 600 nm for the SMT1-deficient subculture strain, high cholesterol-producing strains 1, 2, and 4. [Figure 6] Figure 6 is a graph showing the dry weights after culture for the wild type strain, SMT1-deficient subculture strain, high cholesterol-producing strains 1, 2, and 4. [Figure 7] Figure 7 shows the results of Western blot analysis for confirming the presence or absence of the expression of modified DGAT2C. [Figure 8] Figure 8 is a graph showing the production amounts of DHA and DPA which are cellulose esters. [Figure 9] Figure 9 is a graph showing the production amount of provitamin D3.

Mode for Carrying Out the Invention

[0013] Hereinafter, the present invention will be described in detail. The following description may be made based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. In this specification, the numerical range represented by "~" means a range including the numerical values described before and after "~" as the lower limit value and the upper limit value.

[0014] In this specification, the 20 amino acid residues in an amino acid sequence may be represented by a single-letter abbreviation. In this case, glycine (Gly) is G, alanine (Ala) is A, valine (Val) is V, leucine (Leu) is L, isoleucine (Ile) is I, phenylalanine (Phe) is F, tyrosine (Tyr) is Y, tryptophan (Trp) is W, serine (Ser) is S, threonine (Thr) is T, cysteine ​​(Cys) is C, methionine (Met) is M, aspartic acid (Asp) is D, glutamic acid (Glu) is E, asparagine (Asn) is N, glutamine (Gln) is Q, lysine (Lys) is K, arginine (Arg) is R, histidine (His) is H, and proline (Pro) is P. Also, in this specification, the amino acid sequence shown has the N-terminus on the left and the C-terminus on the right.

[0015] (Labyrinthula microorganisms) This embodiment relates to a Labyrinthula microorganism modified to have reduced or lost sterol 24-C-methyltransferase (SMT1) activity compared to an unmodified strain, and to a Labyrinthula microorganism modified to express a modified diacylglycerol acyltransferase 2C (modified DGAT2C) gene. In this embodiment, the modified diacylglycerol acyltransferase 2C (modified DGAT2C) is modified such that presumed transmembrane regions 1 to 8 of the presumed transmembrane regions 1 to 12 in diacylglycerol acyltransferase 2C (DGAT2C) are missing.

[0016] In this embodiment, a Labyrinthula microorganism is provided in which the activity of sterol 24-C-methyltransferase (SMT1) is reduced or lost compared to an unmodified strain, and which is modified to express a modified diacylglycerol acyltransferase 2C (modified DGAT2C) gene. Such a Labyrinthula microorganism can selectively produce cholesterol and has high sterol ester (cholesterol ester) production capacity.

[0017] "Labyrinthuloid microorganisms" refers to microorganisms classified as Labyrinthuloids. Examples of Labyrinthuloid microorganisms include those belonging to genera such as Aurantiochytrium, Schizochytrium, Thraustochytrium, Parietichytrium, Labyrinthula, Althornia, Aplanochytrium, Japonochytrium, Labyrinthuloides, Ulkenia, Oblongichytrium, Botryochytrium, and Sicyoidochytrium. In particular, the Labyrinthula microorganisms of this embodiment are preferably Aurantiochytrium, Schizochytrium, Thraustochytrium, or Parietichytrium, and are especially preferably Aurantiochytrium.

[0018] Examples of microorganisms belonging to the genus Aurantiochytrium include Aurantiochytrium limacinum and Aurantiochytrium sp.2. Examples of microorganisms belonging to the genus Schizochytrium include Schizochytrium aggregatum. Examples of microorganisms belonging to the genus Thraustochytrium include Thraustochytrium aureum. Examples of microorganisms belonging to the genus Parietichytrium include Parietichytrium sarkarianum. Examples of microorganisms belonging to the genus Botryochytrium include Botryochytrium radiatum. Labyrinthuloid microorganisms include, for example, Aurantiochytrium limacinum mh0186 (FERM BP-11311), Aurantiochytrium limacinum ATCC MYA-1381, Thraustochytrium aureum ATCC34304, Thraustochytrium sp. ATCC26185, Schizochytrium aggregatum ATCC28209, Schizochytrium sp. AL1Ac, Schizochytrium sp. SEK210(NBRC 102615), Schizochytrium sp. SEK345(NBRC 102616), Parietichytrium sarkarianum SEK364(FERM BP-11298), Ulkenia sp. ATCC 28207, Botryochytrium radiatum SEK353 (NBRC 104107) is one example.

[0019] Aurantiochytrium limacinum mh0186 is the strain previously known as Schizochytrium sp. M-8. The genus previously known as Schizochytrium has been reorganized into the genera Schizochytrium, Aurantiochytrium, and Oblongichytrium (Rinka Yokoyama, Daiske Honda (2007) MycoscienceTaxonomic rearrangement of the genus Schizochytrium sensu lato based on morphology, chemotaxonomic characteristics, and 18S rRNA gene phylogeny (Thraustochytriaceae, Labyrinthulomycetes): emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen. nov. Mycoscience, 48, 199-21). Aurantiochytrium limacinum mh0186 was originally deposited on March 29, 2004, with accession number FERM P-19755 at the Patent Microbial Depository Center of the National Institute of Advanced Industrial Science and Technology (now the Patent Microbial Depository Center of the National Institute of Technology and Evaluation, postal code: 292-0818, address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan). It was then transferred to international deposit and assigned accession number FERM BP-11311.

[0020] Labyrinthulomycetes microorganisms have the ability to produce sterols. Microorganisms with the ability to produce sterols refer to microorganisms that have the ability to generate the target sterol and accumulate it in the cells to such an extent that it can be recovered when cultured in a medium. For example, in wild-type Labyrinthulomycetes microorganisms without modification, animal sterols, plant sterols, and fungal sterols are produced. Examples of animal sterols include cholesterol and 7-dehydrocholesterol. Examples of plant sterols include 7-dehydropolypherasterol, stigmasterol, 4-methyl-7,22-stigmastadienol, β-sitosterol, brassicasterol, campesterol, diosgenin, oleanolic acid, betulinic acid, ursolic acid, hecogenin, sarsasapogenin, isofucosterol, avenasterol, Δ7-stigmastenol, Δ7-campestenol, fucosterol, and sargasterol. Examples of fungal sterols include ergosterol and 7-dihydroergosterol.

[0021] The Labyrinthulomycetes microorganisms of the present embodiment can be obtained by modifying the Labyrinthulomycetes microorganisms as exemplified above as described below. That is, the Labyrinthulomycetes microorganisms of the present embodiment are modified strains derived from the Labyrinthulomycetes microorganisms as exemplified above. Among them, the Labyrinthulomycetes microorganisms of the present embodiment are preferably modified strains of microorganisms belonging to the genus Aurantiochytrium, and particularly preferably modified strains of microorganisms derived from Aurantiochytrium limacinum.

[0022] <SMT1 activity> The Labyrinthula microorganisms of this embodiment have been modified so that the activity of sterol 24-C-methyltransferase (SMT1) is reduced or lost compared to the unmodified strain (wild strain). Sterol 24-C-methyltransferase (SMT1) is a protein (enzyme) that has the activity to catalyze the reaction of introducing a methyl group at the C24 position of a sterol. The enzymatic activity of this protein is called "sterol 24-C-methyltransferase activity (SMT1 activity)". The gene encoding SMT1 is called the "sterol 24-C-methyltransferase gene (SMT1 gene)".

[0023] Sterol 24-C-methyltransferase (SMT1) is an enzyme that synthesizes plant sterols and fungal sterols in unmodified Labyrinthuria microorganisms by transferring a methyl group to the C24 position of cycloartenol, a sterol precursor. Therefore, reducing or eliminating the activity of sterol 24-C-methyltransferase (SMT1) suppresses the production capacity of plant sterols and fungal sterols in Labyrinthuria microorganisms. As a result, in the Labyrinthuria microorganisms of this embodiment, only animal sterols are selectively produced, and the production capacity of animal sterols is enhanced. In particular, in this embodiment, it is preferable that only the production capacity of cholesterol is selectively enhanced. In the Labyrinthuria microorganisms of this embodiment, the amount of cholesterol produced relative to the total amount of sterols produced is preferably, for example, 90% (w / w) or more, more preferably 95% (w / w) or more, and even more preferably 97% (w / w) or more. There is no particular upper limit to the amount of cholesterol produced relative to the total amount of sterols produced, and it may be 100% (w / w). Furthermore, if the amount of cholesterol produced in unmodified Labyrinthuria microorganisms is set to 100, the amount of cholesterol produced in the Labyrinthuria microorganisms of this embodiment is preferably 80 or more, more preferably 90 or more, even more preferably 100 or more, even more preferably 110 or more, and particularly preferably 120 or more.

[0024] SMT1 activity can be measured, for example, by incubating the enzyme with a substrate (sterol) in the presence of a methyl group donor and measuring the production of enzyme and substrate-dependent products (sterols with a methyl group introduced at the C24 position). Examples of methyl group donors include S-adenosyl-L-methionine. Examples of substrates and products include zymosterol and fecosterol, respectively.

[0025] As mentioned above, a decrease in SMT1 activity can be confirmed by measuring SMT1 activity. Furthermore, a decrease in SMT1 activity can be confirmed by an increase in the production of the target sterol (animal sterol), or by a decrease in the production of other sterols (plant sterols or fungal sterols).

[0026] The nucleotide sequences of the SMT1 gene possessed by Labyrinthula microorganisms, as well as the amino acid sequences of SMT1 encoded by them, can be obtained from publicly available databases such as NCBI (National Center for Biotechnology Information).

[0027] "Decreased or lost SMT1 activity compared to the unmodified strain" means that the activity of SMT1 per cell is decreased or lost compared to the unmodified strain. "Unmodified strain" here refers to a control strain that has not been modified to reduce the activity of the target protein. Examples of unmodified strains include wild-type strains and parental strains. Specifically, examples of unmodified strains include the strains exemplified in the description of Labyrinthula microorganisms. In one embodiment, the activity of SMT1 can be compared to that of Aurantiochytrium limacinum mh0186. "Decreased SMT1 activity" more specifically means that, compared to the unmodified strain, the number of molecules of the protein per cell is decreased, and / or the function of the protein per molecule is decreased. In this case, "activity" is not limited to the catalytic activity of the protein, but may also refer to the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. The activity of SMT1 is preferably 50% or less of that of the unmodified strain, more preferably 20% or less, even more preferably 10% or less, and even more preferably 5% or less. If the activity of SMT1 is lost, the activity of SMT1 becomes 0% of that of the unmodified strain.

[0028] Modifications that reduce or eliminate SMT1 activity can be achieved, for example, by reducing the expression of the SMT1 gene that encodes SMT1. "Reduced gene expression" means that the expression level of the gene is lower compared to the unmodified strain. More specifically, "reduced gene expression" may mean a decrease in the amount of gene transcription (mRNA) and / or a decrease in the amount of gene translation (protein). The expression level of the SMT1 gene is preferably 50% or less of that of the unmodified strain, more preferably 20% or less, even more preferably 10% or less, and even more preferably 5% or less. When SMT1 activity is lost, it is preferable that the expression level of the SMT1 gene be 0% of that of the unmodified strain.

[0029] A decrease in SMT1 gene expression may be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination of both. A decrease in SMT1 gene expression can be achieved, for example, by modifying the gene's expression regulatory sequence. The "expression regulatory sequence" is a general term for the regions that affect the expression of the SMT1 gene, such as the promoter. When modifying the expression regulatory sequence, preferably one or more bases, more preferably two or more bases, and particularly preferably three or more bases are modified. Alternatively, the entire region of the expression regulatory sequence may be deleted (deleted). A decrease in the gene's transcription efficiency can be achieved, for example, by replacing the promoter of the SMT1 gene on the chromosome with a weaker promoter. A "weaker promoter" means a promoter that weakens the transcription of the SMT1 gene compared to the originally present wild-type promoter. An example of a weaker promoter is an inductive promoter. That is, an inductive promoter can function as a weaker promoter under non-inductive conditions (for example, in the absence of an inductive substance). Furthermore, a decrease in SMT1 gene expression can also be achieved, for example, by manipulating factors involved in expression regulation. Factors involved in gene expression regulation include small molecules (inducers, inhibitors, etc.), proteins (transcription factors, etc.), and nucleic acids (siRNA, etc.) involved in transcription and translation control. Furthermore, a decrease in SMT1 gene expression can also be achieved, for example, by introducing mutations in the coding region of the SMT1 gene that reduce gene expression. For example, SMT1 gene expression can be reduced by replacing the codons in the coding region of the SMT1 gene with synonymous codons that are used less frequently in the host.

[0030] Furthermore, modifications that reduce SMT1 activity can be achieved, for example, by disrupting the SMT1 gene that codes for the protein. "Disruption of the SMT1 gene" means that the gene is modified so that it does not produce a normally functioning protein (SMT1). "Not producing a normally functioning protein" includes cases where no protein is produced at all from the gene, or where the gene produces a protein with reduced or absent function (activity or properties) per molecule.

[0031] Disruption of the SMT1 gene can be achieved, for example, by deleting (removing) the SMT1 gene on a chromosome. "Genetic deletion" refers to the deletion of part or all of the coding region of the gene. Furthermore, the entire gene may be deleted, including the sequences before and after the coding region of the gene on the chromosome. The sequences before and after the coding region of the gene may include, for example, gene expression regulatory sequences. As long as a reduction in SMT1 activity is achieved, the deleted region may be any of the following regions: the N-terminal region (the region that codes for the N-terminal side of the protein), the internal region, or the C-terminal region (the region that codes for the C-terminal side of the protein). Generally, a longer deleted region ensures more reliable gene inactivation. The deleted region may be, for example, a region that is 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, or 95% or more in length of the total coding region of the SMT1 gene. Furthermore, it is preferable that the sequences before and after the region to be deleted do not have matching reading frames. A mismatch in reading frames may cause a frame shift downstream of the region to be deleted.

[0032] Furthermore, disruption of the SMT1 gene can also be achieved, for example, by introducing amino acid substitutions (missense mutations), stop codons (nonsense mutations), or the addition or deletion of 1-2 bases (frameshift mutations) into the coding region of the gene on the chromosome (Journal of Biological Chemistry 272:8611-8617 (1997), Proceedings of the National Academy of Sciences, USA 95 5511-5515 (1998), Journal of Biological Chemistry 26 116, 20833-20839 (1991)).

[0033] Furthermore, disruption of the SMT1 gene can also be achieved, for example, by inserting another base sequence into the coding region of the gene on a chromosome. The insertion site can be any region of the gene, but a longer base sequence ensures more reliable gene inactivation. It is also preferable that the sequences before and after the insertion site do not have matching reading frames. Mismatch in reading frames can cause a frameshift downstream of the insertion site. Other base sequences are not particularly limited as long as they reduce or eliminate the activity of the encoded SMT1, but examples include marker genes such as antibiotic resistance genes and genes useful for the production of target substances.

[0034] Disruption of the SMT1 gene may be carried out in such a way that the amino acid sequence of the encoded protein (SMT1) is deleted (missing). Modifications that reduce SMT1 activity can be achieved, for example, by deleting the amino acid sequence of SMT1 (part or all of the amino acid sequence), specifically by modifying the gene to encode SMT1 with a deleted amino acid sequence (part or all of the amino acid sequence). Note that "deletion of the amino acid sequence of a protein" refers to the deletion of part or all of the amino acid sequence of a protein. Furthermore, "deletion of the amino acid sequence of a protein" means that the original amino acid sequence is no longer present in the protein, and includes cases where the original amino acid sequence is changed to a different amino acid sequence. That is, for example, a region that has been changed to a different amino acid sequence due to a frameshift may be considered a deleted region. Typically, deletion of the amino acid sequence of a protein shortens the total length of the protein, but it is also possible that the total length of the protein does not change or is lengthened.

[0035] Modifying the SMT1 gene on the chromosome as described above can be achieved, for example, by creating a disruption gene that does not produce a normally functioning protein, transforming the host with recombinant DNA containing the disruption gene, and inducing homologous recombination between the disruption gene and the wild-type gene on the chromosome, thereby replacing the wild-type gene on the chromosome with the disruption gene. In this case, it is easier to manipulate if the recombinant DNA contains marker genes according to the host's nutritional requirements and other traits. Examples of disruption genes include genes in which part or all of the coding region of the gene is deleted, genes into which missense mutations are introduced, genes into which nonsense mutations are introduced, genes into which frameshift mutations are introduced, and genes into which insertion sequences such as transposons or marker genes are inserted. Even if the protein encoded by the disruption gene is produced, it will have a different three-dimensional structure from the wild-type protein and its function will be reduced or lost. The structure of the recombinant DNA used for homologous recombination is not particularly limited as long as homologous recombination occurs in the desired manner. For example, by transforming a host with linear DNA containing a disruptive gene, which has the upstream and downstream sequences of the wild-type gene on the chromosome at both ends, homologous recombination occurs upstream and downstream of the wild-type gene, respectively, allowing the wild-type gene to be replaced with the disruptive gene in a single step.

[0036] Transformation can be carried out, for example, by methods commonly used for the transformation of Labyrinthula microorganisms. One such method is electroporation.

[0037] Furthermore, modifications that reduce the activity of SMT1 may be carried out, for example, by mutagenesis. Mutagenesis methods include irradiation with X-rays, irradiation with ultraviolet light, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS). The above methods for reducing the activity of the protein may be used individually or in any combination.

[0038] <Modified DGAT2C> The Labyrinthula microorganisms of this embodiment are modified to express a modified diacylglycerol acyltransferase 2C (modified DGAT2C) gene, in addition to the modifications that reduce or eliminate SMT1 activity as described above. Modified diacylglycerol acyltransferase 2C (modified DGAT2C) is modified in such a way that presumed transmembrane regions 1 to 8 of the presumed transmembrane regions 1 to 12 in diacylglycerol acyltransferase 2C (DGAT2C) are deleted. In this specification, modified DGAT2C modified in such a way that presumed transmembrane regions 1 to 8 are deleted may also be called N-terminally deleted DGAT2C.

[0039] Diacylglycerol acyltransferase (DGAT) is a protein (enzyme) that catalyzes the reaction in which an acyl group is transferred from acyl-CoA to diacylglycerol, thereby producing triacylglycerol (EC2.3.1.20). This activity is also called "diacylglycerol acyltransferase activity (DGAT activity)." The gene encoding DGAT is also called the "diacylglycerol acyltransferase gene (DGAT gene)." 1,2-diacylglycerol is an example of diacylglycerol.

[0040] The DGAT gene and the base and amino acid sequences of DGAT found in various organisms such as Labyrinthula microorganisms can be obtained from publicly available databases such as NCBI (National Center for Biotechnology Information). Known DGATs include diacylglycerol acyltransferase 1 (DGAT1) and diacylglycerol acyltransferase 2 (DGAT2), and examples of DGAT2 include DGTT4, DGAT2A, DGAT2B, DGAT2C, DGAT2D, DGAT2E, DGAT2F, DGAT2G, DGAT2H, DGAT2I, DGAT2J, and DGAT2K. In this embodiment, it is preferable to use DGAT2C from Aurantiochytrium limacinum mh0186.

[0041] As shown in Figure 1, DGAT2C of Aurantiochytrium limacinum mh0186 has 12 presumed transmembrane regions on the N-terminal side. In this specification, the first presumed transmembrane region from the N-terminal side is numbered presumed transmembrane region 1, the second presumed transmembrane region is numbered presumed transmembrane region 2, and so on, with the 12 presumed transmembrane regions being referred to as presumed transmembrane regions 1 to 12. In this embodiment, the modified diacylglycerol acyltransferase 2C (modified DGAT2C) is modified so that presumed transmembrane regions 1 to 8 are missing from the presumed transmembrane regions 1 to 12 in diacylglycerol acyltransferase 2C (DGAT2C). That is, the modified diacylglycerol acyltransferase 2C (modified DGAT2C) has presumed transmembrane regions 9 to 12 (indicated by * in Figure 1). Furthermore, the inventors have revealed that sequences containing presumed transmembrane regions 9 and 10 are essential for the SE synthase activity of DGAT2C (Ishibashi, Y., Sadamitsu, S., Fukahori, Y., Yamamoto, Y., Tanogashira, R., Watanabe, T., Hayashi, M., Ito, M., & Okino, N. (2023). Characterization of thraustochytrid-specific sterol O-acyltransferase: modification of DGAT2-like enzyme to increase the sterol production in Aurantiochytrium limacinum mh0186. Applied and environmentalmicrobiology, 89(11)).

[0042] The amino acid sequences of the estimated transmembrane regions 1 to 12 are as follows. Specifically, the modified diacylglycerol acyltransferase 2C (modified DGAT2C) in this embodiment is modified to lack the amino acid sequences represented by SEQ ID NOs. 2 to 9 in diacylglycerol acyltransferase 2C (DGAT2C), and the modified diacylglycerol acyltransferase 2C (modified DGAT2C) has the amino acid sequences of SEQ ID NOs. 10 to 13. Estimated transmembrane region 1: AWLPYIKITVLWTLTALVLFVVA (SEQ ID NO: 2) Estimated transmembrane region 2: VGFGFLAACGGASLFLAIPATHW (SEQ ID NO: 3) Estimated transmembrane region 3: FVVLQAISWTFYGITAVIVMGGV (SEQ ID NO: 4) Estimated transmembrane region 4: MLASAGVIGILSQVFMVSSLLTY (SEQ ID NO: 5) Estimated transmembrane region 5: FVHMNTGLVVIGIALGLLS (SEQ ID NO: 6) Estimated transmembrane region 6: PILGLLSLMCCIVAISLTHGIGG (SEQ ID NO: 7) Estimated transmembrane region 7: FVFLQAFGWCFFGISILAQGAFV (SEQ ID NO: 8) Estimated transmembrane region 8: MYIGALAAVVSHYAIVSSLP (SEQ ID NO: 9) Estimated transmembrane region 9: ILFCNLQFMSFVVYMVLFTIPYF (SEQ ID NO: 10) Estimated transmembrane region 10: WWILMWAVAFWTFFYSALNNWGI (SEQ ID NO: 11) Estimated transmembrane region 11: ISLSLAAVAAYGGVLSAF (SEQ ID NO: 12) Estimated transmembrane region 12: HYMMLLVICTANLVYISTTF (SEQ ID NO: 13)

[0043] A modified DGAT2C gene, which is modified to delete the presumptive transmembrane regions 1-8, may have the entire N-terminal region containing the presumptive transmembrane regions 1-8 deleted (deleted), or it may have only the gene encoding the presumptive transmembrane regions 1-8 deleted (deleted).

[0044] In this embodiment, the modified diacylglycerol acyltransferase 2C (modified DGAT2C) is preferably the protein described in any of (a) to (c) below. (a) A protein consisting of the amino acid sequence shown in Sequence ID No. 1. (b) A protein having diacylglycerol acyltransferase activity, comprising one or more amino acid residue substitutions, additions, insertions and / or deletions in the amino acid sequence shown in Sequence ID No. 1. (c) A protein that has 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1 and has diacylglycerol acyltransferase activity.

[0045] The modified DGAT2C is preferably the protein described in (a) above, and the amino acid sequence shown in Sequence ID No. 1 is an amino acid sequence modified so that the putative transmembrane region 1-8 of diacylglycerol acyltransferase 2C (DGAT2C) is deleted.

[0046] Modified DGAT2C may be the protein described in (b) above. That is, modified DGAT2C may be a protein that includes substitution, addition, insertion and / or deletion of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 1, and has diacylglycerol acyltransferase activity. "One or more" means different depending on the position of the amino acid residues in the three-dimensional structure of the protein and the type of amino acid residues, but specifically it means, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3. In addition, the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 1 may be extended or shortened.

[0047] Substitutions, additions, insertions, and / or deletions of one or more amino acid residues are preferably conservative mutations that maintain normal protein function. Specifically, these are conservative mutations that maintain normal diacylglycerol acyltransferase activity. A typical example of a conservative mutation is a conservative substitution. A conservative substitution is a mutation in which the substitution site is between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid; between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid; between Gln and Asn when the substitution site is a polar amino acid; between Lys, Arg, and His when the substitution site is a basic amino acid; between Asp and Glu when the substitution site is an acidic amino acid; and between Ser and Thr when the amino acid has a hydroxyl group. Substitutions considered conservative include, specifically, substitutions from Ala to Ser or Thr, from Arg to Gln, His or Lys, from Asn to Glu, Gln, Lys, His or Asp, from Asp to Asn, Glu or Gln, from Cys to Ser or Ala, from Gln to Asn, Glu, Lys, His, Asp or Arg, from Glu to Gly, Asn, Gln, Lys or Asp, from Gly to Pro, from His to Asn, Lys, Gln, Arg or Tyr, and Il Examples of substitutions include e to Leu, Met, Val, or Phe; Leu to Ile, Met, Val, or Phe; Lys to Asn, Glu, Gln, His, or Arg; Met to Ile, Leu, Val, or Phe; Phe to Trp, Tyr, Met, Ile, or Leu; Ser to Thr or Ala; Thr to Ser or Ala; Trp to Phe or Tyr; Tyr to His, Phe, or Trp; and Val to Met, Ile, or Leu. Furthermore, such amino acid substitutions, deletions, insertions, or additions may also result from naturally occurring mutations (mutants or variants) based on individual differences or species differences in the organisms from which the genes originate.

[0048] Modified DGAT2C may be the protein described in (c) above. That is, modified DGAT2C may be a protein that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and has diacylglycerol acyltransferase activity. The sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1 is preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more.

[0049] The percentage of sequence identity in amino acid sequences can be determined, for example, using a mathematical algorithm. Examples of such mathematical algorithms include the algorithm in Myers and Miller (1988) CABIOS 4:11-17, the local homology algorithm in Smith et al (1981) Adv. Appl. Math. 2:482, the homology alignment algorithm in Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, the similarity search method in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448, and improved versions such as the algorithm in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, as described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

[0050] In this embodiment, a modified DGAT2C gene, in which the putative transmembrane regions 1-8 are deleted, is expressed in Labyrinthula microorganisms, and the production of sterol esters has been successfully increased by expressing the modified DGAT2C gene. More specifically, by modifying Labyrinthula microorganisms so that the activity of sterol 24-C-methyltransferase (SMT1) is reduced or lost compared to the unmodified strain (wild strain), the selective production capacity of cholesterol is enhanced. Furthermore, by expressing a modified DGAT2C gene in which the putative transmembrane regions 1-8 are deleted in Labyrinthula microorganisms, cholesterol esters are synthesized from cholesterol, and as a result, the production capacity of cholesterol esters is increased.

[0051] In order to express a modified DGAT2C gene in which the putative transmembrane regions 1-8 are deleted in Labyrinthula microorganisms, it is preferable to employ a method of transforming the host with an expression vector containing the modified DGAT2C gene with the putative transmembrane regions 1-8 deleted. Transformation can be carried out, for example, by methods commonly used for the transformation of Labyrinthula microorganisms. One such method is electroporation.

[0052] Modified DGAT2C can only be expressed by introducing its expression vector into Labyrinthullidae microorganisms. When unmodified (full-length) DGAT2C is expressed, no band of the same size as that of modified DGAT2C appears in Western blot analysis, thus ruling out the possibility that unmodified DGAT2C is converted to modified DGAT2C within the cells of Labyrinthullidae microorganisms (Figure 2). Furthermore, when modified DGAT2C is expressed, the expression of unmodified (full-length) DGAT2C is suppressed (Figure 2).

[0053] In this embodiment, the Labyrinthula microorganism is preferably a microorganism with accession number FERM P-22511 that has been modified to express the modified diacylglycerol acyltransferase 2C (modified DGAT2C) gene. Accession number FERM P-22511 is a Labyrinthula microorganism that has been modified so that the activity of sterol 24-C-methyltransferase (SMT1) is reduced or lost compared to the unmodified strain. The growth capacity of accession number FERM P-22511 is preferably 80% or more of the growth capacity of the unmodified strain.

[0054] The Labyrinthula microorganism in this embodiment is preferably the microorganism with accession number: FERM P-22512. Accession number: FERM P-22512 is a Labyrinthula microorganism modified to have reduced or lost sterol 24-C-methyltransferase (SMT1) activity compared to the unmodified strain, and is a microorganism modified to express a modified DGAT2C gene. The growth capacity of accession number: FERM P-22512 is preferably 80% or more of the growth capacity of the unmodified strain.

[0055] Accession numbers FERM P-22511 and FERM P-22512 are deposited with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (Postal Code: 292-0818, Address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan).

[0056] <Proliferative ability> The growth ability of the Labyrinthuria microorganisms in this embodiment is preferably 80% or more of the growth ability of the unmodified strain, more preferably 85% or more, even more preferably 90% or more, and particularly preferably 95% or more. Furthermore, the growth ability of the Labyrinthuria microorganisms in this embodiment may be 100% or more of the growth ability of the unmodified strain, and may even be higher than the growth ability of the modified strain. In other words, it is preferable that the growth ability of the Labyrinthuria microorganisms in this embodiment is equivalent to or greater than the growth ability of the unmodified strain.

[0057] The growth capacity of Labyrinthula microorganisms can be evaluated by collecting 1 ml of culture medium, centrifugating at 5,000 x g for 3 minutes to collect the cells, cooling in a -80°C freezer, and measuring the dry weight of the freeze-dried sample. The growth capacity can be calculated using the following formula. Growth capacity (%) = Dry weight of the modified Labyrinthull microorganism / Dry weight of the unmodified Labyrinthull microorganism × 100

[0058] Conventionally, modified Labyrinthula microorganisms have sometimes exhibited inferior growth ability, and there have been concerns that Labyrinthula microorganisms modified in such a way that the activity of sterol 24-C-methyltransferase (SMT1) is reduced or lost compared to unmodified strains (wild strains) may have significantly inferior growth ability. However, the Labyrinthula microorganisms of this embodiment do not experience a decrease in growth ability even when modified to reduce or lose the activity of sterol 24-C-methyltransferase (SMT1), and furthermore, their growth ability does not decrease even when modified to express a modified DGAT2C) gene, thus enabling enhanced sterol ester production.

[0059] In this embodiment, the reason why the growth ability of the modified Labyrinthula microorganisms was restored is thought to be due to improved expression levels of genes that have amino acid sequence homology to ferroptosis-suppressing protein 1, which suppresses cell death, and genes involved in the suppression of lipid peroxidation, which affects cell survival and proliferation. The expression levels of the above genes can be analyzed by transcriptome analysis, etc.

[0060] <Sterol ester production capacity> As described above, the Labyrinthula microorganisms of this embodiment have the ability to produce sterol esters. It can also be said that the Labyrinthula microorganisms of this embodiment are microorganisms for sterol ester production. Furthermore, this embodiment may also relate to the use of Labyrinthula microorganisms for the production of sterol esters.

[0061] A "sterol ester" is an acyl ester of a free sterol, a compound having a structure in which a free sterol and a fatty acid are linked by an ester bond. In other words, a sterol ester has a part corresponding to a free sterol and a part corresponding to a fatty acid, linked by an ester bond. The part corresponding to the free sterol is called the "sterol part," and the part corresponding to the fatty acid is called the "fatty acid part" or "acyl group." The ester bond is formed between the hydroxyl group at the C3 position of the sterol and the carboxyl group of the fatty acid. Therefore, the hydroxyl group at the C3 position does not need to remain in the sterol ester.

[0062] The free sterols constituting the sterol ester are preferably animal sterols, and particularly preferably cholesterol. Furthermore, the type of acyl group constituting the sterol ester is not particularly limited, and the chain length and degree of unsaturation of the acyl group are variable. The chain length of the acyl group may be, for example, C14-C26 (e.g., C14, C16, C18, C20, C22, C24, or C26). The acyl group may be saturated or unsaturated. The acyl group may have one or more (e.g., one, two, three, four, five, or six) unsaturated double bonds. Specific examples of fatty acids that correspond to the acyl group include myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), arachidic acid (20:0), behenic acid (22:0), lignoceric acid (24:0), cerotic acid (26:0), myristoleic acid (14:1), palmitoleic acid (16:1), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4), eicosapentaenoic acid (EPA, 20:5), docosapentaenoic acid (DPA, 22:5), and docosahexaenoic acid (DHA, 22:6). The sterol ester obtained in this embodiment preferably contains an acyl group derived from at least one selected from the group consisting of docosahexaenoic acid (DHA, 22:6), docosapentaenoic acid (DPA, 22:5), eicosapentaenoic acid (EPA, 20:5), and palmitic acid (16:0), and more preferably contains an acyl group derived from at least one selected from docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA).

[0063] The sterol ester production capacity of the Labyrinthura microorganisms of this embodiment is preferably 2 times or more, more preferably 3 times or more, even more preferably 4 times or more, even more preferably 5 times or more, even more preferably 6 times or more, even more preferably 7 times or more, particularly preferably 8 times or more, and most preferably 9 times or more, compared to the sterol ester production capacity of the unmodified strain. The upper limit of the sterol ester production capacity of the Labyrinthura microorganisms of this embodiment is not particularly limited, but it is preferably 100 times or less compared to the sterol ester production capacity of the unmodified strain. The Labyrinthura microorganisms of this embodiment preferably have the ability to produce cholesterol esters, and it is also preferable that the cholesterol ester production capacity is within the above range. Thus, the Labyrinthura microorganisms of this embodiment have a high sterol ester (cholesterol ester) production capacity, and sterol esters (cholesterol esters) can be recovered from the Labyrinthura microorganisms in high yield.

[0064] <Other production capacity> The Labyrinthula microorganisms of this embodiment may have the ability to produce sterols in addition to the sterol esters described above, and it is preferable that they have the ability to produce provitamin D sterols. Examples of provitamin D sterols include 7-dehydrocholesterol (provitamin D3) and ergosterol (provitamin D2). Among these, it is preferable that the Labyrinthula microorganisms of this embodiment have the ability to produce 7-dehydrocholesterol (provitamin D3).

[0065] The provitamin D3 production capacity of the Labyrinthula microorganisms of this embodiment is preferably 1.5 times or more, more preferably 2 times or more, even more preferably 3 times or more, and even more preferably 5 times or more, compared to the provitamin D3 production capacity of the unmodified strain. The upper limit of the provitamin D3 production capacity of the Labyrinthula microorganisms of this embodiment is not particularly limited, but it is preferably 100 times or less compared to the provitamin D3 production capacity of the unmodified strain.

[0066] Furthermore, the Labyrinthula microorganisms of this embodiment may also have the ability to produce functional substances such as astaxanthin, which has antioxidant properties, in addition to the sterol esters and sterols mentioned above. The sterol esters, sterols, and functional substances produced by the Labyrinthula microorganisms of this embodiment can be used, for example, alone, in combination with other components, or as raw materials for other components. In addition, the sterol esters, sterols, and functional substances produced by the Labyrinthula microorganisms of this embodiment can be used as food additives, feed additives (e.g., livestock feed and aquatic feed), health foods, pharmaceuticals, chemical products, and cosmetic ingredients.

[0067] (Method for producing sterol esters) This embodiment relates to a method for producing sterol esters, comprising culturing the modified Labyrinthula microorganisms described above and recovering sterol esters from the microbial cells produced by the culturing. Examples of sterol esters produced include the sterol esters described above, but it is preferable that the produced sterol esters contain an acyl group derived from at least one selected from docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA).

[0068] The culture medium used in the method for producing sterol esters according to this embodiment is not particularly limited, as long as the Labyrinthurida microorganisms of this embodiment can grow and sterol esters, etc., can be produced. As the culture medium, for example, a conventional culture medium used for culturing heterotrophic microorganisms such as Labyrinthurida can be used. The culture medium may contain, as necessary, a carbon source, a nitrogen source, a phosphate source, a sulfur source, and other components selected from various organic and inorganic components. Specifically, as the culture medium, for example, GY medium prepared with 1-3% artificial seawater can be used.

[0069] Specific examples of carbon sources include sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, molasses, starch hydrolysates, and biomass hydrolysates; organic acids such as acetic acid and citric acid; alcohols such as ethanol, glycerol, and crude glycerol; and fatty acids. One carbon source may be used, or two or more carbon sources may be used in combination.

[0070] Specific examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; peptone; organic nitrogen sources such as yeast extract, meat extract, and soy protein hydrolysate; ammonia; and urea. Ammonia gas or ammonia water used for pH adjustment may also be used as a nitrogen source. One nitrogen source may be used, or two or more nitrogen sources may be used in combination.

[0071] Examples of phosphate sources include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate. One phosphate source may be used, or two or more phosphate sources may be used in combination.

[0072] Examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. A single sulfur source may be used, or a combination of two or more sulfur sources may be used.

[0073] Other various organic and inorganic components include, specifically, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components containing these, such as peptone, casamino acid, yeast extract, and soy protein hydrolysate. These other organic and inorganic components may be used individually, or in combination of two or more components.

[0074] The culture conditions are not particularly limited, as long as the Labyrinthuridae microorganisms of this embodiment can grow and sterol esters can be produced. Culture can be carried out, for example, under the usual conditions used for culturing heterotrophic microorganisms such as Labyrinthuridae.

[0075] Culturing can be carried out using a liquid medium. When culturing, the Labyrinthuria microorganisms of this embodiment may be cultured on a solid medium such as agar medium and then directly inoculated into the liquid medium, or the Labyrinthuria microorganisms of this embodiment may be cultured as a seed culture on a liquid medium and then inoculated into the liquid medium for the main culture. Thus, cultivation may be carried out separately as a seed culture and a main culture. In this case, the culture conditions for the seed culture and the main culture may be the same or different.

[0076] Culture can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. The culture medium used at the start of culture is also called the "initial culture medium." The culture medium supplied to the culture system (fermenter) in fed-batch or continuous culture is also called the "fed-batch medium." The act of supplying the fed-batch medium to the culture system in fed-batch or continuous culture is also called "fed-batch." When culture is carried out separately as seed culture and main culture, for example, both seed culture and main culture may be carried out by batch culture. Alternatively, for example, seed culture may be carried out by batch culture and main culture by fed-batch or continuous culture.

[0077] Culturing can be carried out, for example, under aerobic conditions. Aerobic conditions mean that the dissolved oxygen concentration in the liquid medium is 0.33 ppm or higher, which is the detection limit of the oxygen membrane electrode, and preferably 1.5 ppm or higher. The oxygen concentration may be controlled, for example, to 5-50% of the saturated oxygen concentration, preferably to about 10%. Culturing under aerobic conditions can be carried out by aeration culture, shaking culture, stirring culture, or a combination thereof. The pH of the medium may be, for example, pH 3-10, preferably pH 4.0-9.5. The pH of the medium can be adjusted as needed during cultivation. The pH of the medium can be adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide, hydrochloric acid, sulfuric acid, etc. The culture temperature may be, for example, 20°C-35°C, preferably 25°C-35°C. The culture period may be, for example, 10-120 hours. The culture may be continued, for example, until the carbon source in the culture medium is consumed, or until the activity of the Labyrinthula microorganisms of this embodiment is lost. By culturing the Labyrinthula microorganisms of this embodiment under such conditions, cells that produce sterol esters can be obtained.

[0078] Sterol esters can be recovered from bacterial cells as appropriate. That is, sterol esters can be extracted and recovered from bacterial cells. Bacterial cells may be subjected to sterol extraction while still contained in the culture medium, or they may be recovered from the culture medium before being subjected to sterol ester extraction. In addition, bacterial cells (for example, the culture medium containing bacterial cells or bacterial cells recovered from the culture medium) may be subjected to treatments such as dilution, concentration, freezing, thawing, or drying as appropriate before being subjected to sterol ester extraction. These treatments may be performed individually or in appropriate combinations. These treatments can be appropriately selected according to various conditions such as the type of sterol ester extraction method.

[0079] The method for recovering bacterial cells from the culture medium is not particularly limited, and known methods (Grima, EM et al. 2003. Biotechnol. Advances 20: 491-515) can be used, for example. Specifically, bacterial cells can be recovered from the culture medium by methods such as natural sedimentation, centrifugation, and filtration. A flocculant may also be used in this process. The recovered bacterial cells can be washed as appropriate using a suitable medium. The recovered bacterial cells can also be resuspended as appropriate using a suitable medium. Examples of media that can be used for washing and suspension include aqueous media (aqueous solvents) such as water and aqueous buffer solutions, organic media (organic solvents) such as methanol, and mixtures thereof. The medium can be appropriately selected according to various conditions such as the type of sterol ester extraction method.

[0080] The method for extracting sterol esters is not particularly limited, and known methods can be used, for example. Such methods include, for example, methods for extracting lipids from the cells of microorganisms such as common algae. Specifically, such methods include, for example, organic solvent treatment, ultrasonic treatment, bead crushing treatment, acid treatment, alkali treatment, enzyme treatment, hydrothermal treatment, supercritical treatment, microwave treatment, electromagnetic field treatment, and compression treatment. These methods can be used individually or in appropriate combinations.

[0081] The organic solvent used in the organic solvent treatment is not particularly limited as long as it can extract sterol esters from the bacterial cells. Examples of organic solvents include alcohols such as methanol, ethanol, 2-propanol, butanol, pentanol, hexanol, heptanol, and octanol; ketones such as acetone; ethers such as dimethyl ether and diethyl ether; esters such as methyl acetate and ethyl acetate; alkanes such as n-hexane; and chloroform. Methods for extracting lipids with organic solvents include the Bligh-Dyer method and the Folch method. One type of organic solvent may be used, or a combination of two or more organic solvents may be used.

[0082] When alkaline treatment is performed during the extraction of sterol esters, the pH of the alkaline treatment is not particularly limited as long as it is a pH at which sterol esters can be extracted from the bacterial cells. The pH of the alkaline treatment is usually 8.5 or higher, preferably 10.5 or higher, more preferably 11.5 or higher, and may be 14 or lower. The temperature of the alkaline treatment is usually 30°C or higher, preferably 50°C or higher, and more preferably 70°C or higher. The temperature of the alkaline treatment may preferably be 120°C or lower. The time of the alkaline treatment is usually 10 minutes or more, preferably 30 minutes or more, and more preferably 50 minutes or more. The time of the alkaline treatment may preferably be 150 minutes or less. Alkaline substances such as NaOH and KOH can be used for the alkaline treatment.

[0083] The extracted sterol esters can be recovered using known methods used for the separation and purification of compounds. Examples of such methods include ion exchange resin methods and membrane treatment methods. These methods can be used individually or in appropriate combinations.

[0084] The recovered material may contain other products besides sterol esters, such as free sterols and functional substances. Furthermore, the recovered material may contain bacterial cells, culture medium components, water, and components used in the extraction process.

[0085] If necessary, sterol esters may be purified from the recovered material. The purity of the purified sterol esters may be, for example, 1% (w / w) or more, 2% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, 30% (w / w) or more, 50% (w / w) or more, 70% (w / w) or more, 90% (w / w) or more, or 95% (w / w) or more.

[0086] The type and amount of sterol esters can be determined by known methods used for the detection or identification of compounds. Such methods include, for example, HPLC, LC / MS, GC / MS, and NMR. These methods can be used individually or in appropriate combinations. [Examples]

[0087] The features of the present invention will be further described below with reference to examples and comparative examples. The materials, amounts used, proportions, processing content, and processing procedures shown in the following examples can be modified as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be interpreted as being limited by the following specific examples.

[0088] (culture medium) • PDA medium (*Hyg+, **Neo+) PDA 0.8% (w / v) SEALIFE 1.7% (w / v) Agar 1.2% (w / v) *Hygromycine 2 mg / μl **G418 0.5 mg / μl

[0089] GY medium D-Glucose 3.1% (w / v) Yeast Extract 1.1% (w / v) SEALIFE 1.8% (w / v) After autoclaving, 1 / 1000 volume of Vitamin mix and 1 / 500 volume of 5 x Element solution were added.

[0090] Vitamin Mix Vitamin B10 2% (w / v) Vitamin B2 0.001% (w / v) Vitamin B 21 0.001% (w / v)

[0091] • 5 x Element solution EDTA di-sodium 3% (w / v) FeCl3·6H2O 0.15% (w / v) H3BO 33.4% (w / v) MnCl2·4H2O 0.43% (w / v) ZnSO4·7H2O 0.13% (w / v) CoCl2·6H2O 0.013% (w / v) NiSO4·6H2O 0.026% (w / v) CuSO4·5H2O 0.001% (w / v) Na2MoO4·2H2O 0.0025% (w / v)

[0092] (cell line) As the cell line, we used Aurantiochytrium limacinum mh0186 (mh0186 strain) isolated from seawater. Aurantiochytrium limacinum mh0186 was originally deposited on March 29, 2004, with accession number FERM P-19755 at the Patent Microorganism Depository Center of the National Institute of Advanced Industrial Science and Technology (now the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation, postal code: 292-0818, address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan), and was subsequently transferred to international depository, where it was assigned accession number FERM BP-11311.

[0093] (Creation of SMT1-deficient strains) PCR was performed using the genomic DNA of strain mh0186 as a template, and 3,006 bp were amplified from 1,010 bp upstream of the start codon to the middle of the SMT1 sequence. Primers 1 and 2 (Table 1) and Tks Gflex DNA polymerase (Takara bio) were used for PCR, and the program was 94°C for 1 min, (98°C for 10 sec, 55°C for 15 sec, 68°C for 2 min) x 35 cycles, followed by 68°C for 2 min. The amplified sequence and the pUC19 linearized vector were subjected to an in-fusion reaction using the In-Fusion HD Cloning Kit (Takara bio) to transform E. coli (JM109). Selection was performed on LB agar medium containing 50 μg / ml ampicillin, and the SMT1 KO pUC19 vector was obtained by plasmid extraction. Next, using the SMT1 KO pUC19 vector as a template, PCR was performed with primers 3 and 4 (Table 1), Tks Gflex DNA polymerase, and a program of 94°C for 1 min, (98°C for 10 sec, 55°C for 15 sec, 68°C for 2.5 min) x 35 cycles, followed by 68°C for 2 min to amplify the SMT1 KO pUC19 linearized vector. In addition, hygromycin cassettes were amplified by PCR using primers 5 and 6 (Table 1) (doi:10.1194 / jlr.M024935). The PCR enzyme used was Tks Gflex DNA polymerase, and the PCR program was 94°C for 1 min, (98°C for 10 sec, 55°C for 15 sec, 68°C for 1.5 min) x 35 cycles, followed by 68°C for 2 min. After gel purification, the SMT1 KO pUC19 linearized vector and fragments of a hygromycin cassette containing loxP sequences at both ends were subjected to an in-fusion reaction using the In-Fusion HD Cloning Kit, and Escherichia coli (JM109) was transformed. SMT1 KO vectors were obtained by plasmid extraction using ampicillin resistance as a selection method.

[0094] Using an SMT1 KO vector as a template, the SMT1-deficient construct was amplified by PCR using primers 5 and 6 (Table 1). The PCR enzyme used was Tks Gflex DNA polymerase, and the PCR program was 94°C for 1 min, (98°C for 10 sec, 55°C for 15 sec, 68°C for 2.25 min) x 35 cycles, followed by 68°C for 2 min. The obtained SMT1-deficient construct was purified by ethanol precipitation and introduced into the mh0186 strain using electroporation. Electroporation was performed using a 1 mm gap cuvette, 750 V, 25 μF, and 200 Ω, with two pulses applied. 700 μl of GY medium was added, and the cells were recovered overnight at 25°C. Candidate SMT1-deficient strains were selected based on hygromycin resistance genes. Colonies grown on hygromycin-containing PDA plates were picked, cultured in 3 ml of GY medium for 2 days, and then the cells were harvested. Genomic DNA was extracted by resuspending the cells in 20 mM NaOH solution and treating at 37°C for 10 minutes. Using this genome as a template, the deletion of the SMT1 gene was confirmed by PCR using primers 7 and 8, and 9 and 10 (Table 1). The PCR enzyme used was Tks Gflex DNA polymerase, and the PCR program was 94°C for 1 min, (98°C for 10 sec, 55°C for 15 sec, 68°C for 1.5 min) x 35 cycles, and 68°C for 2 min. Furthermore, the deletion of the SMT1 gene was confirmed by PCR using primers 9 and 10 (Table 1). The PCR enzyme used was Tks Gflex DNA polymerase, and the PCR program was 94°C for 1 min, (98°C for 10 sec, 55°C for 15 sec, 68°C for 3.0 min) x 35 cycles, and 68°C for 2 min.

[0095] The names and sequences of the primers used to create SMT1-deficient strains and analyze SMT1 are as follows.

[0096] [Table 1]

[0097] Figure 3 shows the production levels of various sterols in the wild-type strain, the DHCR24-deficient strain, and the SMT1-deficient strain obtained above. As shown in Figure 3, only cholesterol was selectively produced in the SMT1-deficient strain.

[0098] (Subculturing of SMT1-deficient strains and acquisition of proliferating, recovered strains) The SMT1-deficient strains obtained above were subcultured in 3 ml of GY medium with 10 μl of culture solution once a week for 19 weeks (4.5 months), and cultured at 25°C. This process was repeated to obtain SMT1-deficient strains with restored growth ability (SMT1-deficient subcultivated strains). The growth ability of the SMT1-deficient subcultivated strains was evaluated by measuring turbidity (OD600) (Figure 4). As shown in Figure 4, the growth ability of the SMT1-deficient subcultivated strains was comparable to that of the wild-type strain.

[0099] (Acquisition of an N-terminally deficient DGAT2C overexpressing strain) An expression vector of DGAT2C with a FLAG tag attached to the C-terminus was constructed using the following method. The ORF of DGAT2C was amplified by PCR using the genomic DNA of strain mh0186 as a template. For PCR, DGAT2C-FLAG-S and DGAT2C-FLAG-A (Table 2), designed to attach the FLAG tag, and Tks Gflex DNA polymerase (Takara bio) were used as primers. Inverse PCR was performed using pEF-Neor / Ubi-mCherry as a template and primers (A.L_vector_Rv and A.L_vector_Fw) designed to remove mCherry. These two PCR products were subjected to an in-fusion reaction using the In-Fusion HD Cloning Kit, and E. coli (JM109) was transformed. This resulted in obtaining a vector in which DGAT2C is expressed under the control of the ubiquitin promoter (DGAT2C-FLAG expression vector). Next, using this vector as a template, a vector expressing a product with eight deletions in the N-terminal presumed transmembrane region of DGAT2C was constructed using the following method. Specifically, PCR was performed using Tks Gflex DNA polymerase with pAlim-TMd4-2C-infu-S and A.L_vector_Rv (Table 2) as primers, and the amplified product was subjected to an infusion reaction to construct a vector with sequences corresponding to eight deletions in the N-terminal transmembrane region. Using this vector as a template, PCR was performed with the primer combination of pENUM transfection-S and transfection PCR-A (Table 2). After ethanol precipitation of the amplified product, it was introduced into SMT1-deficient passages (strains 1-8) by electroporation. Electroporation was performed using a 1 mm gap cuvette, 750 V, 25 μF, and 200 Ω, with two pulses applied. 700 μl of GY medium was added, and recovery culture was performed overnight at 25°C. Candidate strains overexpressing N-terminally knocked DGAT2C were selected based on the neomycin resistance gene. The culture medium after recovery culture was spread onto G418-containing PDA plates, and high-cholesterol-producing strains (strains 1, 2, and 4) that were SMT1-deficient and overexpressed N-terminally knocked DGAT2C were established from the resulting colonies. Strain 3 was an SMT1-deficient strain but did not express N-terminally knocked DGAT2C.

[0100] [Table 2]

[0101] (Confirmation of growth characteristics) The cholesterol-high-producing strains (strains 1, 2, and 4) and the SMT1-deficient subculturing strain (strain 3) obtained above were cultured at 25°C for 4 days with shaking at 150 rpm so that the OD600 was 0.02. 1 ml of culture medium was collected, cells were collected by centrifugation at 5,000 xg for 3 min, and cooled in a -80°C freezer. The samples were frozen using a freeze-dryer, and the growth capacity was evaluated by measuring turbidity (OD600) (Figure 5) and dry weight (using samples cultured for 3 days) (Figure 6). For the dry weight shown in Figure 6, the wild-type mh0186 cultured for the same period was used as a comparison. As shown in Figures 5 and 6, the growth capacity of the cholesterol-high-producing strains (strains 1, 2, and 4) was almost the same as that of the wild-type strain.

[0102] (lipid extraction) 500 μl of the sample cultured as described above (confirmation of growth characteristics) was centrifuged at 5,000 xg for 3 min to precipitate the cells. 200 μl of water and a 0.2 ml tube full of 0.6 mm diameter glass beads (AS ONE Corp) were added, and the mixture was crushed at 3,000 rpm for 1 minute using a BEADS CRUSHER μT-12 (TAITEC), followed by 1 minute of ice cooling, repeated three times. Of the resulting cell lysate, 50 μl was mixed with 200 μl of chloroform / methanol = 2 / 1 (v / v) solution, and lipid extraction was performed at 37°C and 2,000 rpm with shaking for 30 minutes. Subsequently, the mixture was centrifuged at 20,000 xg for 3 min, and the lower 50 μl of the resulting two-layer sample was mixed with 550 μl of IPA. Lipid analysis was performed using the mass spectrometer described below.

[0103] (Confirmation of protein expression) The cell lysate obtained by the above (lipid extraction) was mixed with sample buffer (for SDS-PAGE, 6-fold concentrated, containing reducing agent), heated at 95°C for 3 minutes, and then the proteins were separated by SDS-PAGE and transferred to a PVDF membrane using Trusblot turbo (Bio-Rad). After blocking with TBS-T containing 5% BSA for 30 minutes, the cells were reacted with a 4000-fold diluted anti-DYKDDDDK tag antibody (Cell signaling technology) at 4°C for 20 hours. After washing three times with TBS-T, the cells were reacted with a 10000-fold diluted Rabbit IgG HRP (Cell signaling technology) for 1 hour, washed three times with TBS-T, and luminescence was induced using Luminata Western HRP substrate (Merck). The target protein (N-terminally deficient DGAT2C) was detected by Ez-Capture II (ATTO) (Figure 7). Of these, strain 3 did not express the target protein, indicating that it can be used as a non-expression control. On the other hand, bands of the expected size were detected in strains 1, 2, and 4, confirming that strains expressing the target protein could be established by introducing the construct.

[0104] (Lipid analysis using mass spectrometry) The extracted lipids were analyzed by LC-MS (3200 Q TRAP, AB SCIEX). LC-MS measurements were performed using Multiple Reaction Monitoring (MRM) with various Q1 / Q3 combinations to detect SEs (sterol esters) with different fatty acid compositions. Here, the precursor ion of the target lipid molecular species was set as Q1, and the characteristic product ion obtained by collisional dissociation was set as Q3. The solvent was flowed at a rate of 200 μl / min, and TGs and SEs were separated using an InertSustain C18 column (2.1 x 150 mm, 5 μm, GL Sciences). Solvents used were solvent A: acetonitrile / methanol / water = 19 / 19 / 2 (v / v / v) (containing 0.1% formic acid and 0.028% ammonia) and solvent B: isopropyl alcohol (0.1% formic acid and 0.028% ammonia). The MRM conditions and solvent gradient conditions are shown in Table 3. As shown in Figure 8, the high cholesterol-producing strains (strains 1, 2, and 4) showed a significant increase in the production of cholesterol esters with DHA and n-6DPA, while strain 3, which does not express the N-terminally deficient DGAT2C, showed remarkably low production of cholesterol esters with DHA and n-6DPA. Furthermore, as shown in Figure 9, the high cholesterol-producing strains (strains 1, 2, and 4) were shown to be able to produce provitamin D3 at high levels, while strain 3 showed remarkably low provitamin D3 production.

[0105] [Table 3]

[0106] Sequence ID 1: MRFDQKEDNKATDPRRYRVLESFTFWEHLSAWPIMILFCNLQFMSFVVYMVLFTIPYFMPDEWQPWLAQNTLDLIPDLNAYESNDFVHTFKLWWILMWAVAFWTFFYSALNNWGIEGWRQSISLSLAAVAAYGGVLSAFRDSPHYMMLLVICTANLVYISTTFTKRPEFNACREWDLFRRLQFLPRIVEKYFGLRVELTEEMQKFAPYLGDENAKDPRMHQVLLLFHPHGIFPVTHAVLPLSLTWRKLFPKLQVNSLSATINHIIPVMRDVIQWMGVCDVSRATVMNLIRMGRNLQIVCGGQTEMFESRSWDKKIAIVRKRRRGIFKIAIQQGLGIVPMYSFGEPQIFDNVYMPRTQAFFKRLLGFPFPFFMIGKFGLPIPCRVPVTVAIDAPVFPVKQNSNPTPEEVTEFQYRYFEKLEALFERYKHDNGHGEHELDFIDY

Claims

1. Labyrinthula microorganisms modified to have reduced or lost sterol 24-C-methyltransferase (SMT1) activity compared to unmodified strains, The aforementioned Labyrinthula microorganisms have been modified to express the modified diacylglycerol acyltransferase 2C (modified DGAT2C) gene. The modified diacylglycerol acyltransferase 2C (modified DGAT2C) is a Labyrinthula microorganism in which presumed transmembrane regions 1 to 8 of the presumed transmembrane regions 1 to 12 in diacylglycerol acyltransferase 2C (DGAT2C) are deficient.

2. The Labyrinthula microorganism according to claim 1, wherein the modified diacylglycerol acyltransferase 2C (modified DGAT2C) is a protein described in any of (a) to (c) below; (a) A protein consisting of the amino acid sequence shown in Sequence ID No. 1; (b) A protein having diacylglycerol acyltransferase activity, comprising one or more amino acid residue substitutions, additions, insertions and / or deletions in the amino acid sequence shown in Sequence ID No. 1; (c) A protein having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1, and possessing diacylglycerol acyltransferase activity.

3. The labyrinthula microorganism according to claim 1, wherein the labyrinthula microorganism is a microorganism belonging to the genus Aurantiochytrium.

4. The labyrinthula microorganism according to claim 3, wherein the labyrinthula microorganism is a microorganism derived from Aurantiochytrium limacinum.

5. The Labyrinthuria microorganism according to claim 1, wherein the growth ability of the Labyrinthuria microorganism is 80% or more of the growth ability of the unmodified strain.

6. A Labyrinthula microorganism according to claim 1, having the ability to produce sterol esters.

7. The Labyrinthula microorganism according to claim 6, further possessing the ability to produce provitamin D3.

8. The Labyrinthula microorganism according to claim 1, wherein the microorganism with accession number: FERM P-22511 is modified to express the modified diacylglycerol acyltransferase 2C (modified DGAT2C) gene.

9. The Labyrinthula microorganism according to claim 1, which is the microorganism with accession number: FERM P-22512.

10. A method for producing sterol esters, comprising culturing a Labyrinthula microorganism according to any one of claims 1 to 9, and recovering sterol esters from the microbial cells produced by the culturing.

11. The method for producing a sterol ester according to claim 10, wherein the sterol ester comprises an acyl group derived from at least one selected from docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA).

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