JP7151279B2 - Production method of sterols - Google Patents

Description

IPOD FERM BP-22342

TECHNICAL FIELD The present invention relates to a method for producing sterols using Labyrinthulid microorganisms.

Sterols are steroid derivatives found in various organisms such as animals, plants and fungi. Sterols have been produced, for example, by extraction from biological resources that contain them. Labyrinthula, which are heterotrophic microorganisms, are known to produce sterols such as cholesterol (Non-Patent Document 1). A method for producing sterols using modified strains of Labyrinthulid microorganisms has also been reported.

Sterols can also be present in the form of various derivatives such as sterol esters.

In higher animals such as humans, it is known that cholesterol ester is produced from cholesterol by the action of acyl-coenzyme A: cholesterol acyltransferase (ACAT). Also, in yeast, acyl-coenzyme A: sterol acyltransferase (ASAT) is known to produce sterol esters from sterols such as ergosterol and lanosterol (non-patent literature 2).

In addition, it has been reported that cholesterol esters were produced by enhancing the expression of the PSAT1 gene, which encodes phospholipid sterol acyltransferase (PSAT), in Labyrinthulid microorganisms capable of producing cholesterol (Non-Patent Document 3). .

Diacylglycerol acyltransferase (DGAT) is an enzyme that catalyzes the reaction that transfers an acyl group from acyl-CoA to diacylglycerol to produce triacylglycerol (EC 2.3.1.20). As DGAT, two families (DGAT1 and DGAT2) are known (Non-Patent Document 4). The relationship between DGAT and sterol production such as sterol esters is unknown.

Weete JD, et al., Lipids and ultrastructure of Thraustochytrium sp. ATCC 26185., Lipids. 1997 Aug;32(8):839-45. Zweytick D, et al., Contribution of Are1p and Are2p to steryl ester synthesis in the yeast Saccharomyces cerevisiae. Eur J Biochem. 2000 Feb; 267(4): 1075-82. Ishimaru et al., Mechanism of synthesis of PUFA-containing sterol esters in marine microorganism Labyrinthulids, Abstracts of the 2016 Annual Meeting of the Japan Society for Bioscience, Biotechnology and Agrochemistry Chi-Liang Eric Yen, et al., DGAT enzymes and triacylglycerol biosynthesis. J Lipid Res. 2008 Nov; 49(11): 2283-2301.

An object of the present invention is to provide a method for producing sterols.

As a result of intensive research to solve the above problems, the present inventor found that by modifying a Labyrinthulea microorganism to increase the activity of DGAT, the sterol-producing ability of the microorganism can be improved. , completed the present invention.

That is, the present invention can be exemplified as follows.
[1]
A method for producing a sterol, comprising:
culturing a Labyrinthulea microorganism having sterol-producing ability in a medium;
recovering sterols from the cells produced by the culture;
including
A method, wherein the microorganism has been modified to have an increased diacylglycerol acyltransferase (DGAT) activity compared to an unmodified strain.
[2]
The above method, wherein the DGAT is DGAT2.
[3]
The method, wherein the DGAT is a protein according to (a), (b), or (c) below:
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 ;
(b) a protein comprising an amino acid sequence containing substitutions, deletions, insertions, and/or additions of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 and having DGAT activity;
(c) A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having DGAT activity.
[4]
The above method, wherein the activity of the DGAT is increased by increasing the expression of the gene encoding the DGAT.
[5]
The above method, wherein the expression of the gene is increased by increasing the copy number of the gene and/or by modifying the expression regulatory sequence of the gene.
[6]
The above method, wherein the sterol is cholesterol.
[7]
The above method, wherein the sterol is a sterol ester.
[8]
The method, wherein the microorganism further has the following properties (A) and/or (B):
(A) the microorganism has been modified such that the activity of sterol 24-C-methyltransferase is reduced compared to an unmodified strain;
(B) the microorganism is modified such that the activity of 24-dehydrocholesterol reductase is increased compared to an unmodified strain;
[9]
The above method, wherein the microorganism belongs to the genus Aurantiochytrium, Schizochytrium, Thraustochytrium, or Parietichytrium.
[10]
The above method, wherein the microorganism is Aurantiochytrium sp.1.

INDUSTRIAL APPLICABILITY According to the present invention, the sterol-producing ability of Labyrinthulid microorganisms can be improved, and sterols can be produced efficiently.

The present invention will be described in detail below.

<1> Microorganism of the Present Invention The microorganism of the present invention is a sterol-producing Labyrinthulid microorganism that has been modified to increase DGAT activity.

<1-1> Labyrinthulea Microorganism The term “Labyrinthulan microorganism” refers to a microorganism classified into the Labyrinthulea. Labyrinthula microorganisms include the genus Aurantiochytrium, the genus Schizochytrium, the genus Thraustochytrium, the genus Parietichytrium, the genus Labyrinthula, the genus Althornia , Aplanochytrium, Japonochytrium, Labyrinthuloides, Ulkenia, Oblongichytrium, Botryochytrium, and Examples thereof include microorganisms belonging to genera such as the genus Sicyoidochtrium. Labyrinthulid microorganisms include, in particular, Aurantiochytrium, Schizochytrium, Thraustochytrium, and Parietichytrium microorganisms. Labyrinthulid microorganisms more particularly include Aurantiochytrium microorganisms. Aurantiochytrium microorganisms include, for example, Aurantiochytrium limacinum, Aurantiochytrium sp.1, and Aurantiochytrium sp.2. Aurantiochytrium genus microorganisms include, in particular, Aurantiochytrium sp.1. Schizochytrium microorganisms include, for example, Schizochytrium aggregatum. Thraustochytrium microorganisms include, for example, Thraustochytrium aureum. Parietichytrium genus microorganisms include, for example, Parietichytrium sarkarianum. Botryochytrium microorganisms include, for example, Botryochytrium radiatum.

"Aurantiochytrium sp.1" refers to a specific species of the genus Aurantiochytrium, specifically to the species to which Aurantiochytrium sp.1 strain AJ7869 (FERM BP-22342) belongs. In other words, "Aurantiochytrium sp.1" specifically refers to a group of microorganisms composed of the Aurantiochytrium sp.1 strain AJ7869 and strains belonging to the same species. As a strain belonging to the same species as the Aurantiochytrium sp.1 AJ7869 strain, the 18S rDNA nucleotide sequence has 97% or more identity to the 18S rDNA nucleotide sequence (SEQ ID NO: 9) of the Aurantiochytrium sp.1 AJ7869 strain. is mentioned. Specific examples of Aurantiochytrium sp.1 include, in addition to the AJ7869 strain, the AJ7867 strain (FERM BP-22304), the AJ7868 strain (FERM BP-22290), the BURABG162 strain, the SKE217 strain, and the SKE218 strain. In addition, all of the strains (for example, modified strains) derived from the above-exemplified strains belonging to Aurantiochytrium sp.1 are included in Aurantiochytrium sp.1.

On August 8, 2017, the AJ7869 strain was deposited with the Patent Organism Depositary Center, National Institute of Technology and Evaluation (zip code 292-0818, room 120, 2-5-8 Kisarazu Kazusa Kamatari, Chiba Prefecture, Japan). Deposited as FERM P-22342 and transferred to the international deposit under the Budapest Treaty on July 8, 2018, with accession number FERM BP-22342.

In addition, specific examples of Labyrinthulid microorganisms include Aurantiochytrium limacinum mh0186 (FERM BP-11311), Aurantiochytrium limacinum ATCC MYA-1381, Thraustochytrium aureum ATCC34304, Thraustochytrium sp. SEK210 (NBRC 102615), Schizochytrium sp. SEK345 (NBRC 102616), Pariatichytrium sarkarianum SEK364 (FERM BP-11298), Ulkenia sp. ATCC 28207, Botryochytrium radiatum SEK353 (NBRC 104107).

These strains can be obtained, for example, from the American Type Culture Collection (Address: 12301 Parklawn Drive, Rockville, Maryland 20852 PO Box 1549, Manassas, VA 20108, United States of America). That is, a registration number corresponding to each strain is assigned, and this registration number can be used to obtain distribution (see http://www.atcc.org/). The accession number corresponding to each strain can be found in the catalog of the American Type Culture Collection. In addition, these strains can be obtained, for example, from the depository to which each strain has been deposited.

The microorganism of the present invention can be obtained by appropriately modifying the Labyrinthulid microorganisms exemplified above. That is, the microorganism of the present invention may be a modified strain derived from Labyrinthulid microorganisms as exemplified above. The microorganisms of the invention are in particular Aurantiochytrium
It may be a modified strain derived from the sp.1 AJ7869 strain.

<1-2> Sterol-producing ability The microorganism of the present invention has sterol-producing ability. The term “microorganism capable of producing sterol” refers to a microorganism capable of producing a desired sterol when cultured in a medium and accumulating it within the cell to the extent that it can be recovered. The microorganism of the present invention may have one type of sterol-producing ability, or may have two or more types of sterol-producing ability.

The microorganism of the present invention may be one that inherently has sterol-producing ability, or one that has been modified to have sterol-producing ability. For example, Labyrinthulid microorganisms such as those exemplified above can be used as microorganisms having sterol-producing ability as they are or after imparting or enhancing sterol-producing ability. A method for imparting or enhancing sterol-producing ability is not particularly limited. For example, sterol-producing ability can be imparted or enhanced by introducing various modifications such as modifications that increase DGAT activity and other modifications described later into Labyrinthulea microorganisms.

"Sterol" is a general term for steroid derivatives having a hydroxyl group at the C3 position. "Steroid" is a general term for compounds having a cyclopentanohydrophenanthrene ring skeleton. Specifically, "sterol" is a general term for compounds having a cyclopentanohydrophenanthrene ring skeleton having a hydroxyl group at the C3 position. This skeleton is also referred to as the "basic skeleton" of sterols.

The sterol may have various substituents on the basic skeleton. Representative substituents of sterols include a methyl group at the C10-position, a methyl group at the C13-position, and an alkyl group at the C17-position. Alkyl groups can be, for example, linear, branched or cyclic, saturated or unsaturated. Also, the cyclopentanohydrophenanthrene ring skeleton may contain one or more double bonds.

Representative skeletons of steroids include cholestane, cholane, pregnane, androstane, and estrane. That is, representative sterols include compounds having a skeleton in which a hydroxyl group is introduced at the C3 position of cholestane, cholane, pregnane, androstane, or estrane.

Sterols include animal sterols, plant sterols and fungal sterols. "Animal sterol", "plant sterol" and "fungal sterol" refer to sterols found in animals, plants and fungi, respectively.

Animal sterols include cholesterol, 7-dehydrocholesterol, glycocholic acid, taurocholic acid, cholic acid, estradiol, estrone, ethinylestradiol, estriol, dehydroepiandrosterone, methylandrostenediol, 5β-pregnane-3α,20α- Diol, pregnenolone, chenodeoxycholic acid, dehydrocholic acid, dihydroxycholesterol, digitoxygenin, desmosterol, lathosterol. Animal sterols include, inter alia, cholesterol and 7-dehydrocholesterol. Animal sterols more particularly include cholesterol.

Plant sterols include 7-dehydropolyferasterol, stigmasterol, 4-methyl-7,22-stigmasteradienol, β-sitosterol, brassicasterol, campesterol, diosgenin, oleanolic acid, betulinic acid, ursolic acid, hecogenin, sarsasapogenin, isofucosterol, avenasterol, Δ7-stigmastenol, Δ7-campestenol, fucosterol, sargasterol. Plant sterols particularly include those in which the C24 position is methyl or ethyl. Plant sterols more particularly include stigmasterol and brassicasterol.

Fungal sterols include ergosterol and 7-dihydroergosterol. Fungal sterols include, inter alia, ergosterol.

Moreover, a provitamin D is mentioned as a sterol. Provitamin Ds include 7-dehydrocholesterol (provitamin D3) and ergosterol (provitamin D2). Pro-vitamin Ds include, inter alia, 7-dehydrocholesterol.

Sterols are not limited to free forms, but can also exist in the form of various derivatives. Therefore, unless otherwise specified, "sterol" may collectively refer to free sterols and derivatives thereof. Thus, "sterol" may mean sterol in free form, derivatives thereof, or mixtures thereof, unless otherwise specified. Sterol derivatives include sterol esters, steryl glucosides, acyl steryl glucosides. That is, "sterol" may specifically mean, for example, free sterols, sterol esters, steryl glucosides, acyl steryl glucosides, or mixtures thereof. Derivatives of sterols include in particular sterol esters. Derivatives of sterols more particularly include cholesterol esters.

The term “sterol ester” refers to an acyl ester of free sterol, that is, a compound having a structure in which a free sterol and a fatty acid are linked via an ester bond. In other words, a sterol ester has a site corresponding to a free sterol and a site corresponding to a fatty acid bound together by an ester bond. A site corresponding to a free sterol is also referred to as a "sterol site". A site corresponding to a fatty acid is also called a “fatty acid site” or an “acyl group”. An ester bond can be formed between the C3 hydroxyl group of a sterol and the carboxyl group of a fatty acid. Therefore, when the sterol exists in the form of an ester, the hydroxyl group at the C3 position may not remain. The type of acyl group is not particularly limited. That is, the chain length and degree of unsaturation of the acyl group are variable. The chain length of the acyl group can be, for example, C14-C26 (eg, C14, C16, C18, C20, C22, C24, or C26). Acyl groups may be saturated or unsaturated. Acyl groups can have one or more (eg, 1, 2, 3, 4, 5, or 6) unsaturated double bonds. Specific examples of fatty acids corresponding to acyl groups 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), docosahexaenoic acid (DHA, 22:6). Fatty acids corresponding to acyl groups include, inter alia, docosahexaenoic acid (DHA, 22:6), docosapentaenoic acid (DPA, 22:5), palmitic acid (16:0).

"Steryl glucoside" refers to glycosides of free sterols. "Acylsteryl glucoside" refers to glycosides of sterol esters.

<1-3> Enhancement of DGAT Activity The microorganism of the present invention is modified to enhance the activity of DGAT. Specifically, the microorganism of the present invention is modified such that the DGAT activity is increased compared to the unmodified strain. By modifying a Labyrinthulea microorganism to increase the activity of DGAT, the ability of the microorganism to produce sterols can be improved, ie, the production of sterols by the microorganism can be increased. Increased sterol production includes increased sterol production, increased sterol production rate, and increased sterol yield. An increase in the sterol production amount includes an increase in the sterol production amount per cell weight and an increase in the sterol production amount per culture medium volume.

The microorganism of the present invention can be obtained by modifying a Labyrinthulea microorganism capable of producing sterols so as to increase the activity of DGAT. The microorganism of the present invention can also be obtained by modifying a Labyrinthulea microorganism to increase DGAT activity and then imparting sterol-producing ability. In addition, the microorganism of the present invention may be endowed with sterol-producing ability by modification that increases DGAT activity or by a combination of modification that increases DGAT activity and another modification. In the present invention, modifications for constructing the microorganism of the present invention can be performed in any order.

DGAT and the gene encoding it are described below.

The term "diacylglycerol acyltransferase (DGAT)" refers to a protein (enzyme) that has the activity of catalyzing the reaction of transferring an acyl group from acyl-CoA to diacylglycerol to produce triacylglycerol (EC 2.3.1.20 ). The same activity is also referred to as "diacylglycerol acyltransferase activity (DGAT activity)". A gene encoding DGAT is also referred to as a "diacylglycerol acyltransferase gene (DGAT gene)" or "dgat gene". Diacylglycerols include 1,2-diacylglycerols.

DGAT activity can be measured, for example, by incubating the enzyme with a substrate (diacylglycerol) in the presence of an acyl-CoA and measuring the formation of an enzyme- and substrate-dependent product (triacylglycerol) (Lardizabal KD, et al. al., DGAT2 is a new diacylglycerol acyltransferase gene family: purification, cloning, and expression in insect cells of two polypeptides from Mortierella ramanniana with diacylglycerol acyltransferase activity. J Biol Chem. 2001 Oct 19;276(42):38862-9.) .

DGAT genes and DGATs include those of various organisms such as Labyrinthulid microorganisms. The DGAT gene possessed by various organisms such as Labyrinthulid microorganisms and the nucleotide and amino acid sequences of DGAT can be obtained, for example, from the NCBI (National Center for Biotechnology Information
) and other public databases. DGATs include diacylglycerol acyltransferase 1 (DGAT1) and diacylglycerol acyltransferase 2 (DGA
T2). DGATs include, in particular, DGAT2. DGAT2 specifically includes DGAT2B of Aurantiochytrium sp.1 AJ7869 strain. As DGAT2, specifically, Chlamydomonas reinhardtii DGTT4, Nannochloropsis sp. DGAT2 of tauri can also be mentioned (WO2015/137449
). The nucleotide sequence of the DGAT2B gene of Aurantiochytrium sp.1 AJ7869 strain and the amino acid sequence of DGAT2B encoded by the gene are shown in SEQ ID NOs: 1 and 2, respectively. That is, the DGAT gene may be, for example, a gene having the nucleotide sequence shown in SEQ ID NO:1. DGAT may also be a protein having the amino acid sequence shown in SEQ ID NO:2, for example. The expression "having a (amino acid or base) sequence" means "including the (amino acid or base) sequence" unless otherwise specified, and includes the case of "consisting of the (amino acid or base) sequence". do.

The DGAT gene may be a variant of the DGAT gene exemplified above (for example, the gene having the nucleotide sequence shown in SEQ ID NO: 1) as long as the original function is maintained. Similarly, DGAT may be a variant of the DGAT exemplified above (for example, a protein having the amino acid sequence shown in SEQ ID NO: 2) as long as the original function is maintained. Note that such a variant in which the original function is maintained is sometimes referred to as a "conservative variant". The term "DGAT gene" shall encompass the DGAT genes exemplified above as well as conservative variants thereof. Similarly, the term "DGAT" shall encompass the DGATs exemplified above, as well as conservative variants thereof. Conservative variants include, for example, the above-exemplified DGAT genes, DGAT homologues, and artificial variants.

“Original function is maintained” means that a gene or protein variant has a function (activity or property) corresponding to the function (activity or property) of the original gene or protein. "Maintaining the original function" of a gene means that a variant of the gene encodes a protein that retains the original function. That is, the expression "maintaining the original function" of the DGAT gene means that the variant of the gene encodes a protein having DGAT activity. In addition, "original function is maintained" with respect to DGAT means that a protein variant has DGAT activity.

Examples of conservative variants are given below.

DGAT genes or DGAT homologs can be easily identified from public databases by, for example, BLAST searches or FASTA searches using the DGAT gene nucleotide sequences exemplified above or the DGAT amino acid sequences exemplified above as query sequences. DGAT gene homologues can be obtained by PCR using, for example, the chromosomes of organisms such as Labyrinthulid microorganisms as templates and oligonucleotides prepared based on the known base sequences of DGAT genes as primers. can.

The DGAT gene has one or several amino acid substitutions, deletions, or insertions at one or several positions in the above amino acid sequence (for example, the amino acid sequence shown in SEQ ID NO: 2), as long as the original function is maintained. , and/or genes encoding proteins with added amino acid sequences. For example, the encoded protein may be lengthened or truncated at its N-terminus and/or C-terminus. The above-mentioned "one or several" varies depending on the position of the amino acid residue in the three-dimensional structure of the protein and the type of the amino acid residue. to 30, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, particularly preferably 1 to 3.

Substitutions, deletions, insertions and/or additions of one or several amino acids described above are conservative mutations that maintain normal protein function. A typical conservative mutation is a conservative substitution. Conservative substitutions are between Phe, Trp and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile and Val when the substitution site is a hydrophobic amino acid, which is a polar amino acid. between Gln and Asn, between Lys, Arg, and His when it is a basic amino acid, and between Asp and Glu when it is an acidic amino acid, when it is an amino acid having a hydroxyl group are mutations that replace each other between Ser and Thr. Substitutions regarded as conservative substitutions specifically include substitutions of Ala to Ser or Thr, substitutions of Arg to Gln, His or Lys, substitutions of Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln substitution, Cys to Ser or Ala substitution, Gln to Asn, Glu, Lys, His, Asp or Arg substitution, Glu to Gly, Asn, Gln
, Lys or Asp, Gly to Pro, His to Asn, Lys, Gln, Arg or Tyr, Ile to Leu, Met, Val or Phe, Leu to Ile, Met, Val or Phe substitution, Lys to Asn, Glu, Gln, His or Arg substitution, Met to Ile, Leu, Val or Phe substitution, Phe to Trp, Tyr, Met, Ile or Leu substitution, 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 mentioned. In addition, the substitution, deletion, insertion, or addition of amino acids as described above may be caused by naturally occurring mutations (mutants or variants) such as those based on individual differences in organisms from which genes are derived, or differences in species. included.

In addition, as long as the original function is maintained, the DGAT gene is, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more of the entire amino acid sequence. ,
More preferably, it may be a gene encoding a protein having an amino acid sequence with 97% or more homology, particularly preferably 99% or more homology. As used herein, "homology" means "identity".

In addition, the DGAT gene can be prepared from the above base sequence (for example, the base sequence shown in SEQ ID NO: 1), such as a complementary sequence to all or part of the above base sequence, and a string, as long as the original function is maintained. DNA that hybridizes under gentle conditions. “Stringent conditions” refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. As an example, highly homologous DNAs, for example, 50% or higher, 65% or higher, 80% or higher, preferably 90% or higher, more preferably 95% or higher, still more preferably 97% or higher, particularly preferably 99% DNAs with more than 100% homology hybridize but DNAs with lower homology do not hybridize, or normal Southern hybridization washing conditions of 60°C, 1×SSC, 0.1% SDS , preferably 60 ° C., 0.1 × SSC, 0.1% SDS, more preferably at a salt concentration and temperature corresponding to 68 ° C., 0.1 × SSC, 0.1% SDS, washing once, preferably 2 to 3 times. be able to.

As mentioned above, the probes used for the hybridization may be part of the complementary sequence of the gene. Such probes can be prepared by PCR using oligonucleotides prepared based on known gene sequences as primers and a DNA fragment containing the gene described above as a template. For example, a DNA fragment with a length of about 300 bp can be used as a probe. When using a DNA fragment with a length of about 300 bp as a probe, washing conditions for hybridization include 50° C., 2×SSC, and 0.1% SDS.

Also, the DGAT gene may be one in which any codon is replaced with an equivalent codon. That is, the DGAT gene may be a variant of the DGAT gene exemplified above due to the degeneracy of the genetic code. For example, the DGAT gene may be modified to have optimal codons according to the codon usage of the host used.

The “identity” between amino acid sequences is defined by default Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional
Adjustments: means identity between amino acid sequences calculated using Conditional compositional score matrix adjustment). In addition, "identity" between base sequences means the identity between base sequences calculated by blastn using the default Scoring Parameters (Match/Mismatch Scores = 1, -2; Gap Costs = Linear). do.

Note that the above descriptions regarding variants of genes and proteins can also be applied mutatis mutandis to arbitrary proteins and genes encoding them.

<1-4> Other Modifications The microorganism of the present invention may further have other modifications as long as the sterol-producing ability is not impaired. Other modifications include modifications that enhance sterol-producing ability. Other modifications, specifically, modification of the activity of sterol 24-C-methyltransferase (sterol 24-C-methyltransferase; also referred to as "SMT1"), 24-dehydrocholesterol reductase (24-dehydrocholesterol reductase; "DHCR24") and modification of the activity of 7-dehydrocholesterol reductase (also referred to as “DHCR7”). Modification of activity includes:
An increase or decrease in activity can be mentioned. These modifications may enhance sterol production capacity. These modifications can be appropriately selected according to various conditions such as the type of sterol to be produced. These modifications can be used singly or in combination as appropriate.

The microorganism of the present invention may be modified so that the activity of SMT1 is decreased or increased. Specifically, the microorganism of the present invention may be modified such that the activity of SMT1 is decreased or increased compared to the unmodified strain. SMT1 activity may be reduced, for example, when producing animal sterols such as cholesterol. Also, the activity of SMT1 may be increased, for example, when producing plant sterols or fungal sterols.

The microorganisms of the invention may be modified to decrease or increase the activity of DHCR24. The microorganisms of the present invention may specifically be modified such that the activity of DHCR24 is reduced or increased compared to unmodified strains. The activity of DHCR24 may be reduced, for example, when producing plant sterols or fungal sterols. Also, the activity of DHCR24 may be increased when producing some animal sterols, eg cholesterol, lathosterol, 7-dehydrocholesterol.

The microorganism of the present invention may be modified to have reduced or increased DHCR7 activity. Specifically, the microorganism of the present invention may be modified such that the DHCR7 activity is decreased or increased compared to the unmodified strain. DHCR7 activity may be reduced, for example, when producing provitamin Ds. Also, the activity of DHCR7 may be increased when producing, for example, cholesterol, 7-dihydroergosterol, or stigmasterol.

The microorganisms of the invention may be modified to have one or more properties selected from, among others, reduced SMT1 activity, increased DHCR24 activity, and increased DHCR7 activity. The microorganism of the present invention may be modified to have at least the property of decreased SMT1 activity and/or increased DHCR24 activity.

“Sterol 24-C-methyltransferase (SMT1)” refers to a protein (enzyme) having the activity of catalyzing a reaction that introduces a methyl group at the C24 position of sterol (EC 2.1.1.41). The same activity is also referred to as "sterol 24-C-methyltransferase activity (SMT1 activity)". The gene encoding SMT1 is also called "sterol 24-C-methyltransferase gene (SMT1 gene)" or "smt1 gene". Sterols that serve as substrates for SMT1 include sterols having a double bond (C24-C25 double bond) at the C24 position. Introduction of the methyl group may be accompanied by reduction of the double bond at the C24 position. Moreover, the introduced methyl group may remain after being converted to a methylene group. In other words, the product of SMT1 may be a sterol in which the double bond at the C24 position has been reduced and a methylene group has been introduced at the C24 position. The “C24 position” as used herein means the carbon corresponding to the C24 position of zymosterol.

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

The term "24-dehydrocholesterol reductase (DHCR24)" refers to a protein (enzyme) that has the activity of catalyzing the reduction of the double bond (C24-C25 double bond) at the C24 position of sterol (EC 1.3.1.72 ). The same activity is also referred to as "24-dehydrocholesterol reductase activity (DHCR24 activity)". The gene encoding DHCR24 is also referred to as "24-dehydrocholesterol reductase gene (DHCR24 gene)" or "dhcr24 gene". The “C24 position” as used herein means the carbon corresponding to the C24 position of zymosterol.

DHCR24 activity is determined, for example, by incubating the enzyme with a substrate (a sterol with a double bond at the C24 position) in the presence of an electron donor to produce an enzyme- and substrate-dependent product (a sterol with a reduced double bond at the C24 position). ) can be measured. Electron donors include, for example, NADPH. Substrates and products include, for example, zymosterol and 5a-cholest-8-en-3b-ol, respectively.

"7-dehydrocholesterol reductase (DHCR7)" refers to a protein (enzyme) that has the activity of catalyzing the reduction of the double bond (C7-C8 double bond) at the C7 position of sterol (EC 1.3.1.21 ). The same activity is also referred to as "7-dehydrocholesterol reductase activity (DHCR7 activity)". The gene encoding DHCR7 is also referred to as "7-dehydrocholesterol reductase gene (DHCR7 gene)" or "dhcr7 gene". The “C7 position” as used herein means the carbon corresponding to the C7 position of the basic skeleton of the sterol described above.

DHCR7 activity is induced by, for example, incubating the enzyme with a substrate (a sterol with a double bond at the C7 position) in the presence of an electron donor to produce an enzyme- and substrate-dependent product (a sterol with a reduced double bond at the C7 position). ) can be measured. Electron donors include, for example, NADPH. Substrates and products include, for example, cholesterol and 7-dehydrocholesterol, respectively.

These genes and proteins include those of various organisms such as Labyrinthulid microorganisms. The nucleotide sequences and amino acid sequences of these genes and proteins possessed by various organisms such as Labyrinthulid microorganisms can be obtained from public databases such as NCBI (National Center for Biotechnology Information). The nucleotide sequence of the SMT1 gene of Aurantiochytrium sp.1 AJ7869 strain and the amino acid sequence of SMT1 encoded by the gene are shown in SEQ ID NOs: 3 and 4, respectively. The nucleotide sequence of the DHCR24 gene of Aurantiochytrium sp.1 AJ7869 strain and the amino acid sequence of DHCR24 encoded by the gene are shown in SEQ ID NOs: 5 and 6, respectively. The nucleotide sequence of the DHCR7 gene of Aurantiochytrium sp.1 AJ7869 strain and the amino acid sequence of DHCR7 encoded by the gene are shown in SEQ ID NOs: 7 and 8, respectively.

These genes and proteins may be genes and proteins having the nucleotide sequences and amino acid sequences exemplified above, respectively. These genes and proteins may also be conservative variants of the genes and proteins exemplified above (eg, conservative variants of genes and proteins having the nucleotide sequences and amino acid sequences exemplified above), respectively. Specifically, for example, these genes have one or several amino acid substitutions, deletions, or insertions at one or several positions in the amino acid sequences exemplified above, as long as the original functions are maintained. , and/or genes encoding proteins with added amino acid sequences. Also, specifically, for example, these genes are, for example, 50% or more, 65% or more, 80% or more, preferably It may be a gene encoding a protein having an amino acid sequence with a homology of 90% or more, more preferably 95% or more, more preferably 97% or more, and particularly preferably 99% or more. Regarding conservative variants of genes and proteins, the descriptions of the DGAT gene and conservative variants of DGAT can be applied mutatis mutandis.

<1-5> Method for increasing protein activity Methods for increasing protein activity such as DGAT will be described below.

By "the activity of a protein is increased" is meant that the activity of the same protein is increased compared to an unmodified strain. "Increased activity of a protein" specifically means that the activity of the same protein per cell is increased relative to an unmodified strain. As used herein, "unmodified strain" means a control strain that has not been modified to increase the activity of the target protein. Unmodified strains include wild strains and parental strains. Specific examples of unmodified strains include the strains exemplified in the description of Labyrinthulid microorganisms. Thus, in one aspect, the activity of the protein may be increased compared to Aurantiochytrium sp.1 strain AJ7869. In addition, "the activity of the protein is increased" is also referred to as "the activity of the protein is enhanced". More specifically, "the activity of the protein is increased" means that the number of molecules of the same protein per cell is increased compared to an unmodified strain, and/or It may mean that the function is increased. That is, the "activity" in the case of "increase in protein activity" is not limited to the catalytic activity of the protein, but means the transcription level (mRNA level) or translation level (protein level) of the gene encoding the protein. may In addition, "increasing the activity of the protein" includes not only increasing the activity of the protein in a strain that originally has the activity of the target protein, but also increasing the activity of the same protein in a strain that does not originally have the activity of the target protein. Conferring activity is also included. In addition, as long as the activity of the protein is increased as a result, the activity of the target protein originally possessed by the host may be reduced or eliminated, and then the activity of the preferred target protein may be imparted.

The degree of increase in protein activity is not particularly limited as long as the protein activity is increased compared to the unmodified strain. The activity of the protein may be increased, for example, by 1.5-fold or more, 2-fold or more, or 3-fold or more over the unmodified strain. In addition, if the unmodified strain does not have the activity of the target protein, the protein may be produced by introducing the gene encoding the protein. It should be produced as much as possible.

Modifications that increase the activity of a protein can be achieved, for example, by increasing the expression of the gene encoding the same protein. “Increase in gene expression” means that the expression of the same gene is increased compared to unmodified strains such as wild strains and parent strains. “Increase in gene expression” specifically means that the expression level of the same gene per cell is increased compared to an unmodified strain. “Increase in gene expression” more specifically means an increase in the transcription level (mRNA level) of the gene and/or an increase in the translation level (protein level) of the gene. you can In addition, "gene expression is increased" is also referred to as "gene expression is enhanced". Expression of the gene may be increased, for example, by 1.5-fold or more, 2-fold or more, or 3-fold or more over the unmodified strain. In addition, "increased gene expression" means not only increasing the expression level of the gene in a strain that originally expresses the target gene, but also in a strain that does not originally express the target gene, Expression of the same gene is also included. That is, "increasing the expression of a gene" may mean, for example, introducing the target gene into a strain that does not retain the target gene and expressing the same gene.

Increased gene expression can be achieved, for example, by increasing the copy number of the gene.

An increase in gene copy number can be achieved by introducing the same gene into the host chromosome. Introduction of a gene into a chromosome can be performed using, for example, homologous recombination. Specifically, by transforming a host with recombinant DNA containing a target gene and causing homologous recombination with the target site on the chromosome of the host, the gene can be introduced onto the chromosome of the host. 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 a linear DNA containing a target gene and having homologous nucleotide sequences upstream and downstream of the target site on the chromosome at both ends of the gene, and downstream, respectively, the target site can be replaced with the gene. The recombinant DNA used for homologous recombination may have a marker gene for selecting transformants. Marker genes include antibiotic resistance genes such as hygromycin resistance gene (HygR), neomycin resistance gene (NeoR) and blasticidin resistance gene (BlaR). The gene may be introduced in only one copy, two copies or more. For example, multiple copies of a gene can be introduced into the chromosome by performing homologous recombination targeting a nucleotide sequence of which multiple copies exist on the chromosome. It should be noted that such a technique for modifying chromosomes using homologous recombination is not limited to the introduction of target genes, and can be used for arbitrary modification of chromosomes such as modification of expression regulatory sequences.

Confirmation that the target gene has been introduced onto the chromosome is performed by Southern hybridization using a probe having a sequence complementary to all or part of the gene, or by using a primer prepared based on the sequence of the gene. It can be confirmed by PCR or the like.

An increase in gene copy number can also be achieved by introducing a vector containing the same gene into a host. For example, a DNA fragment containing a target gene is ligated to a vector that functions in a host to construct an expression vector for the same gene, and the host is transformed with the expression vector to increase the copy number of the same gene. can. A DNA fragment containing a target gene can be obtained, for example, by PCR using the genomic DNA of a microorganism containing the target gene as a template. As a vector, a vector capable of autonomous replication in host cells can be used. A vector may be a single-copy vector or a multi-copy vector. The vector may contain a marker gene for selecting transformants.

When introducing a gene, the gene may be retained in the host so as to be expressible. Specifically, the gene may be retained so as to be expressed under the control of a promoter that functions in the host. The promoter is not particularly limited as long as it functions in the host. A “promoter that functions in the host” refers to a promoter that has promoter activity in the host. The promoter may be a host-derived promoter or a heterologous promoter. The promoter may be the intrinsic promoter of the gene to be introduced, or the promoter of another gene. Specific examples of promoters include actin gene promoter, glyceraldehyde-3-phosphate dehydrogenase gene promoter, pyruvate kinase gene promoter, elongation factor 1α (EF1α) gene promoter, ubiquitin promoter (UbiP), simian virus 4
0 promoter (SV40P), tubulin gene promoter, viral immediate early gene promoter (Smp1P) (JP 2018-064551, JP 2006-304686, WO2016/056610, WO02/083869). Moreover, as the promoter, for example, a stronger promoter as described later may be used.

A terminator for terminating transcription can be placed downstream of the gene. The terminator is not particularly limited as long as it functions in the host. The terminator may be a host-derived terminator or a heterologous terminator. The terminator may be a terminator specific to the gene to be introduced, or may be a terminator of another gene. Specific examples of terminators include actin gene terminator, glyceraldehyde-3-phosphate dehydrogenase gene terminator, pyruvate kinase gene terminator, elongation factor 1α (EF1α) gene terminator, ubiquitin terminator (UbiT), and simian virus 40. A terminator (SV40T) can be mentioned (JP 2018-064551, JP 2006-304686, WO2016/056610).

Vectors, promoters, and terminators that can be used in various microorganisms are described in detail, for example, in "Microbiology Basic Course 8 Genetic Engineering, Kyoritsu Shuppan, 1987" and can be used.

Also, when introducing two or more genes, each gene may be retained in the host so as to be expressible. For example, each gene may be all maintained on a single expression vector, or all may be maintained on a chromosome. In addition, each gene may be separately maintained on a plurality of expression vectors, or may be separately maintained on a single or multiple expression vectors and on the chromosome. Alternatively, two or more genes may be introduced to form an operon.

The introduced gene is not particularly limited as long as it encodes a protein that functions in the host. The introduced gene may be a host-derived gene or a heterologous gene. The gene to be introduced can be obtained by PCR using, for example, primers designed based on the base sequence of the gene and genomic DNA of an organism having the gene or a plasmid carrying the gene as a template. Also, the introduced gene may be totally synthesized based on the base sequence of the same gene (Gene, 60(1), 115-127 (1987)). The obtained gene can be used as it is or after being modified as appropriate. That is, by modifying the gene, its variants can be obtained. Gene modification can be performed by a known technique. For example, site-directed mutagenesis can be used to introduce a desired mutation at a desired site in DNA. Thus, for example, by site-directed mutagenesis, the coding region of a gene can be altered such that the encoded protein contains substitutions, deletions, insertions, and/or additions of amino acid residues at specific sites. . As site-directed mutagenesis, a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and methods using phage (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 ( 1987)). Alternatively, all variants of the gene may be synthesized.

In addition, an increase in gene expression can be achieved by improving the transcription efficiency of the gene. In addition, an increase in gene expression can be achieved by improving the translation efficiency of the gene. Improving gene transcription efficiency and translation efficiency can be achieved, for example, by modifying an expression regulatory sequence. “Expression control sequence” is a general term for sites that affect gene expression, such as promoters. Expression regulatory sequences can be determined, for example, using promoter search vectors and gene analysis software such as GENETYX.

Improving the transcription efficiency of a gene can be achieved, for example, by replacing the promoter of the gene on the chromosome with a stronger promoter. By "stronger promoter" is meant a promoter that improves transcription of a gene over the naturally occurring wild-type promoter. As a stronger promoter, for example, a natural high expression promoter can be used. As a stronger promoter, for example, a highly active type of a conventional promoter may be obtained and used. A highly active version of a conventional promoter can be obtained, for example, by using various reporter genes.

Improving the efficiency of gene translation can also be achieved, for example, by modifying codons. For example, gene translation efficiency can be improved by replacing rare codons present in a gene with synonymous codons that are used more frequently. That is, the introduced gene may be modified so as to have optimal codons according to the codon usage of the host to be used, for example. Codon substitution can be performed, for example, by site-directed mutagenesis. Alternatively, a gene fragment in which codons are replaced may be totally synthesized. The frequency of codon usage in various organisms can be found in "Codon Usage Database" (http://www.kazusa.or.jp/codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292 (2000)). disclosed in

Gene expression can also be increased by amplifying a regulator that increases gene expression, or by deleting or weakening a regulator that decreases gene expression.

The techniques for increasing gene expression as described above may be used alone or in any combination.

Modifications to increase the activity of a protein can also be achieved, for example, by enhancing the specific activity of the protein. Proteins with enhanced specific activity can be obtained by searching various organisms, for example. Alternatively, a highly active protein may be obtained by introducing a mutation into a conventional protein. The mutations introduced may be, for example, substitutions, deletions, insertions or additions of one or several amino acids at one or several positions of the protein. Mutations can be introduced, for example, by site-directed mutagenesis as described above. In addition, mutation may be introduced by, for example, mutagenesis. Mutation treatments include X-ray irradiation, ultraviolet irradiation, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS). ) and other mutating agents. DNA may also be treated directly with hydroxylamine in vitro to induce random mutations. Enhancement of specific activity may be used alone, or may be used in combination with any of the methods for enhancing gene expression as described above.

Transformation can be performed, for example, by a technique commonly used for transformation of Labyrinthulid microorganisms. Such techniques include electroporation.

An increase in protein activity can be confirmed by measuring the activity of the protein.

Increased protein activity can also be confirmed by confirming increased expression of the gene encoding the protein. An increase in gene expression can be confirmed by confirming that the amount of transcription of the gene has increased or by confirming that the amount of protein expressed from the gene has increased.

An increase in the transcription level of a gene can be confirmed by comparing the amount of mRNA transcribed from the same gene with that of a wild strain or an unmodified strain such as a parent strain. Methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, microarray, RNA-seq and the like (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual/Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The amount of mRNA may be increased, for example, by 1.5-fold or more, 2-fold or more, or 3-fold or more over the unmodified strain.

Confirmation of increased protein abundance can be performed by Western blotting using antibodies (Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of protein (eg, number of molecules per cell) may be increased, for example, by 1.5-fold or more, 2-fold or more, or 3-fold or more over the unmodified strain.

The technique for increasing the activity of the protein described above can be used to enhance the activity of any protein or enhance the expression of any gene, in addition to enhancing the activity of DGAT.

<1-6> Technique for Reducing Protein Activity Techniques for reducing protein activity will be described below.

"The activity of a protein is decreased" means that the activity of the same protein is decreased compared to an unmodified strain. "The activity of a protein is decreased" specifically means that the activity of the same protein per cell is decreased compared to an unmodified strain. As used herein, "unmodified strain" means a control strain that has not been modified to reduce the activity of the target protein. Unmodified strains include wild strains and parental strains. Specific examples of unmodified strains include strains exemplified in the description of Labyrinthulid microorganisms. Thus, in one aspect, the activity of the protein may be reduced compared to Aurantiochytrium sp.1 strain AJ7869. It should be noted that "the activity of the protein is decreased" includes the case where the activity of the protein is completely lost. "The activity of the protein is reduced" means, more specifically, that the number of molecules of the same protein per cell is reduced as compared with an unmodified strain, and/or It may mean that the function is degraded. That is, the "activity" in the case of "the activity of the protein is decreased" is not limited to the catalytic activity of the protein, but means the transcription level (mRNA level) or translation level (protein level) of the gene encoding the protein. may It should be noted that "the number of protein molecules per cell is reduced" includes the case where the protein does not exist at all. In addition, "the function per molecule of the protein is reduced" also includes the case where the function per molecule of the same protein is completely lost. The degree of reduction in protein activity is not particularly limited as long as the protein activity is reduced compared to the unmodified strain. The activity of the protein may be reduced, eg, to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

A modification that reduces the activity of a protein can be achieved, for example, by reducing the expression of the gene encoding the same protein. "Gene expression is decreased" means that the expression of the same gene is decreased compared to the unmodified strain. “Reduced expression of a gene” specifically means that the expression level of the same gene per cell is reduced compared to an unmodified strain. More specifically, the term "gene expression is decreased" means that the transcription level (mRNA level) of the gene is decreased and/or the translation level (protein level) of the gene is decreased. you can "The expression of a gene is decreased" includes cases where the same gene is not expressed at all. In addition, "the expression of a gene is decreased" is also referred to as "the expression of a gene is attenuated". Expression of the gene may be reduced to, eg, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

A decrease in gene expression may be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination thereof. Reduction of gene expression can be achieved, for example, by modifying the expression regulatory sequence of the gene. “Expression control sequence” is a general term for sites that affect gene expression, such as promoters. Expression regulatory sequences can be determined, for example, using promoter search vectors and gene analysis software such as GENETYX. When modifying an expression regulatory sequence, preferably 1 or more bases, more preferably 2 or more bases, and particularly preferably 3 or more bases are modified. A decrease in transcription efficiency of a gene can be achieved, for example, by replacing the promoter of the gene on the chromosome with a weaker promoter. By "weaker promoter" is meant a promoter whose gene transcription is weaker than the naturally occurring wild-type promoter. Weaker promoters include, for example, inducible promoters. That is, an inducible promoter can function as a weaker promoter under non-inducing conditions (eg, in the absence of an inducer). In addition, part or all of the expression control sequence may be deleted (deleted). In addition, reduction of gene expression can also be achieved, for example, by manipulating factors involved in expression control. Factors involved in expression control include low-molecular-weight molecules (inducing substances, inhibitors, etc.), proteins (transcription factors, etc.), nucleic acids (siRNA, etc.), etc., involved in transcription and translation control. In addition, the reduction of gene expression can also be achieved, for example, by introducing a mutation that reduces gene expression into the coding region of the gene. For example, expression of a gene can be reduced by replacing codons in the coding region of the gene with synonymous codons that are used less frequently in the host. In addition, for example, gene expression itself can be reduced by disruption of the gene as described later.

Also, modification that reduces the activity of a protein can be achieved, for example, by disrupting the gene encoding the same protein. By "disrupted in a gene" is meant that the same gene is altered so that it no longer produces a protein that functions normally. "Does not produce protein that functions normally" includes cases where no protein is produced from the same gene, and cases where the same gene produces a protein with reduced or lost function (activity or properties) per molecule. be done.

Gene disruption can be achieved, for example, by deleting (deleting) the gene on the chromosome. "Gene deletion" refers to deletion of a part or all of the coding region of a gene. Furthermore, the entire gene may be deleted, including 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, expression regulatory sequences of the gene. As long as the protein activity can be reduced, the region to be deleted includes the N-terminal region (region encoding the N-terminal side of the protein), internal region, C-terminal region (region encoding the C-terminal side of the protein), etc. Any region may be used. Generally, the longer the region to be deleted, the more reliably the gene can be inactivated. The region to be deleted is, 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 of the entire coding region of the gene, Or it may be a region with a length of 95% or more. Moreover, it is preferable that the sequences before and after the region to be deleted do not have the same reading frame. A reading frame mismatch can result in a frameshift downstream of the deleted region.

In addition, gene disruption includes, for example, introducing an amino acid substitution (missense mutation) into the coding region of the gene on the chromosome, introducing a stop codon (nonsense mutation), or adding or deleting 1 to 2 bases ( (Journal of Biological Chemistry 272:8611-8617 (1997), Proceedings of the Nati
National Academy of Sciences, USA 95 5511-5515 (1998), Journal of Biological Chemistry 26 116, 20833-20839 (1991)).

Gene disruption can also be achieved, for example, by inserting other nucleotide sequences into the coding region of the gene on the chromosome. The insertion site may be any region of the gene, but the longer the base sequence to be inserted, the more reliably the gene can be inactivated. Moreover, it is preferable that the sequences before and after the insertion site do not have the same reading frame. A reading frame mismatch can result in a frameshift downstream of the insertion site. Other nucleotide sequences are not particularly limited as long as they reduce or eliminate the activity of the encoded protein, and examples thereof include marker genes such as antibiotic resistance genes and genes useful for the production of target substances.

Disruption of the gene may in particular be carried out such that the amino acid sequence of the encoded protein is deleted (deleted). In other words, modifications that reduce the activity of a protein are, for example, deletion of the amino acid sequence of the protein (a region of part or all of the amino acid sequence). This can be achieved by modifying the gene to encode a protein that lacks a partial or complete region. In addition, "deletion of the amino acid sequence of a protein" refers to deletion of a part or the entire region of the amino acid sequence of the protein. "Deletion of the amino acid sequence of a protein" means that the original amino acid sequence does not exist in the protein, and includes cases in which the original amino acid sequence is changed to another amino acid sequence. That is, for example, a region changed to a different amino acid sequence by frameshifting may be considered a deleted region. Deletion of the amino acid sequence of a protein typically shortens the overall length of the protein, but may leave the overall length of the protein unchanged or may lengthen it. For example, deletion of part or all of the coding region of a gene can result in deletion of the region encoded by the deleted region in the amino acid sequence of the encoded protein. In addition, for example, by introducing a stop codon into the coding region of a gene, it is possible to delete the region encoded by the region downstream from the introduction site in the amino acid sequence of the encoded protein. Also, for example, by frameshifting in the coding region of a gene, the region encoded by the frameshift site can be deleted. Regarding the position and length of the region to be deleted in amino acid sequence deletion, the description of the position and length of the region to be deleted in gene deletion can be applied mutatis mutandis.

Modifying a gene on a chromosome as described above involves, for example, preparing a disrupted gene that is modified so as not to produce a protein that functions normally, and transforming a host with recombinant DNA containing the disrupted gene. This can be achieved by causing homologous recombination between the disrupted gene and the wild-type gene on the chromosome to replace the wild-type gene on the chromosome with the disrupted gene. In this case, the manipulation is facilitated if the recombinant DNA contains a marker gene according to the traits such as auxotrophy of the host. Disruption-type genes include genes in which part or all of the coding region of a gene is deleted, genes introduced with missense mutations, genes introduced with nonsense mutations, genes introduced with frameshift mutations, transposons, marker genes, etc. and a gene into which the insertion sequence of is inserted. Even if the protein encoded by the disrupted gene is produced, it has a three-dimensional structure different from that of the wild-type protein, and its function is 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, a host is transformed with a linear DNA containing a disrupted gene, the linear DNA having sequences upstream and downstream of the wild-type gene on the chromosome at both ends, respectively. The wild-type gene can be replaced with the disrupted gene by causing homologous recombination, respectively. It should be noted that such a chromosomal modification technique using homologous recombination is not limited to target gene disruption, and can be used for arbitrary modification of chromosomes such as modification of expression regulatory sequences.

Further, modifications that reduce protein activity may be performed, for example, by mutagenesis. Mutation treatments include X-ray irradiation, ultraviolet irradiation, and N-methyl-N'
-treatment with mutating agents such as nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

Techniques for reducing protein activity as described above may be used alone or in any combination.

A decrease in protein activity can be confirmed by measuring the activity of the same protein.

Decreased protein activity can also be confirmed by confirming decreased expression of the gene encoding the same protein. Decreased gene expression can be confirmed by confirming that the amount of transcription of the gene has decreased or by confirming that the amount of protein expressed from the gene has decreased.

Decrease in gene transcription level can be confirmed by comparing the amount of mRNA transcribed from the same gene with an unmodified strain. Methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, microarray, RNA-seq and the like (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of mRNA (eg, number of molecules per cell) may be reduced, eg, to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

Confirmation that the amount of protein has decreased can be performed by performing SDS-PAGE and confirming the intensity of the separated protein bands. In addition, confirmation that the amount of protein has decreased can be performed by Western blotting using an antibody (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of protein (eg, number of molecules per cell) may be reduced, eg, to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

Disruption of a gene can be confirmed by determining the base sequence, restriction enzyme map, full length, or the like of part or all of the gene, depending on the means used for the disruption.

Transformation can be performed, for example, by a technique commonly used for transformation of Labyrinthulid microorganisms. Such techniques include electroporation.

The techniques for reducing protein activity described above can be used to reduce the activity of any protein or to reduce the expression of any gene.

<2> Method for Producing Sterol The method of the present invention is a method for producing a sterol, comprising culturing the microorganism of the present invention in a medium and recovering the sterol from the cells produced by the culture. In the process of the invention, one sterol may be produced, two or more sterols may be produced.

The medium to be used is not particularly limited as long as the microorganism of the present invention can grow and sterols can be produced. As the medium, for example, a normal medium used for culturing heterotrophic microorganisms such as Labyrinthula can be used. The medium may optionally contain components selected from carbon sources, nitrogen sources, phosphoric acid sources, sulfur sources, and various other organic and inorganic components. Specific examples of the medium include GY medium prepared with 0-1× artificial seawater.

Specific examples of carbon sources include sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, blackstrap molasses, starch hydrolysates, and biomass hydrolysates, and organic acids such as acetic acid and citric acid. , ethanol, glycerol, crude glycerol, and fatty acids. As the carbon source, one type of carbon source may be used, or two or more types of carbon sources may be used in combination.

Specific examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate; organic nitrogen sources such as peptone, yeast extract, meat extract and soy protein hydrolyzate; ammonia; and urea. Ammonia gas or ammonia water used for pH adjustment may be used as the nitrogen source. As the nitrogen source, one type of nitrogen source may be used, or two or more types of nitrogen sources may be used in combination.

Specific examples of the phosphate source include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate. As the phosphoric acid source, one phosphoric acid source may be used, or two or more phosphoric acid sources may be used in combination.

Specific 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. As the sulfur source, one sulfur source may be used, or two or more sulfur sources may be used in combination.

Specific examples of other organic and inorganic components include inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; vitamin B1, vitamin B2, vitamin B6, and nicotine. Acids, nicotinamide, vitamins such as vitamin B12; amino acids; nucleic acids; As other various organic components and inorganic components, one component may be used, or two or more components may be used in combination.

Culture conditions are not particularly limited as long as the microorganism of the present invention can grow and sterols can be produced. Cultivation can be performed, for example, under normal conditions used for culturing heterotrophic microorganisms such as Labyrinthulids.

Cultivation can be performed using a liquid medium. When culturing, the microorganism of the present invention may be cultured in a solid medium such as an agar medium and directly inoculated into a liquid medium. The medium may be inoculated. That is, the culture may be divided into seed culture and main culture. In that case, the culture conditions for seed culture and main culture may or may not be the same. The amount of the microorganism of the present invention contained in the medium at the start of culture is not particularly limited. The main culture may be performed, for example, by inoculating 1 to 50% (v/v) of the seed culture into the medium for the main culture.

Cultivation can be performed by batch culture, fed-batch culture, continuous culture, or a combination thereof. In addition, the medium at the start of culture is also referred to as "starting medium". In addition, the medium supplied to the culture system (fermentor) in fed-batch culture or continuous culture is also referred to as "fed-batch medium". Also, supplying a feed medium to a culture system in fed-batch culture or continuous culture is also referred to as "fed-batch". In addition, when the culture is performed separately into a seed culture and a main culture, both the seed culture and the main culture may be performed by batch culture, for example. Also, for example, the seed culture may be performed by batch culture, and the main culture may be performed by fed-batch culture or continuous culture.

Culturing can be performed, for example, under aerobic conditions. The aerobic condition means that the dissolved oxygen concentration in the liquid medium is 0.33 ppm or more, which is the detection limit of an oxygen membrane electrode, preferably 1.5 ppm or more. The oxygen concentration may be controlled to, for example, 5 to 50% of the saturated oxygen concentration, preferably about 10%. Cultivation under aerobic conditions can be specifically performed by aerobic culture, shaking culture, agitation culture, or a combination thereof. The pH of the medium may be, for example, pH 3-10, preferably pH 4.0-9.5. During cultivation, the pH of the medium can be adjusted as needed. The pH of the medium can be adjusted to 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, and sulfuric acid. can be adjusted using The culture temperature may be, for example, 20°C to 35°C, preferably 25°C to 35°C. The culture period may be, for example, 10 hours to 120 hours. Cultivation may be continued, for example, until the carbon source in the medium is consumed, or until the activity of the microorganism of the present invention is lost. By culturing the microorganism of the present invention under such conditions, sterol-containing cells are produced.

Sterols can be appropriately recovered from the cells. That is, sterols can be extracted and recovered from the cells. The cells may be subjected to sterol extraction as they are contained in the culture solution, or may be recovered from the culture solution and then subjected to sterol extraction. In addition, the cells (for example, the culture medium containing the cells or the cells collected from the culture medium) are appropriately subjected to treatments such as dilution, concentration, freezing, thawing, drying, etc., and then subjected to sterol extraction. may These treatments may be performed singly or in combination as appropriate. These treatments can be appropriately selected according to various conditions such as the type of sterol extraction method.

The technique for recovering the cells from the culture medium is not particularly limited, and for example, a known technique (Grima, E. M. et al. 2003. Biotechnol. Advances 20: 491-515) can be used. Specifically, the cells can be recovered from the culture solution by, for example, natural sedimentation, centrifugation, filtration, or the like. A flocculant may also be used at that time. The recovered cells can be appropriately washed using an appropriate medium. In addition, the collected cells can be appropriately resuspended using an appropriate medium. Examples of media that can be used for washing and suspension include aqueous media (aqueous solvents) such as water and aqueous buffers, 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 extraction method.

A technique for extracting sterols is not particularly limited, and for example, a known technique can be used. Examples of such a technique include a technique for extracting lipids from the cells of microorganisms such as common algae. Specific examples of such techniques include organic solvent treatment, ultrasonic treatment, bead crushing treatment, acid treatment, alkali treatment, enzymatic treatment, hydrothermal treatment, supercritical treatment, microwave treatment, electromagnetic field treatment, and compression treatment. is mentioned. These techniques can be used alone or in combination as appropriate.

The organic solvent used for the organic solvent treatment is not particularly limited as long as it can extract sterols from the 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, methyl acetate and ethyl acetate. alkanes such as n-hexane, and chloroform. The Bligh-Dyer method and the Folch method are examples of lipid extraction methods using organic solvents. As the organic solvent, one organic solvent may be used, or two or more organic solvents may be used in combination.

The pH of the alkaline treatment is not particularly limited as long as the pH allows sterols to be extracted from the cells. The pH of the alkaline treatment is usually pH 8.5 or higher, preferably pH 10.5 or higher, more preferably pH 11.5 or higher, and may be pH 14 or lower. The temperature of alkaline treatment is usually 30
°C or higher, preferably 50°C or higher, more preferably 70°C or higher. The temperature of the alkaline treatment may preferably be 120°C or lower. The alkali treatment time is usually 10 minutes or longer, preferably 30 minutes or longer, and more preferably 50 minutes or longer. The alkali treatment time may preferably be 150 minutes or less. Alkaline substances such as NaOH and KOH can be used for alkali treatment.

Recovery of the extracted sterol can be performed by a known method used for separation and purification of compounds. Such techniques include, for example, an ion exchange resin method and a membrane treatment method. These techniques can be used alone or in combination as appropriate.

The collected sterols may contain components such as bacterial cells, medium components, moisture, components used in the extraction process, metabolic by-products of the microorganism of the present invention, in addition to sterols. The sterols may be purified to the desired degree. Purity of sterols, e.g. 1% (w/w) or more, 2% (w/w) or more, 5% (w/w) or more, 10% (w/w) or more, 30% (w/w) ≥ 50% (w/w) ≥ 70
% (w/w) or greater, 90% (w/w) or greater, or 95% (w/w) or greater.

The type and amount of sterol can be determined by known techniques used for detection or identification of compounds. Such techniques include, for example, HPLC, LC/MS, GC/MS, NMR. These techniques can be used alone or in combination as appropriate.

The recovered sterol can be, for example, sterol in free form, derivatives thereof (sterol esters, steryl glucosides, acyl steryl glucosides, etc.), or mixtures thereof. Also, when sterols are present in the form of their derivatives, hydrolysis can also produce free sterols. Hydrolysis can be carried out by conventional methods. For example, sterol esters can be enzymatically hydrolyzed by esterases, and steryl glucosides and acyl steryl glucosides can be enzymatically hydrolyzed by glycosidases.

Sterols have a variety of uses. Uses of sterols are not particularly limited. The sterols can be used, for example, as they are, alone, in combination with other ingredients, or as raw materials for other ingredients. Uses of sterols include food additives, feed additives, health foods, pharmaceuticals, chemical products, cosmetic ingredients, and raw materials thereof. Feeds include livestock feeds and aquatic feeds.

EXAMPLES The present invention will now be described in greater detail by way of non-limiting examples.

[Example 1] Construction of smt1 gene-deficient strain
Using the Aurantiochytrium sp.1 strain AJ7869 (FERM BP-22342) as the parent strain, a strain lacking the smt1 gene (SEQ ID NO: 3) was constructed by the following procedure.

PCR was performed using the genomic DNA of the AJ7869 strain as a template and the primers of SEQ ID NOS: 10 and 11 to amplify a DNA fragment containing the upstream region of the smt1 gene. PCR was performed using the genomic DNA of the AJ7869 strain as a template and the primers of SEQ ID NOs: 12 and 13 to amplify a DNA fragment containing the downstream region of the smt1 gene. Blasticidin S resistance gene expression cassette (Keishi Sakaguchi, et al., Versatile Transformation system that is Applicable to both
multiple transgene expression and gene targeting for thraustochytrids.
and Environmental Microbiology. 2012;78(9):3193-3202.) as a template, PCR was performed using the primers of SEQ ID NOs: 14 and 15 to amplify a DNA fragment containing the blasticidin S resistance gene. Using the plasmid pUC19 as a template, PCR was performed using the primers of SEQ ID NOs: 16 and 17 to amplify the pUC19 DNA fragment. These DNA fragments were ligated using In-fusion (Takara), and the resulting plasmid was named pUC19smt1-bla. Using pUC19smt1-bla as a template, PCR was performed using primers of SEQ ID NOS: 18 and 19 to obtain a DNA fragment containing the smt1 gene into which the blasticidin S resistance gene was inserted. The DNA fragment was electroporated (Keishi Sakaguchi, et al., Versatile Transformation system that is Applicable to both multiple transgene expression and gene targeting for thraustochytrids., Applied and Environmental Microbiology. 2012;78(9):3193-3202). was introduced into the AJ7869 strain to obtain a blasticidin S-resistant strain. This strain was named AJ7870 strain. This strain is an smt1 gene-deficient strain in which the smt1 gene is replaced with a blasticidin S-resistant gene.

[Example 2] Construction of smt1 gene-deficient + dhcr24 gene expression-enhanced strain
Using the AJ7869 strain as a parent strain, a strain lacking the smt1 gene (SEQ ID NO: 3) and having enhanced expression of the dhcr24 gene (SEQ ID NO: 5) was constructed by the following procedure.

Using pUC19smt1-bla as a template, PCR was performed using primers of SEQ ID NOs: 20 and 21 to amplify a DNA fragment containing the upstream region and downstream region of the smt1 gene. Using the plasmid (SEQ ID NO: 22) containing the zeocin resistance gene-dhcr24 gene expression cassette as a template, PCR was performed using the primers of SEQ ID NOs: 23 and 24 to amplify a DNA fragment containing the zeocin resistance gene-dhcr24 gene expression cassette. did. These DNA fragments were ligated using In-fusion (Takara), and the resulting plasmid was named pUC19dhcr24-zeo. Using pUC19dhcr24-Zeo as a template, PCR was performed using primers of SEQ ID NOS: 25 and 26 to obtain a DNA fragment containing the zeocin resistance gene-dhcr24 gene expression cassette to which the smt1 disruption site was added. The DNA fragment was electroporated (Keishi Sakaguchi, et al., Versatile Transformation system that is Applicable to both multiple transgene expression and gene targeting for thraustochytrids., Applied and Environmental Microbiology. 2012;78(9):3193-3202). was introduced into the AJ7869 strain to obtain a zeocin-resistant strain. This strain was named AJ7876 strain. This strain is a smt1 gene-deficient strain with enhanced expression of the dhcr24 gene, in which the smt1 gene is replaced with a zeocin-resistant gene-dhcr24 gene expression cassette.

[Example 3] Construction of dagt2B gene expression-enhanced strain
Using the AJ7869 strain, the AJ7870 strain, and the AJ7876 strain (Δsmt: dhcr24) as parent strains, strains with enhanced expression of the dgat2B gene (SEQ ID NO: 1) were constructed according to the following procedure.

Using the plasmid (SEQ ID NO: 27) containing the neomycin resistance gene-dgat2B gene expression cassette as a template, PCR was performed using the primers of SEQ ID NOS: 28 and 29 to obtain a DNA fragment containing the neomycin resistance gene-dgat2B gene expression cassette. rice field. The DNA fragment was electroporated (Keishi Sakaguchi, et al., Versatile Transformation system that is Applicable to both multiple transgene expression and gene targeting for thraustochytrids., Applied and Environmental Microbiology. 2012;78(9):3193-3202). was introduced into strains AJ7869, AJ7870, and AJ7876 (Δsmt: dhcr24) to obtain neomycin-resistant strains. The acquired strains were named AJ7874 strain, AJ7875 strain, and AJ7877 strain, respectively. These strains are strains with enhanced expression of the Dgat2B gene in which the neomycin resistance gene-Dgat2B gene expression cassette was randomly inserted into the genome.

[Example 4] Culture evaluation of AJ7869 strain and its modified strain
AJ7869 strain, AJ7870 strain, AJ7876 strain, AJ7874 strain, AJ7875 strain, and AJ7877 strain were added to 0.2× Reihimani storage medium (Nissui) prepared with 0.5× artificial seawater to final concentrations of 2 mg/L and 0.0, respectively.
1 mg/L, 0.01 mg/L of vitamin B1 (Nacalai Tesque), vitamin B2 (Nacalai Tesque), and vitamin B12 (Nacalai Tesque) were added to the medium plate, and 28.5
℃ for 60 hours. Scrape off 5 platinum loops of cells on the plate medium and add 0.25 of the following composition.
The cells were inoculated into an Erlenmeyer flask with a 500 mL capacity baffle containing 50 mL of GY medium prepared with × artificial seawater, and cultured for 24 hours at a culture temperature of 28.5°C and stirring at 120 rpm (rotary). 1.5 mL of the obtained culture solution was inoculated into an Erlenmeyer flask with a 500 mL capacity baffle containing 48.5 mL of GTY medium prepared with 0.25 × artificial seawater, and the culture temperature was 28.5 ° C and the stirring was performed at 120 rpm (rotary) for 48 A time culture was performed.

<Composition of GY medium>
(A Ward)
Glucose 30 g/L
(B Ward)
Yeast extract 10 g/L
(C Ward)
Vitamin B1 2 mg/L
Vitamin B2 0.01 mg/L
Vitamin B12 0.01 mg/L
(District D)
PIPES 13.6g/L
Section B was adjusted to pH 6.5 using HCl, and section A was autoclaved at 120°C for 10 minutes without pH adjustment. After adjusting pH to 6.5 using NaOH in section D, filter filtration was performed in section C without pH adjustment. After Cooling Sections A and B to room temperature, Section 4 was mixed.

<Composition of GTY medium>
(A Ward)
Glucose 50 g/L
(B Ward)
Tryptone 10 g/L
Yeast extract 5 g/L
(C Ward)
Vitamin B1 2 mg/L
Vitamin B2 0.01 mg/L
Vitamin B12 0.01 mg/L
(District D)
PIPES 13.6g/L
Section B was adjusted to pH 6.5 using HCl, and section A was autoclaved at 120°C for 10 minutes without pH adjustment. After adjusting pH to 6.5 using NaOH in section D, filter filtration was performed in section C without pH adjustment. After Cooling Sections A and B to room temperature, Section 4 was mixed.

After completion of the culture, 0.5 mL of the culture medium was centrifuged, and lipids were extracted from the obtained precipitates using the Bligh-Dyer method to obtain a lipid extract. To hydrolyze the sterol esters in the lipid extract, a fatty acid methylation kit (Nacalai Tesque) was used, which can hydrolyze sterol esters while methylating fatty acids. The resulting hexane layer was applied to gas chromatography to quantify each sterol. The dry alga body weight (DCW) was calculated by centrifuging 0.5 mL of the culture solution, drying the resulting precipitate at 50°C for 3 days, and then subtracting the weight of the microtube from the measured weight.

Table 1 shows the results. In all Dgat2B expression-enhanced strains, an increase in cholesterol accumulation was observed as compared to the corresponding control strain (Dgat2B expression non-enhanced strain). In particular, strains AJ7875 (smt1-deficient-dgat2B-enhanced strain) and AJ7877 (smt1-deficient-dhcr24 + dgat2B-enhanced strain), Dgat2B-enhanced strains with increased cholesterol biosynthetic flux, showed significantly higher levels of cholesterol than corresponding control strains. Accumulated amount (/DCW(%)) increased by 0.4% to 0.5%.
From the above, it was suggested that the production of sterols such as cholesterol is improved by enhancing DGAT activity.

<Description of Sequence Listing>
SEQ ID NO: 1: Aurantiochytrium sp.1 AJ7869 strain DGAT2B gene nucleotide sequence SEQ ID NO: 2: Aurantiochytrium sp.1 AJ7869 strain DGAT2B amino acid sequence SEQ ID NO: 3: Aurantiochytrium sp.1 AJ7869 strain SMT1 gene nucleotide sequence SEQ ID NO: 4 : amino acid sequence of SMT1 of Aurantiochytrium sp.1 AJ7869 strain SEQ ID NO:5: base sequence of DHCR24 gene of Aurantiochytrium sp.1 AJ7869 strain SEQ ID NO:6: amino acid sequence of DHCR24 of Aurantiochytrium sp.1 AJ7869 strain 1 AJ7869 strain DHCR7 gene nucleotide sequence SEQ ID NO: 8: Aurantiochytrium sp.1 AJ7869 strain DHCR7 amino acid sequence SEQ ID NO: 9: Aurantiochytrium sp.1 AJ7869 strain 18S rDNA region nucleotide sequence SEQ ID NOS: 10 to 21: Primer SEQ ID NOS 22: Plasmid SEQ ID NO: 23-26: Primer SEQ ID NO: 27: Plasmid SEQ ID NO: 28-29: Primer

Claims (9)

A method for producing a sterol, comprising:
culturing a Labyrinthulea microorganism having sterol-producing ability in a medium;
recovering sterols from the cells produced by the culture;
including
The microorganism has been modified to have an increased diacylglycerol acyltransferase (DGAT) activity compared to an unmodified strain ,
A method, wherein the DGAT is a protein according to (a), (b), or (c) below:
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2;
(b) a protein comprising an amino acid sequence containing substitutions, deletions, insertions, and/or additions of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 and having DGAT activity;
(c) A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having DGAT activity. 2. The method of claim 1, wherein said DGAT is DGAT2. 3. The method of claim 1 or 2 , wherein the activity of said DGAT is increased by increasing the expression of the gene encoding said DGAT. 4. The method of claim 3 , wherein the expression of said gene is increased by increasing the copy number of said gene and/or by modifying the expression regulatory sequence of said gene. A method according to any one of claims 1 to 4 , wherein said sterol is cholesterol. A method according to any one of claims 1 to 5 , wherein said sterol is a sterol ester. The method according to any one of claims 1 to 6 , wherein said microorganism further has the following properties (A) and/or (B):
(A) the microorganism has been modified such that the activity of sterol 24-C-methyltransferase is reduced compared to an unmodified strain;
(B) the microorganism is modified such that the activity of 24-dehydrocholesterol reductase is increased compared to an unmodified strain; Any one of claims 1 to 7 , wherein the microorganism belongs to the genus Aurantiochytrium, Schizochytrium, Thraustochytrium, or Parietichytrium. The method described in section. The method according to any one of claims 1 to 8 , wherein said microorganism is Aurantiochytrium sp.1.