JP2019071860A - Method for producing sterol - Google Patents

Abstract

To provide a method for producing sterol .SOLUTION: Sterol is produced by culturing a Labyrinthula microorganism having a sterol production ability modified so as to reduce the activity of 24-dehydrocholesterol reductase (DHCR24) or sterol 24-C-methyltransferase (SMT1), and recoverying sterol from the culture.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing sterol using a labyrinthula microorganism.

  Sterols are steroid derivatives found in various organisms such as animals, plants and fungi. Sterols are produced, for example, by extraction from biological resources with these sterols. Further, among labyrinthulas which are heterotrophic microorganisms, those producing sterols such as cholesterol are known (Non-patent Document 1). In addition, methods for producing sterols using modified strains of Labyrinthula microorganisms have also been reported.

  For example, a method of producing 7-dehydrocholesterol using a labyrinthula microorganism such as Aurantiochytrium limacinum having a reduced activity of 7-dehydrocholesterol reductase has been reported (Patent Document 1).

  Also, for example, 7-dehydrocholesterol using a labyrinthines microorganism such as Aurantiochytrium limansena that has reduced the activity of sterol 24-C-methyltransferase (Sterol 24-C-methyltransferase; also referred to as "SMT1"). The manufacturing method of is reported (patent document 2). According to Patent Document 2, it is shown that the decrease of the activity of SMT1 increases the accumulation of 7-dehydrocholesterol but decreases the accumulation of cholesterol.

  SMT1 is an enzyme that introduces a methyl group at the C24 position of sterol (EC 2.1.1.41). SMT1 is one of the enzymes involved in sterol biosynthesis and specifically involved in plant sterol and fungal sterol biosynthesis. There is no known method for producing cholesterol using a Labyrinth microbe which has reduced the activity of SMT1.

  24-dehydrocholesterol reductase (also referred to as "DHCR24") is an enzyme that reduces the double bond at the C24 position of sterol (EC 1.3.1. 72). DHCR24 is one of the enzymes involved in sterol biosynthesis and specifically involved in the biosynthesis of animal sterols such as cholesterol and 7-dehydrocholesterol. There is no known method for producing plant sterols or fungal sterols using a Labyrinth microbe having a reduced activity of DHCR24.

WO 2015/108058 WO 2016/056610

Weete JD, et al., Lipids and ultrastructure of Thraustochytrium sp. ATCC 26185., Lipids. 1997 Aug; 32 (8): 839-45.

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

  As a result of conducting earnest studies to solve the above problems, the present inventor can improve the sterol producing ability of the labyrinthula by modifying the labyrinthula microorganism so that the activity of DHCR24 or SMT1 decreases. And completed the present invention.

That is, the present invention can be exemplified as follows.
[1]
A method of producing sterols,
Cultivating a labyrinthula microorganism having sterol-producing ability in a culture medium,
Recovering sterol from cells produced by the culture;
Including
Said microorganism has been modified such that the activity of 24-dehydrocholesterol reductase is reduced compared to the unmodified strain;
The sterol is at least one sterol selected from plant sterol and fungal sterol,
However, the method except that the microorganism is Aurantiochytrium sp.
[2]
The method wherein the 24-dehydrocholesterol reductase is a protein according to (a), (b) or (c) below:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 18;
(B) A protein comprising an amino acid sequence comprising substitution, deletion, insertion and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 18 and having 24-dehydrocholesterol reductase activity ;
(C) A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 18 and having 24-dehydrocholesterol reductase activity. [3]
The method wherein the activity of 24-dehydrocholesterol reductase is reduced by reducing the expression of the gene encoding 24-dehydrocholesterol reductase or by disrupting said gene.
[4]
The method wherein the activity of 24-dehydrocholesterol reductase is reduced by deletion of part or all of the amino acid sequence of 24-dehydrocholesterol reductase.
[5]
The method, wherein the sterol is at least one sterol selected from 7-dehydropolyferasterol, stigmasterol, 4-methyl-7, 22-stigmastadienol, ergosterol, and 7-dihydroergosterol.
[6]
A method of producing sterols,
Cultivating a labyrinthula microorganism having sterol-producing ability in a culture medium,
Recovering sterol from cells produced by the culture;
Including
Said microorganism has been modified to reduce the activity of sterol 24-C-methyltransferase compared to the unmodified strain;
The sterol is cholesterol,
However, the method except that the microorganism is Aurantiochytrium sp.
[7]
The method wherein the sterol 24-C-methyltransferase is a protein according to (a), (b) or (c) below:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 20;
(B) an amino acid sequence comprising substitution, deletion, insertion and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 20, and sterol 24-C-methyltransferase activity Proteins possessed;
(C) A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 20 and having sterol 24-C-methyltransferase activity.
[8]
The method wherein the activity of sterol 24-C-methyltransferase is reduced by reducing the expression of the gene encoding sterol 24-C-methyltransferase or by disrupting said gene.
[9]
The method wherein the activity of sterol 24-C-methyltransferase is reduced by deletion of part or all of the amino acid sequence of sterol 24-C-methyltransferase.
[10]
The above method wherein the sterol is in free form sterol, sterol ester, steryl glucoside, acyl steryl glucoside or mixtures thereof.
[11]
The method according to the above, wherein the microorganism belongs to the genus Aurantiochytrium, Schizochytrium, Thraustochytrium, or Parietichytrium.
[12]
The method, wherein the microorganism is Aurantiochytrium limacinum.

  According to the present invention, it is possible to improve the sterol producing ability of a labyrinthula microorganism and to efficiently produce sterol.

(A) Schematic of DHCR24 gene deficient construct. (B) Schematic of confirmation of DHCR24 gene defect by PCR. (A) Schematic of SMT1 gene deletion construct. (B) Schematic of confirmation of SMT1 gene deletion by PCR. The figure which shows the result of sterol analysis of a wild strain and a DHCR24 gene deletion strain. (A) free sterols, (B) sterol derivatives. The vertical axis represents Peak Area. Bar graph left: wild strain; bar graph middle and right: two DHCR24 gene-deficient strains. The results are shown as Mean ± SD (n = 3). The figure which shows the result of sterol analysis of a wild strain and a SMT1 gene deletion strain. (A) free sterols, (B) sterol derivatives. The vertical axis represents Peak Area. Bar graph left represents wild strain, and bar graph right represents SMT1 gene deficient strain. The results are shown as Mean ± SD (n = 3).

  Hereinafter, the present invention will be described in detail.

<1> Microorganism of the Present Invention The microorganism of the present invention is a labyrinthula having sterol-producing ability, which has been modified so as to reduce the activity of DHCR24 or SMT1.

<1-1> Labyrinthula microorganism "Labyrinthula microorganism" refers to a microorganism classified into labyrinthula. As the Labyrinth microbes, there are aurantiochytrium, Schizochytrium, Thraustochytrium, Parietichytrium, Labyrinthula, and Altrunia. Aplanochytrium, Japonochytrium, Labyrinthuloides, Ulkenia, Oblongichytrium, Botryochytrium, and A microorganism belonging to a genus such as the genus Sicyoidochytrium can be mentioned. Examples of the Labyrinth microbe include, in particular, Aurantiochytrium microbe, Schizochytrium microbe, Thraustochytrium microbe, and Parietichytrium microbe. As the labyrinthula microorganism, more particularly, a microorganism of the genus Aurantiochytrium can be mentioned. As a microorganism of the genus Aurantiochytrium, for example, Aurantiochytrium limacinum and Aurantiochytrium sp. 2 can be mentioned. As a Schizochytrium genus microorganism, Schizochytrium aggregatum is mentioned, for example. As the Thraustochytrium genus microorganism, for example, Thraustochytrium aureum can be mentioned. Examples of the microorganism of the genus Parietichytrium include, for example, Parietichytrium sarkarianum. As a Botryochytrium genus microorganism, for example, Botryochytrium radiatum is mentioned. Specific examples of the labyrinthula microorganism include, for example, Aurantiocyclium limacinum mh0186 (FERM BP-11311), Aurantiochytrium limacinum ATCC MYA-1381, Thraustochytrium aureum ATCC 34304, Thraustochytrium sp. SEK 210 (NBRC 102615), Schizochytrium sp. SEK 345 (NBRC 102616), Parietiechytrium sarkarianum SEK 364 (FERM BP-11298), Ulkenia sp. ATCC 28207, Botryochtrium radiatum SEK 353 (NBRC 104107).

  Aurantiochytrium limacinum mh0186 is a strain conventionally called Schizochytrium sp. M-8. The genus Schizochytrium, which was conventionally called Schizochytrium, has been reorganized into Schizochytrium, Aurantiochytrium, and Oblongichytrium (Rinka Yokoyama, Daiske Honda (2007) Mycoscience Taxonomic rearrangement of the genus Schizochytrium sensu lato based on biology, , 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, March 29, 2004, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary (National Institute of Technology and Evaluation, National Institute of Technology and Evaluation, Patent Organism Depositary, Japan Postal Code: 292-0818, Address: After being originally deposited under Accession No. FERM P-19755 in Kazusa Tsuji, Kisarazu City, Chiba Prefecture, Japan, as Accession No. FERM P-19755, it was transferred to an international deposit, and Accession No. FERM BP-11311 is assigned.

  These strains can be distributed, 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 given, and it is possible to obtain a sale using this registration number (see http://www.atcc.org/). The registration number corresponding to each strain is listed in the catalog of American Type Culture Collection. Also, these strains can be obtained, for example, from the depository where each strain has been deposited.

The microorganism of the present invention can be obtained by appropriately modifying a Labyrinthula microorganism as exemplified above. That is, the microorganism of the present invention may be a modified strain derived from a Labyrinthula microorganism as exemplified above. In one aspect, the present invention may exclude the case where the microorganism of the present invention is a modified strain derived from Aurantiochytrium limacinum ATCC MYA-1381. Specifically, for example, the present invention may exclude the case where the microorganism of the present invention is a disrupted strain (eg, a deficient strain) of the SMT1 gene of Aurantiochytrium limacinum ATCC MYA-1381.

  In the present invention, the Labyrinthula microorganism is selected from Labyrinthula microorganisms other than Aurantiochytrium sp. That is, in the present invention, the case where the labyrinthula microorganism is Aurantiochytrium sp. 1 is excluded. "Aurantiochytrium sp. 1" refers to a specific species of the genus Aurantiochytrium, specifically to the species to which Aurantiochytrium sp. 1 strain AJ7869 (FERM P-22342) belongs. In other words, "Aurantiochytrium sp. 1" specifically refers to a group of microorganisms consisting of a strain belonging to the same strain as the Aurantiochytrium sp. 1 AJ7869 strain. As a strain belonging to the same species as the Aurantiochytrium sp. 1 AJ7869 strain, a strain in which the nucleotide sequence of 18S rDNA has 97% or more identity to the nucleotide sequence of 18S rDNA of Aurantiochytrium sp. 1 AJ7869 strain (SEQ ID NO: 21) Can be mentioned. Specific examples of Aurantiochytrium sp. 1 include, in addition to AJ7869 strain, AJ7867 strain (FERM BP-22304), AJ7868 strain (FERM BP-22290), BURABG162 strain, SKE217 strain and SKE218 strain. In addition, all strains (for example, modified strains) derived from the above-exemplified strains of Aurantiochytrium sp. 1 are encompassed in Aurantiochytrium sp.

<1-2> Sterol-Producing Ability The microorganism of the present invention has a sterol-producing ability. The “microorganism having sterol-producing ability” refers to a microorganism having the ability to produce the desired sterol when cultured in a culture medium and accumulate it in the cells to the extent that it can be recovered.

  The microorganism of the present invention may inherently have sterol producing ability, or may be modified to have sterol producing ability. For example, a labyrinthula microorganism as exemplified above can be used as a microorganism having sterol producing ability, as it is or after imparting or enhancing sterol producing ability. The method for imparting or enhancing the sterol producing ability is not particularly limited. For example, as described later, sterol-producing ability can be imparted or enhanced by modifying a Labyrinthula microorganism so as to decrease the activity of DHCR24 or SMT1. In addition, for example, the ability to produce cholesterol can be imparted or enhanced by, for example, enhancing expression of an enzyme involved in biosynthesis of cholesterol (WO2016 / 056610).

  The target sterols when the microorganism of the present invention is modified so as to reduce the activity of DHCR 24 include plant sterols and fungal sterols. The target sterols when the microorganism of the present invention is modified so as to reduce the activity of DHCR24 include, in particular, plant sterols. The total amount of plant sterols and fungal sterols produced relative to the total amount of sterols produced is, for example, 90% (w / w) or more, 95% (w / w) or more, 97% (w / w) or 99 It may be% (w / w) or more. The amount of plant sterol produced relative to the total amount of sterol produced is, for example, 50% (w / w) or more, 70% (w / w) or more, 80% (w / w) or more, or 85% (w) / W) or more. "Plant sterols" and "fungal sterols" refer to sterols found in plants and fungi, respectively. Examples of plant sterols include 7-dehydropolyferasterol, stigmasterol, 4-methyl-7,22-stigmastadienol, β-sitosterol, brassicasterol, campesterol, diosgenin, oleanolic acid, betulinic acid, ursolic acid, hecogenin, There may be mentioned sarsasapogenin, isofucosterol, avenasterol, Δ7-stigmastenol, Δ7-campestenol, fucosterol and salgasterol. Plant sterols include, in particular, 7-dehydropolyferasterol, stigmasterol, 4-methyl-7,22-stigmathienol. Fungal sterols include ergosterol and 7-dihydroergosterol. Fungal sterols include in particular ergosterol.

  The target sterols when the microorganism of the present invention is modified to reduce the activity of SMT1 include cholesterol. The amount of cholesterol produced relative to the total amount of sterols produced is, for example, 90% (w / w) or more, 95% (w / w) or more, 97% (w / w) or more, or 99% (w / w) w) It may be more than.

  The microorganism of the present invention may have the ability to produce one sterol, and may have the ability to produce two or more sterols.

  The term "sterol" generally refers to free sterol and its derivatives unless otherwise specified. That is, "sterol" may mean, if not otherwise specified, sterol in free form, a derivative thereof, or a mixture thereof. Derivatives of sterols include sterol esters, steryl glucoside, acyl steryl glucosides. That is, "sterol" may specifically mean, for example, sterol in free form, sterol ester, steryl glucoside, acyl steryl glucoside, or a mixture thereof. There are no particular restrictions on the type of fatty acid (acyl group) that makes up the sterol ester or the acylsteryl glucoside. As a fatty acid (acyl group), for example, docosahexaenoic acid (DHA, 22: 6) can be mentioned.

<1-3> Decreased Activity of DHCR24 or SMT1 The microorganism of the present invention is modified to reduce the activity of DHCR24 or SMT1. Specifically, the microorganism of the present invention is modified such that the activity of DHCR24 or SMT1 is reduced as compared to the non-modified strain. By modifying the Labyrinthula microorganism so that the activity of DHCR24 or SMT1 is reduced, the sterol producing ability of the same microorganism can be improved, that is, the production of sterol by the same microorganism can be increased. Specifically, the reduced activity of DHCR24 can increase the production of plant sterols and / or fungal sterols. Also, specifically, the decrease in the activity of SMT1 can increase the production of cholesterol.

  The microorganism of the present invention can be obtained by modifying a Labyrinthial microorganism having sterol-producing ability so that the activity of DHCR24 or SMT1 is reduced. The microorganism of the present invention can also be obtained by imparting sterol producing ability after modifying a Labyrinthula microorganism so as to reduce the activity of DHCR24 or SMT1. Alternatively, the microorganism of the present invention may be one to which sterol-producing ability has been imparted by modification so as to reduce the activity of DHCR24 or SMT1. Moreover, the strain before being modified so as to reduce the activity of DHCR24 or SMT1 may have an ability to produce sterols other than the desired sterols. That is, for example, a Labyrinth microbe having the ability to produce animal sterols may be modified to reduce the activity of DHCR24. "Animal sterol" refers to a sterol found in an animal. Animal sterols include cholesterol and 7-dehydrocholesterol. In addition, for example, Labyrinth microbes capable of producing plant sterols and / or fungal sterols may be modified to reduce the activity of SMT1. In the present invention, the modifications for constructing the microorganism of the present invention can be performed in any order.

  Hereinafter, DHCR24 and SMT1, and genes encoding them will be described.

"24-Dehydrocholesterol reductase (DHCR24)" refers to a protein (enzyme) having an activity of catalyzing a reaction for reducing the double bond at the C24 position of sterol. The same activity is also referred to as "24-dehydrocholesterol reductase activity (DHCR24 activity)." In addition, the gene encoding DHCR24 is referred to as “24-dehydrocholesterol reductase gene (DH
CR24 gene).

  DHCR24 activity can be obtained, for example, by incubating the enzyme with a substrate (sterol having a double bond at position C24) in the presence of an electron donor, and producing an enzyme and substrate dependent product (sterol in which the double bond at position C24 is reduced) Can be measured by measuring the formation of Examples of the electron donor include NADPH. Substrates and products include, for example, zymosterol (Zymosterol) and 5a-cholesta-8-en-3b-ol (5a-Cholest-8-en-3b-ol), respectively.

  The “sterol 24-C-methyltransferase (SMT1)” refers to a protein (enzyme) having an activity of catalyzing a reaction of introducing a methyl group at the C24 position of sterol. The same activity is also referred to as "sterol 24-C-methyltransferase activity (SMT1 activity)." In addition, the gene encoding SMT1 is also referred to as "sterol 24-C-methyltransferase gene (SMT1 gene)".

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

  The decrease in the activity of DHCR24 or SMT1 can be confirmed by measuring the activity or the like as described later. In addition, the decrease in the activity of DHCR24 or SMT1 can also be confirmed using an increase in the production of the desired sterol. In addition, the decrease in the activity of DHCR24 or SMT1 can also be indicated by the decrease in the production of sterols other than the target sterols.

  The nucleotide sequences of DHCR24 gene and SMT1 gene possessed by the Labyrinthula microorganism, and the amino acid sequences of DHCR24 and SMT1 encoded by them can be obtained from public databases such as, for example, the National Center for Biotechnology Information (NCBI). The nucleotide sequence of DHCR24 gene of Aurantiochytrium limacinum mh0186 and the amino acid sequence of DHCR24 encoded by the same gene are shown in SEQ ID NOs: 17 and 18, respectively. That is, the DHCR24 gene may be, for example, a gene having the base sequence shown in SEQ ID NO: 17. In addition, DHCR 24 may be, for example, a protein having the amino acid sequence shown in SEQ ID NO: 18. The nucleotide sequence of SMT1 gene of Aurantiochytrium limacinum mh0186 and the amino acid sequence of SMT1 encoded by the same gene are shown in SEQ ID NOs: 19 and 20, respectively. That is, the SMT1 gene may be, for example, a gene having the base sequence shown in SEQ ID NO: 19. In addition, SMT1 may be, for example, a protein having the amino acid sequence shown in SEQ ID NO: 20. In addition, the expression "having an (amino acid or base) sequence" means, unless otherwise stated, including the "(amino acid or base) sequence", including the case of the "(amino acid or base) sequence" Do.

The DHCR24 gene may be a variant of the DHCR24 gene exemplified above (for example, a gene having the nucleotide sequence shown in SEQ ID NO: 17) as long as the original function is maintained. Similarly, DHCR 24 may be a variant of DHCR 24 exemplified above (eg, a protein having the amino acid sequence shown in SEQ ID NO: 18) as long as the original function is maintained. In addition, the SMT1 gene may be a variant of the above-exemplified SMT1 gene (for example, a gene having a nucleotide sequence shown in SEQ ID NO: 19) as long as the original function is maintained. Similarly, SMT1 may be a variant of the above-exemplified SMT1 (for example, a protein having the amino acid sequence shown in SEQ ID NO: 20) as long as the original function is maintained. In addition, the variant in which such original function was maintained may be called "conservative variant." The terms "DHCR24 gene" and "SMT1 gene" are intended to encompass their conservative variants in addition to the DHCR24 gene and SMT1 gene exemplified above, respectively. Similarly, the terms "DHCR24" and "SMT1" are intended to encompass their conservative variants in addition to the DHCR24 and SMT1 exemplified above, respectively. The conservative variants include, for example, the DHCR24 gene and SMT1 gene exemplified above and homologs of DHCR24 and SMT1.

  "The original function is maintained" means that a variant of a gene or protein has a function (activity or property) corresponding to the function (activity or property) of the original gene or protein. "The original function is maintained" for a gene means that a variant of the gene encodes a protein with the original function maintained. That is, "the original function is maintained" for the DHCR24 gene means that a variant of the gene encodes a protein having DHCR24 activity. Also, "the original function is maintained" for DHCR24 means that a variant of the protein has DHCR24 activity. Also, "the original function is maintained" for the SMT1 gene means that a variant of the gene encodes a protein having SMT1 activity. Also, "the original function is maintained" for SMT1 means that a variant of the protein has SMT1 activity.

  The following is an example of conservative variants.

  The homolog of DHCR24 gene or SMT1 gene or the homolog of DHCR24 or SMT1 can be obtained, for example, by BLAST search or FASTA search using the nucleotide sequence of DHCR24 gene or SMT1 exemplified above or the amino acid sequence of DHCR24 or SMT1 exemplified above as a query sequence It can be easily identified from public databases. Furthermore, homologs of the DHCR24 gene or SMT1 gene are obtained, for example, by PCR using an oligonucleotide prepared based on the nucleotide sequence of the known DHCR24 gene or SMT1 gene, using the chromosome of a labyrinthula microorganism as a template, as a primer. Can be identified.

  The DHCR24 gene or SMT1 gene may be one or several in the above amino acid sequence (for example, the amino acid sequence shown in SEQ ID NO: 18 for DHCR24, the amino acid sequence shown in SEQ ID NO: 20 for SMT1) as long as the original function is maintained. It may be a gene encoding a protein having an amino acid sequence in which one or several amino acids at a position are substituted, deleted, inserted and / or added. For example, the encoded protein may be extended or truncated at its N-terminus and / or C-terminus. In addition, although said "one or several" changes also with the position in the three-dimensional structure of protein of an amino acid residue, and the kind of amino acid residue, specifically, 1-50 pieces, 1-40 pieces, 1 It means 30 to 30, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, particularly preferably 1 to 3.

The above-mentioned substitution, deletion, insertion and / or addition of one or several amino acids is a conservative mutation in which the function of the protein is normally maintained. Representatives of conservative mutations are conservative substitutions. Conservative substitution is a polar amino acid 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. In the case where the amino acid has a hydroxyl group between Gln and Asn if it is a basic amino acid, if it is an acidic amino acid if it is an acidic amino acid if it is an acidic amino acid, if it is an amino acid having a hydroxyl group Are mutations that substitute each other between Ser and Thr. Specifically, substitution considered as conservative substitution includes substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from 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 Substitution, substitution of Gly to Pro, substitution of His to Asn, Lys, Gln, Arg or Tyr, substitution of Ile to Leu, Met, Val or Phe, substitution of Leu to Ile, Met, Val or Phe, 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 Examples include substitution, substitution of Thr to Ser or Ala, substitution of Trp to Phe or Tyr, substitution of Tyr to His, Phe or Trp, and substitution of Val to Met, Ile or Leu. In addition, amino acid substitutions, deletions, insertions, or additions as described above may also be those caused by naturally occurring mutations (mutants or variants) based on individual differences of organisms from which the gene is derived, differences in species, etc. included.

  The DHCR24 gene or SMT1 gene is, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 50% or more, based on the entire amino acid sequence as long as the original function is maintained. It may be a gene encoding a protein having an amino acid sequence having homology of 95% or more, more preferably 97% or more, particularly preferably 99% or more. In the present specification, "homology" means "identity".

  In addition, DHCR24 gene or SMT1 gene is prepared from the above base sequence (for example, the base sequence shown in SEQ ID NO: 17 for DHCR24 gene, the base sequence shown in SEQ ID NO: 19 for SMT1 gene) as long as the original function is maintained. The resulting probe may be, for example, DNA that hybridizes with a complementary sequence to all or part of the above base sequence under stringent conditions. "Stringent conditions" refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more of DNAs having high homology. C., 1 × SSC, 0.1% SDS, which is a condition under which DNAs having% or more homology hybridize with each other, and DNAs with lower homology with each other do not hybridize, or under conditions for washing with ordinary Southern hybridization. Conditions which are preferably washed once, preferably 2-3 times, at a salt concentration and temperature corresponding to 60.degree. C., 0.1.times.SSC, 0.1% SDS, more preferably 68.degree. C., 0.1.times.SSC, 0.1% SDS. be able to.

  As mentioned above, the probe used for the above hybridization may be part of a complementary sequence of a gene. Such a probe can be produced by PCR using an oligonucleotide produced based on a known gene sequence as a primer and a DNA fragment containing the aforementioned gene as a template. For example, a DNA fragment about 300 bp in length can be used as a probe. When a DNA fragment of about 300 bp in length is used as a probe, conditions for washing of hybridization include 50 ° C., 2 × SSC, and 0.1% SDS.

  Also, the DHCR24 gene or SMT1 gene may have any codon replaced with an equivalent codon. That is, the DHCR24 gene or SMT1 gene may be a variant of the DHCR24 gene or SMT1 gene exemplified above due to the degeneracy of the genetic code.

  The percentage of sequence identity between two sequences can be determined, for example, using a mathematical algorithm. A non-limiting example of such a mathematical algorithm is the algorithm of Myers and Miller (1988) CABIOS 4: 11-17, Smith et al (1981) Adv. Appl. Math. 2: 482 local homology algorithm, Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453 homology alignment algorithm, Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85: 2444-2448, a method of searching for similarity, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877, and the improved algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. It can be mentioned.

Programs based on these mathematical algorithms can be used to perform sequence comparisons (alignments) to determine sequence identity. The program can be suitably executed by a computer. Such a program is not particularly limited, but
The PC / Gene program CLUSTAL (available from Intelligenetics, Mountain View, Calif.), The ALIGN program (Version 2.0), and the Wisconsin Genetics Software Package, Version 8 (Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis. , GAP, BESTFIT, BLAST, FASTA, and TFASTA). Alignment using these programs can be performed, for example, using initial parameters. For the CLUSTAL program, see Higgins et al. (1988) Gene 73: 237-244, Higgins et al. (1989) CABIOS 5: 151-153, Corpet et al. (1988) Nucleic Acids Res. 16: 10881-90, Huang et al. (1992) CABIOS 8: 155-65, and Pearson et al. (1994) Meth. Mol. Biol. 24: 307-331.

  Specifically, for example, a BLAST nucleotide search can be performed with the BLASTN program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleotide sequence encoding the protein of interest. Specifically, for example, a BLAST protein search can be performed with the BLASTX program, score = 50, word length = 3 to obtain an amino acid sequence homologous to a protein of interest. For BLAST nucleotide searches and BLAST protein searches, see http://www.ncbi.nlm.nih.gov. Also, Gapped BLAST (BLAST 2.0) can be used to obtain gapped alignments for comparison purposes. Also, PSI-BLAST (BLAST 2.0) can be used to perform an iterated search which detects distant relationships between sequences. For Gapped BLAST and PSI-BLAST, see Altschul et al. (1997) Nucleic Acids Res. 25: 3389. When utilizing BLAST, Gapped BLAST, or PSI-BLAST, for example, initial parameters of each program (eg, BLASTN for nucleotide sequences, BLASTX for amino acid sequences) can be used. Alignment may be performed manually.

  Sequence identity between two sequences is calculated as the ratio of residues which match between the two sequences when the two sequences are aligned for maximal match.

  The descriptions of the above-mentioned variants of genes and proteins can be applied to any proteins and genes encoding them.

The method to reduce the activity of <1-4> protein Below, the method to reduce the activity of proteins, such as DHCR24 and SMT1, is demonstrated.

"The activity of the protein is reduced" means that the activity of the protein is reduced as compared to the non-modified strain. "The activity of the protein is reduced" specifically means that the activity per cell of the same protein is reduced as compared to the non-modified strain. As used herein, "non-modified strain" means a control strain that has not been modified to reduce the activity of the target protein. Non-modified strains include wild strains and parent strains. Specific examples of the non-modified strain include the strains exemplified in the description of the Labyrinthula microorganism. Thus, in one aspect, the activity of the protein may be reduced compared to Aurantiochytrium limacinum mh0186. Incidentally, "a decrease in the activity of a protein" also encompasses the case where the activity of the same protein is completely abolished. “The activity of the protein is reduced” more specifically means that the number of molecules per cell of the protein is reduced as compared to the non-modified strain, and / or the molecule per molecule of the protein is reduced. It may mean that the function is degraded. That is, "activity" in the case of "the activity of the protein is reduced" is not limited to the catalytic activity of the protein but means the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. May be The phrase "the number of molecules per cell of protein is reduced" also includes the case where the same protein is not present at all. In addition, "the function of the protein per molecule is reduced" includes the case where the function per molecule of the protein is completely lost. The degree of reduction of the activity of the protein is not particularly limited as long as the activity of the protein is reduced as compared to the non-modified strain. The activity of the protein may be reduced, for example, to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of the non-modified strain.

  Modification that reduces the activity of a protein can be achieved, for example, by reducing the expression of a gene encoding the same protein. "The expression of the gene is reduced" means that the expression of the same gene is reduced as compared to the non-modified strain. “The expression of the gene is reduced” specifically means that the amount of expression of the gene per cell is reduced as compared to the non-modified strain. More specifically, “a decrease in gene expression” means a decrease in gene transcription amount (mRNA amount) and / or a gene translation amount (protein amount). You may The phrase "a decrease in the expression of a gene" includes cases in which the gene is not expressed at all. In addition, that "the expression of a gene is reduced" is also referred to as "the expression of a gene is attenuated". Expression of the gene may be reduced, for example, to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of the non-modified strain.

  The reduction in gene expression may be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination thereof. The reduction of gene expression can be achieved, for example, by modifying the expression regulatory sequence of the gene. "Expression control sequence" is a generic term for a site that affects the expression of a gene, such as a promoter. The expression regulatory sequence can be determined using, for example, a promoter search vector or gene analysis software such as GENETYX. When modifying the expression regulatory sequence, the expression regulatory sequence is preferably modified with one or more bases, more preferably two bases or more, particularly preferably three bases or more. Decreased 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 transcription of a gene is weaker than that of the wild-type promoter originally present. 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 region of the expression regulatory sequence may be deleted (deletion). 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 small molecules (inducers, inhibitors, etc.) involved in transcription and translation control, proteins (transcription factors etc.), nucleic acids (siRNA etc.) and the like. Also, reduction of gene expression can be achieved, for example, by introducing a mutation into the coding region of the gene such that gene expression is reduced. For example, gene expression can be reduced by replacing codons in the coding region of the gene with synonymous codons that are used less frequently in the host. Also, for example, gene expression itself may be reduced by gene disruption as described later.

  In addition, modifications that reduce the activity of a protein can be achieved, for example, by disrupting the gene encoding the same protein. "A gene is disrupted" means that the same gene is altered so as not to produce a normally functioning protein. The phrase "does not produce a normally functioning protein" includes cases where no protein is produced from the same gene, or cases where the same gene produces a protein with a reduced or eliminated function (activity or property) per molecule. Be done.

Disruption of a gene can be achieved, for example, by deleting a gene on a chromosome. "Gene deletion" refers to a deletion of 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. Sequences before and after the coding region of a gene may include, for example, expression regulatory sequences of the gene. The region to be deleted includes an N-terminal region (a region encoding the N-terminal side of the protein), an internal region, a C-terminal region (a region encoding the C-terminal side of the protein), etc. It may be any area. In general, the longer the region to be deleted, the more surely 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 total coding region of the gene. Or it may be an area of 95% or more in length. 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 frame shift downstream of the region to be deleted.

  In addition, gene disruption may be performed, for example, by 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 ( This can also be achieved by introducing a frame shift mutation) (Journal of Biological Chemistry 272: 8611-8617 (1997), Proceedings of the National Academy of Sciences, USA 95 551 1-5515 (1998), Journal of Biological Chemistry 26 116 , 20833-20839 (1991)).

  Disruption of a gene can also be achieved, for example, by inserting another base sequence into the coding region of a gene on a chromosome. The insertion site may be any region of the gene, but the longer the base sequence to be inserted, the more surely 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 frame shift downstream of the insertion site. The other base sequence is not particularly limited as long as it reduces or eliminates the activity of the encoded protein, and includes, for example, marker genes such as antibiotic resistance genes and genes useful for producing a target substance.

  Disruption of the gene may in particular be carried out in such a way that the amino acid sequence of the encoded protein is deleted. In other words, the modification that reduces the activity of the protein may be carried out, for example, by deleting the amino acid sequence (a part or whole region of the amino acid sequence) of the protein. This can be achieved by modifying the gene to encode a protein in which part or the entire region is deleted. The “deletion of the amino acid sequence of a protein” refers to a deletion of a part or the whole region of the amino acid sequence of a protein. In addition, "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 where the original amino acid sequence changes to another amino acid sequence. That is, for example, a region changed to another amino acid sequence by frameshift may be regarded as a deleted region. Although deletion of the amino acid sequence of a protein typically shortens the total length of the protein, the total length of the protein may not change or may be extended. For example, deletion of part or all of the coding region of a gene can delete the region encoded by the deleted region in the amino acid sequence of the encoded protein. Also, for example, by introducing a stop codon into the coding region of a gene, in the amino acid sequence of the encoded protein, the region encoded by the region downstream of the introduction site can be deleted. Also, for example, a frame shift in the coding region of a gene can delete the region encoded by the frame shift site. With regard to the position and length of the deletion region in the deletion of the amino acid sequence, the description of the position and length of the deletion region in the deletion of the gene can be applied correspondingly.

Altering the gene on the chromosome as described above produces, for example, a disrupted gene that has been altered not to produce a normally functioning protein, and transforms the host with the recombinant DNA containing the disrupted gene. This can be achieved by replacing the wild-type gene on the chromosome with the destructive gene by causing homologous recombination between the destructive gene and the wild-type gene on the chromosome. At that time, it is easy to manipulate the recombinant DNA if it contains a marker gene according to the trait such as auxotrophy of the host. The destructive gene includes a gene in which part or all of the coding region of the gene is deleted, a gene in which a missense mutation is introduced, a gene in which a nonsense mutation is introduced, a gene in which a frameshift mutation is introduced, a transposon, a marker gene, etc. And the gene into which the insertion sequence of The protein encoded by the disrupted gene, even if produced, has a three-dimensional structure different from that of the wild-type protein, and its function is reduced or eliminated. 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, the host is transformed with linear DNA containing a disrupted gene and having sequences upstream and downstream of the wild-type gene on the chromosome at both ends, and the upstream and downstream of the wild-type gene are transformed By causing homologous recombination, respectively, the wild type gene can be replaced with a destructive gene in one step.

  In addition, modifications that reduce the activity of a protein may be performed, for example, by a mutation treatment. Mutation treatments include X-ray irradiation, ultraviolet irradiation, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), and methylmethanesulfonate (MMS) Treatment with mutagens such as.

  The methods for reducing the activity of proteins as described above may be used alone or in any combination.

  The reduced activity of the protein can be confirmed by measuring the activity of the protein.

  The reduced activity of the protein can also be confirmed by confirming that the expression of the gene encoding the same protein is reduced. The reduced expression of the gene can be confirmed by confirming that the amount of transcription of the same gene has been reduced, and by confirming that the amount of protein expressed from the same gene has been reduced.

  Confirmation that the amount of transcription of the gene has been reduced can be carried out by comparing the amount of mRNA transcribed from the gene with a non-modified strain. Methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR 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 to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the non-modified strain.

  Confirmation that the amount of protein has been reduced can be performed by performing SDS-PAGE and confirming the strength of the separated protein band. In addition, confirmation that the amount of protein has been reduced can be performed by Western blot using an antibody (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The amount of protein (eg, number of molecules per cell) may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the non-modified strain.

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

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

<2> Method of Producing Sterol The method of the present invention is a method of producing sterol, which comprises culturing the microorganism of the present invention in a culture medium, and recovering sterol from cells produced by the culture. In the method of the present invention, one sterol may be produced, and two or more sterols may be produced. The combination of an enzyme that reduces activity and the sterol produced is
This is as described above in the description of sterol producing ability.

  The medium used is not particularly limited as long as the microorganism of the present invention can grow and sterols are produced. As a culture medium, the usual culture medium used for culture of heterotrophic microorganisms, such as labyrinthulas, can be used, for example. The medium may contain components selected from carbon sources, nitrogen sources, phosphoric acid sources, sulfur sources, and various other organic components and inorganic components as required. As a culture medium, the GY culture medium specifically, prepared with 0-1x artificial seawater is mentioned, for example.

  As a carbon source, specifically, for example, sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, waste molasses, starch hydrolysate, hydrolysate of biomass, etc. Organic acids such as acetic acid and citric acid And alcohols such as ethanol, glycerol and crude glycerol, and fatty acids. As a carbon source, one type of carbon source may be used, and two or more types of carbon sources may be used in combination.

  Specific examples of the nitrogen source include ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, peptone, yeast extract, organic nitrogen sources such as meat extract and soy protein hydrolyzate, ammonia and urea. Ammonia gas or aqueous ammonia used for pH adjustment may be used as a 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 phosphoric acid source include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphoric acid polymers such as pyrophosphoric acid. As a phosphoric acid source, one type of phosphoric acid source may be used, and two or more types of phosphoric acid sources may be used in combination.

  Specific examples of the sulfur source include inorganic sulfur compounds such as sulfates, thiosulfates and sulfites, and sulfur-containing amino acids such as cysteine, cystine and glutathione. As a sulfur source, one type of sulfur source may be used, and two or more types of sulfur sources may be used in combination.

  As various other organic components and inorganic components, specifically, for example, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium and calcium; vitamin B1, vitamin B2, vitamin B6, nicotine Acids, nicotinic acid amide, vitamins such as vitamin B12; amino acids; nucleic acids; and organic components such as peptone containing these, casamino acids, yeast extract, soybean protein degradation products and the like. As various other organic components and inorganic components, one component may be used, or two or more components may be used in combination.

  The culture conditions are not particularly limited as long as the microorganism of the present invention can grow and sterols are produced. Culturing can be performed, for example, under ordinary conditions used for culturing heterotrophic microorganisms such as labyrinthulas.

  The culture can be performed using a liquid medium. At the time of culture, a culture of the microorganism of the present invention in a solid culture medium such as agar may be directly inoculated into a liquid culture medium, or a culture of the microorganism of the present invention in a liquid culture medium may be used as a liquid for main culture. The medium may be inoculated. That is, the culture may be performed separately in the seed culture and the main culture. In that case, the culture conditions of the seed culture and the 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 a 1 to 50% (v / v) seed culture solution into the medium of the main culture.

The culture can be performed in batch culture, fed-batch culture, continuous culture (co
It can be carried out by ntinuous culture) or a combination thereof. The culture medium at the start of culture is also referred to as "initial culture medium". In addition, a medium supplied to a culture system (fermenter) in fed-batch culture or continuous culture is also referred to as "feed medium". Also, feeding a feed medium to a culture system in fed-batch culture or continuous culture is also referred to as "feed". In addition, when culture | cultivation is divided and performed to a seed culture and a main culture, you may carry out both a seed culture and a main culture by batch culture, for example. For example, seed culture may be performed by batch culture, and main culture may be performed by fed-batch culture or continuous culture.

  The culture can be performed, for example, under aerobic conditions. The aerobic condition means that the concentration of dissolved oxygen in the liquid culture medium is 0.33 ppm or more, which is the detection limit by the oxygen film electrode, and preferably 1.5 ppm or more. The oxygen concentration may be controlled, for example, to about 5 to 50%, preferably about 10% of the saturated oxygen concentration. Culture under aerobic conditions can be performed specifically by aeration culture, shake culture, spinner culture, or a combination thereof. The pH of the culture medium may be, for example, pH 3 to 10, preferably pH 4.0 to 9.5. During culture, the pH of the medium can be adjusted as needed. The pH of the culture medium is 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 and the like 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. The culture may be continued, for example, until the carbon source in the medium is consumed or the activity of the microorganism of the present invention is lost. By culturing the microorganism of the present invention under such conditions, bacterial cells containing sterols are produced.

  Sterols can be recovered from the cells as appropriate. 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 solution containing the cells and cells collected from the culture solution) are appropriately subjected to treatments such as dilution, concentration, freezing, thawing, drying and the like, and then subjected to extraction of sterols. May be These treatments may be performed alone or in combination as appropriate. These treatments can be appropriately selected according to various conditions such as the type of sterol extraction method.

  The method for recovering the cells from the culture solution is not particularly limited, and for example, known methods (Grima, E. M. et al. 2003. Biotechnol. Advances 20: 491-515) can be used. Specifically, for example, bacterial cells can be recovered from the culture solution by methods such as natural sedimentation, centrifugation, filtration and the like. At the same time, flocculants may be used. The collected cells can be appropriately washed using an appropriate medium. Also, the recovered cells can be appropriately resuspended using an appropriate medium. Examples of the medium that can be used for washing and suspension include an aqueous medium (aqueous solvent) such as water and an aqueous buffer, an organic medium (organic solvent) such as methanol, and a mixture thereof. The medium can be appropriately selected according to various conditions such as the type of sterol extraction method.

  The method for extracting sterols is not particularly limited, and for example, known methods can be used. As such a method, for example, a method of extracting lipids from microbial cells of general algae and the like can be mentioned. As such a method, specifically, 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, squeeze treatment Can be mentioned. These techniques can be used alone or in combination as appropriate.

The organic solvent used in the organic solvent treatment is not particularly limited as long as it can extract sterols from the cells. Examples of the organic solvent 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, ethyl acetate And esters thereof, alkanes such as n-hexane, and chloroform. Examples of extraction methods of lipids with organic solvents include Bligh-Dyer method and Folch method. 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 alkali treatment is not particularly limited as long as it can extract sterols from cells. The pH of the alkali treatment may be usually pH 8.5 or more, preferably pH 10.5 or more, more preferably pH 11.5 or more, and may be pH 14 or less. The temperature of the alkali treatment may be usually 30 ° C. or more, preferably 50 ° C. or more, more preferably 70 ° C. or more. The temperature of the alkali treatment may preferably be 120 ° C. or less. The time of the alkali treatment may be usually 10 minutes or more, preferably 30 minutes or more, and more preferably 50 minutes or more. The time of the alkali treatment may preferably be 150 minutes or less. For alkaline treatment, alkaline substances such as NaOH and KOH can be used.

  Recovery of the extracted sterols can be carried out by known methods used for separation and purification of compounds. Examples of such a method include an ion exchange resin method and a membrane treatment method. These techniques can be used alone or in combination as appropriate.

  The recovered sterols may contain, in addition to sterols, components such as bacterial cells, medium components, water, components used for extraction processing, metabolic by-products of the microorganism of the present invention, and the like. The sterols may be purified to the desired degree. The purity of the sterol is, 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) The above may be 50% (w / w) or more, 70% (w / w) or more, 90% (w / w) or more, or 95% (w / w) or more.

  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 sterol to be recovered may be, for example, a sterol in free form, a derivative thereof (sterol ester, steryl glucoside, acyl steryl glucoside, etc.), or a mixture thereof. Also, when present in the form of these derivatives, it is also possible to produce free sterols by hydrolysis. The hydrolysis can be carried out by conventional methods. For example, sterol esters can be enzymatically hydrolyzed by esterases, and steryl glucoside and acyl steryl glucosides can be hydrolyzed enzymatically by glycosidases, respectively.

  Sterols can be used for a variety of applications. The use of the sterols is not particularly limited. Sterols can be used, for example, as they are alone, in combination with other components, or as raw materials for other components. Applications of sterols include food additives, feed additives, health foods, pharmaceuticals, chemical products, cosmetic ingredients, and raw materials thereof. The feed includes livestock feed and fish feed.

  Hereinafter, the present invention will be more specifically described by way of examples.

<1> Preparation of gene-deficient strain of Aurantiochytrium limacinum <1-1> Preparation of DHCR24 gene-deficient strain
A DHCR24 gene-deficient strain was prepared by replacing the DHCR24 gene with a hygromycin resistance gene using Aurantiochytrium limacinum mh0186 (FERM BP-11311) as a parent strain (FIG. 1A). The procedure is shown below.

A fragment of 3,878 bp from 1,158 bp upstream of the start codon of the DHCR24 gene to the middle of the DHCR24 gene was amplified by PCR using the genomic DNA of Aurantiochytrium limacinum mh0186 as a template. PCR was performed using primers 1 and 2 (Table 1), Tks Gflex DNA polymerase (TakaraBio), and the PCR program was 94 ° C. 1 min, (98 ° C. 10 sec, 55 ° C. 15 sec, 68 ° C. 2.5 min) x
35 cycles, 68 ° C. 4 min. The amplified fragment and the pUC19 linearized vector were subjected to an In-Fusion reaction using an In-Fusion HD Cloning Kit (Clontech), and transformed into Escherichia coli JM109. The transformant was selected on LB agar medium containing 50 μg / ml of ampicillin, and plasmid extraction was performed to obtain a DHCR24 KO pUC19 vector.

Subsequently, a DHCR24 KO pUC19 linearized vector was amplified by PCR using primers 3 and 4 (Table 1) with the DHCR24 KO pUC19 vector as a template. The PCR enzyme was Tks Gflex DNA polymerase, and the PCR program was 94 ° C. 1 min, (98 ° C. 10 sec, 55 ° C. 15 sec, 68 ° C. 2.5 min) × 35 cycles, 68 ° C. 2 min. The hygromycin resistant gene (derived from pcDNA3.1 / Hygro (+)) is expressed under the control of the ubiquitin promoter (UbiP) and SV40 terminator (SV40T) (derived from pcDNA3.1 Myc-His) derived from Thraustochytrium aureum ATCC 34304 Vector (Matsuda, T., et al. (2012) Analysis of Delta 12-fatty acid desaturase
function revealed that two distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304. J. Lipid Res. 53, 1210-1222) as a template, using PCR with primers 5 and 6 (Table 1). The mycin resistance gene cassette was amplified. The PCR enzyme 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) × 35 cycles, 68 ° C. for 2 min. After gel purification, a DHCR24 KO pUC19 linearized vector and a fragment of the hygromycin resistance gene cassette were in-fusion reacted using In-Fusion HD Cloning Kit and transformed into Escherichia coli JM109. The transformant was isolated using ampicillin resistance, and plasmid extraction was performed to obtain a DHCR24 KO vector.

Subsequently, a DHCR24 gene-deficient construct was amplified by PCR using primers 1 and 2 (Table 1) with the DHCR24 KO vector as a template. The PCR enzyme was Tks Gflex DNA polymerase, and the PCR program was 94 ° C. 1 min, (98 ° C. 10 sec, 55 ° C. 15 sec, 68 ° C. 3 min) × 35 cycles, 68 ° C. 2 min. The resulting DHCR24 gene-deficient construct is purified by ethanol precipitation, and using an electroporation method, Aurantiochytrium limacinum
It was introduced in mh0186. Electroporation was performed in a 1 mm gap cuvette by applying two 750 V, 25 μF, 200 Ω pulses. Hygromycin resistance was used to isolate DHCR24 gene-deficient candidate strains. The colonies of the DHCR24 gene-deficient candidate strain were picked up, cultured in 3 ml of GY medium for 2 days, cells were recovered, suspended in 20 mM NaOH solution, and genome extracted by treating at 37 ° C. for 10 minutes. Using this genome as a template, deletion of the DHCR24 gene was confirmed by PCR using primers 7 and 10, 8 and 9 (Table 1). The PCR enzyme 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) × 35 cycles, 68 ° C. for 2 min. Each of these primer sets is configured to amplify a fragment only in the DHCR24 gene-deficient strain (FIG. 1B). As a result, amplification of fragments of the target size was observed in two candidate strains. Furthermore, deletion of the DHCR24 gene was confirmed by PCR using primers 9 and 10 (Table 1). The PCR enzyme was Tks Gflex DNA polymerase, and the PCR program was 94 ° C. 1 min, (98 ° C. 10 sec, 55 ° C. 15 sec, 68 ° C. 3 min) × 35 cycles, 68 ° C. 2 min. This primer set is configured to amplify a fragment larger in size than the wild strain (parent strain; 4,359 bp) in the DHCR24 gene-deficient strain (4,940 bp) (FIG. 1B). As a result, amplification of a fragment of the desired size was observed in the above two candidate strains. That is, two DHCR24 gene-deficient strains were obtained.

<1-2> Production of SMT1 gene-deficient strain
Using Aurantiochytrium limacinum mh0186 as a parent strain and replacing the SMT1 gene with a hygromycin resistant gene, a SMT1 gene-deficient strain was produced (FIG. 2A). The procedure is shown below.

A fragment of 3,006 bp from 1,010 bp upstream of the start codon of the SMT1 gene to the middle of the SMT1 gene was amplified by PCR using the genomic DNA of Aurantiochytrium limacinum mh0186 as a template. PCR was performed using primers 1 and 2 (Table 2) and 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 min) x 35 cycles, 68 ° C 2
It was min. The amplified fragment and the pUC19 linearized vector were in-fusion reacted using In-Fusion HD Cloning Kit, and transformed into Escherichia coli JM109. The transformants were selected on LB agar medium containing 50 μg / ml of ampicillin, and SMT1 KO pUC19 vector was obtained by plasmid extraction.

  Subsequently, the SMT1 KO pUC19 linearized vector was amplified by PCR using primers 3 and 4 (Table 2) with the SMT1 KO pUC19 vector as a template. The PCR enzyme was Tks Gflex DNA polymerase, and the PCR program was 94 ° C. 1 min, (98 ° C. 10 sec, 55 ° C. 15 sec, 68 ° C. 2.5 min) × 35 cycles, 68 ° C. 2 min. The hygromycin resistant gene (derived from pcDNA3.1 / Hygro (+)) is expressed under the control of the ubiquitin promoter (UbiP) and SV40 terminator (SV40T) (derived from pcDNA3.1 Myc-His) derived from Thraustochytrium aureum ATCC 34304 Vector (Matsuda, T., et al. (2012) Analysis of Delta 12-fatty acid desaturase function released that two distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304. J. Lipid Res. 53, 1210-1222 The hygromycin resistance gene cassette was amplified by PCR using primers 5 and 6 (Table 2) as a template. The PCR enzyme 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) × 35 cycles, 68 ° C. for 2 min. After gel purification, the SMT1 KO pUC19 linearized vector and a fragment of the hygromycin resistance gene cassette were in-fusion reacted using In-Fusion HD Cloning Kit, and transformed into Escherichia coli JM109. Transformants were isolated using ampicillin resistance, and plasmid extraction yielded SMT1 KO vector.

Subsequently, the SMT1 gene-deficient construct was amplified by PCR using primers 5 and 6 (Table 2) with the SMT1 KO vector as a template. The PCR enzyme was Tks Gflex DNA polymerase, and the PCR program was 94 ° C. 1 min, (98 ° C. 10 sec, 55 ° C. 15 sec, 68 ° C. 2.25 min) × 35 cycles, 68 ° C. 2 min. The resulting SMT1 gene-deficient construct was purified by ethanol precipitation and introduced into Aurantiochytrium limacinum mh0186 using electroporation. Electroporation was performed in a 1 mm gap cuvette by applying pulses twice under conditions of 750 V, 25 μF, 200 Ω. Hygromycin resistance was used to isolate SMT1 gene-deficient candidate strains. The colonies of the SMT1 gene-deficient candidate strain were picked up, cultured in 3 ml of GY medium for 2 days, cells were collected, suspended in 20 mM NaOH solution, and treated at 37 ° C. for 10 minutes to extract genomic DNA. Using this genome as a template, deletion of the SMT1 gene was confirmed by PCR using primers 7 and 10 and 8 and 9 (Table 2). The PCR enzyme is Tks Gflex DNA polymerase, and the PCR program is 94 ° C 1 min, (98 ° C 10 sec, 55 ° C 15 sec, 68 ° C 1.5 min) x
35 cycles, 68 ° C. 2 min. Each of these primer sets is configured to amplify a fragment only in the SMT1 gene-deficient strain (FIG. 2B). As a result, amplification of fragments of the target size was observed in two candidate strains. Furthermore, deletion of the SMT1 gene was confirmed by PCR using primers 9 and 10 (Table 2). The PCR enzyme 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) × 35 cycles, 68 ° C. for 2 min. This primer set is a SMT1 gene-deficient strain (4,484
Fragments larger in size than the wild strain (parent strain; 3,339 bp) are configured to be amplified in bp) (FIG. 2B). As a result, amplification of a fragment of the desired size was observed in the above two candidate strains. That is, two SMT1 gene-deficient strains were obtained. One of them was used as an SMT1 gene-deficient strain in the following experiments.

Sterol analysis of sterol production <2-1> DHCR24 gene deficient strain using gene deficient strain of <2> Aurantiochytrium limacinum
The DHCR24 gene-deficient strain and the wild strain were precultured in 3 mL of GY medium. The obtained preculture liquid was inoculated in 50 mL of GY medium in a 200 mL flask so that the OD ₆₀₀ at the start of culture was 0.02, and cultured for 10 days at a culture temperature of 25 ° C. and a stirring speed of 150 rpm. . The cells were collected from the culture solution and subjected to lipid extraction.

  Samples prepared by lipid extraction were subjected to LC-MS (LC-APCI MS) to analyze free sterols. The results are shown in FIG. 3A. In the DHCR24 gene-deficient strain, cholesterol was lost. On the other hand, in the DHCR24 gene-deficient strain, plant sterols (7-dehydropolyferasterol) accumulated to the same extent as in the wild strain, and fungal sterols (ergosterol) accumulated in large amounts as compared to the wild strain.

In addition, samples prepared by lipid extraction are subjected to LC-MS (LC-ESI MS), and analysis of sterol derivatives sterol ester (SE), steryl glucoside (SG), and acyl steryl glucoside (ASG) went. Result (for SE and ASG, fatty acid (acyl group) is
DHA (22: 6) is shown in FIG. 3B. In the DHCR24 gene-deficient strain, cholesteryl ester (CE), cholesteryl glucoside (CG) and acyl cholesteryl glucoside (ACG), which are derivatives of cholesterol, disappeared. On the other hand, in the DHCR24 gene-deficient strain, 7-dehydropolyferasterol ester, 7-dehydropolyferasteryl glucoside, and acyl-7-dehydropolyferasteryl glucoside, which are derivatives of plant sterol (7-dehydropolyferasterol) And ergosterol esters, which are derivatives of fungal sterols (ergosterol), were all accumulated in large amounts as compared to the wild strain.

  From the above, it has become clear that the ability to produce plant sterols or fungal sterols (including derivatives) of Labyrinth microbes is improved by the reduced activity of DHCR24.

<2-2> Sterol analysis of SMT1 gene deficient strain
The SMT1 gene-deficient strain and the wild strain were precultured in 3 mL of GY medium. The obtained preculture liquid was inoculated in 50 mL of GY medium in a 200 mL flask so that the OD ₆₀₀ at the start of culture was 0.02, and cultured for 10 days at a culture temperature of 25 ° C. and a stirring speed of 150 rpm. . The cells were collected from the culture solution and subjected to lipid extraction.

  Samples prepared by lipid extraction were subjected to LC-MS (LC-APCI MS) to analyze free sterols. The results are shown in FIG. 4A. In the SMT1 gene-deficient strain, cholesterol was accumulated in large amounts as compared to the wild-type strain, but plant sterols (7-dehydropolyferasterol) and fungal sterols (ergosterol) disappeared.

  In addition, samples prepared by lipid extraction are subjected to LC-MS (LC-ESI MS), and analysis of sterol derivatives sterol ester (SE), steryl glucoside (SG), and acyl steryl glucoside (ASG) went. The results (for SE and ASG the fatty acid (acyl group) is DHA (22: 6)) are shown in FIG. 4B. In the SMT1 gene-deficient strain, cholesteryl ester (CE), cholesteryl glucoside (CG) and acyl cholesteryl glucoside (ACG), which are derivatives of cholesterol, accumulated in large amounts as compared to the wild-type strain. On the other hand, in the SMT1 gene-deficient strain, 7-dehydropolyferasterol ester which is a derivative of plant sterol (7-dehydropolyferasterol), 7-dehydropolyferasteryl glucoside, and acyl-7-dehydropolyferasteryl glucoside And ergosterol esters, which are derivatives of fungal sterols (ergosterol), have all disappeared.

  From the above, it has become clear that the ability to produce cholesterol (including derivatives) of the Labyrinthula microorganism is improved by the decrease in the activity of SMT1.

<3> Materials and Methods Other materials and methods used in the examples are as follows.

<Composition of GY medium>
(District A)
Glucose 30 g / L
Yeast extract 10 g / L
SEA LIFE 17.5 g / L
(District B)
Vitamin B1 2 mg / L
Vitamin B2 0.01 mg / L
Vitamin B12 0.01 mg / L
(District C)
EDTA 60 mg / L
FeCl ₃ · 6 H ₂ O 2.9 mg / L
H ₃ BO ₃ 68.4 mg / L
MnCl ₂ · 4 H ₂ O 8.6 mg / L
ZnSO ₄ · 7H ₂ O 2.6 mg / L
CoCl ₂ · 6H ₂ O 0.26 mg / L
NiSO ₄ · 6 H ₂ O 0.52 mg / L
CuSO ₄ · 5 H ₂ O 0.02 mg / L
Na ₂ MoO ₄ · 2H ₂ O 0.05 mg / L
The A section was autoclaved at 120 ° C. for 10 minutes. The B and C sections were subjected to filter filtration. After cooling the area A to room temperature, the areas A, B and C were mixed.

<Lipid extraction>
The recovered cells were suspended in 500 μl of ultrapure water, 200 μl of glass beads (0.5 mm in diameter) were added, and the cells were disrupted for 3 minutes with BEADS CRUSHER μT-12 (TAITEC). The mixture was centrifuged at 15,000 × g for 3 minutes at 4 ° C., and the upper layer was collected to obtain a cell disruption solution. A 200 μl aliquot of the cell lysate was added to 800 μl chloroform / methanol (C / M) = 2/1. Incubate at 37 ° C. for 90 minutes to extract lipids. The mixture was centrifuged (20 ° C., 15,000 rpm, 1 min), 200 μl was taken from the lower layer, and dried in nitrogen. The lipid weight was measured and adjusted to 2 mg / ml with IPA (isopropyl alcohol). After sonication, the supernatant was centrifuged (20 ° C., 15,000 rpm, 5 min), and the supernatant was transferred to a vial, which was used as a sample.

<Lipid analysis by high performance liquid chromatograph mass spectrometer (LC-ESI MS)>
Samples prepared by lipid extraction were subjected to LC-MS. Measurement is Multiple Reaction Monitoring (MRM) by combination of various Q1 / Q3 which detects phospholipid with different fatty acid composition, neutral lipid, sterol ester (SE), steryl glucoside (SG), and acyl steryl glucoside (ASG) Went by. In LC-MS, a triple quadrupole 3200 QTRAP (SCIEX) was used in a positive ion mode for a mass spectrometer, and InertSustain C18 (2.1 × 150 mm, 5 μm, GL Sciences) was used for a column. The solvent used is solvent A: methanol / water = 19/19/2 (v / v / v) (0.1% Formic acid, 0.025% Ammonia) and solvent B: isopropyl alcohol (0.1% Formic acid, 0.025% Ammonia) It was.

Sterol analysis by high performance liquid chromatograph mass spectrometer (LC-APCI MS)>
Samples prepared by lipid extraction were subjected to LC-MS. The measurement was performed by Multiple Reaction Monitoring (MRM) with various Q1 / Q3 combinations that detect sterol with different structures. In LC-MS, a triple quadrupole 3200 QTRAP (AB SCIEX) was used in a positive ion mode for a mass spectrometer, and InertSustain C18 (2.1 × 150 mm, 5 μm, GL Sciences) was used for a column. As the solvent, solvent A: acetonitrile / methanol / water = 96/4 (v / v) (0.1% Acetic acid) and solvent B: methanol (0.1% Acetic acid) were used.

<Description of Sequence Listing>
SEQ ID NO: 1 to 16: Primer SEQ ID NO: 17: Nucleotide sequence of DHCR24 gene of Aurantiochytrium limacinum mh0186 SEQ ID NO: 18: Amino acid sequence of DHCR24 of Aurantio chytrium limacinum mh0186 SEQ ID NO: 19 Amino acid sequence of SMT1 of limacinum mh0186 SEQ ID NO: 21: Nucleotide sequence of 18S rDNA region of Aurantiochytrium sp. 1 AJ7869 strain

Claims (12)

A method of producing sterols,
Cultivating a labyrinthula microorganism having sterol-producing ability in a culture medium,
Recovering sterol from cells produced by the culture;
Including
Said microorganism has been modified such that the activity of 24-dehydrocholesterol reductase is reduced compared to the unmodified strain;
The sterol is at least one sterol selected from plant sterol and fungal sterol,
However, the method except that the microorganism is Aurantiochytrium sp. The method according to claim 1, wherein the 24-dehydrocholesterol reductase is a protein according to (a), (b) or (c) below:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 18;
(B) A protein comprising an amino acid sequence comprising substitution, deletion, insertion and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 18 and having 24-dehydrocholesterol reductase activity ;
(C) A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 18 and having 24-dehydrocholesterol reductase activity.   The method according to claim 1 or 2, wherein the activity of 24-dehydrocholesterol reductase is reduced by reducing the expression of the gene encoding 24-dehydrocholesterol reductase or by disrupting the gene.   The method according to any one of claims 1 to 3, wherein the deletion of part or all of the amino acid sequence of 24-dehydrocholesterol reductase reduces the activity of 24-dehydrocholesterol reductase.   The sterol is at least one sterol selected from 7-dehydropolyferasterol, stigmasterol, 4-methyl-7, 22-stigmastadienol, ergosterol, and 7-dihydroergosterol. The method according to any one of ~ 4. A method of producing sterols,
Cultivating a labyrinthula microorganism having sterol-producing ability in a culture medium,
Recovering sterol from cells produced by the culture;
Including
Said microorganism has been modified to reduce the activity of sterol 24-C-methyltransferase compared to the unmodified strain;
The sterol is cholesterol,
However, the method except that the microorganism is Aurantiochytrium sp. The method according to claim 6, wherein the sterol 24-C-methyltransferase is a protein according to (a), (b) or (c) below:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 20;
(B) amino acid sequence containing a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 20, and sterol 24-C—
A protein having methyltransferase activity;
(C) A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 20 and having sterol 24-C-methyltransferase activity.   The method according to claim 6 or 7, wherein the activity of sterol 24-C-methyltransferase is reduced by reducing the expression of the gene encoding sterol 24-C-methyltransferase or by disrupting the gene. .   The method according to any one of claims 6 to 8, wherein the deletion of a part or all of the amino acid sequence of sterol 24-C-methyltransferase reduces the activity of sterol 24-C-methyltransferase.   10. The method according to any one of claims 1 to 9, wherein the sterol is free sterol, sterol ester, steryl glucoside, acyl steryl glucoside, or a mixture thereof.   The microorganism according to any one of claims 1 to 10, wherein the microorganism belongs to the genus Aurantiochytrium, Schizochytrium, Thraustochytrium, or Parietichytrium. Method described in Section.   The method according to any one of claims 1 to 11, wherein the microorganism is Aurantiochytrium limacinum.