JP2018023356A - Method for producing useful substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing useful substance that allows mass production and gives useful substance with high activity.SOLUTION: A method for producing useful substance is a method for making microorganisms secrete and produce useful substance in a culture medium, the culture medium containing a phosphate compound (A) represented by formula (1), where Ris H or a C1-50 monovalent hydrocarbon group, where at any position of the hydrocarbon group, there may be a substituent selected from specific substituents).SELECTED DRAWING: None

Description

  The present invention relates to a method for producing a useful substance and a compound used in this production method.

Bacteria are widely used to produce useful substances such as amino acids and proteins. Gram-negative bacteria (such as Escherichia coli) are frequently used as bacteria for the production of useful substances. With the advancement of genetic engineering technology, genes of proteins that are useful in medicine and industry are introduced into Escherichia coli to efficiently use useful proteins. The manufacturing technology is known.
When proteins are expressed using bacteria, physical crushing methods such as ultrasonic waves, high-pressure homogenizers, and French presses are used to extract target proteins. However, these physical disruption methods have a problem that purity is lowered because a large amount of substances other than the target protein existing in bacterial cells are mixed when the protein is extracted.

As a protein production method that solves this problem, a protein secretion production method using a surfactant is known. In the production of glutamic acid, polyoxyethylene sorbitan monopalmitate, which is a kind of surfactant, is used (Patent Document 1). In addition, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monooleate, and the like are used in the production of proteins such as cellulase and phosphatase (Non-patent Document 1 and Patent Document 2).
When secretory production is performed, high productivity can be maintained, and a longer production time leads to improved productivity. However, the production volume is not sufficient, and there is a problem that mass production is not possible. Moreover, there exists a subject that activity of useful substances, such as obtained protein, is low.

International Publication No. 99/07853 Pamphlet International Publication No. 2010/137624 Pamphlet

Bioindustry Association Fermentation and Metabolism Study Group, “Fermentation Handbook”, Kyoritsu Shuppan, July 2001, page 253

  The problem to be solved by the present invention is to provide a method for producing a useful substance that can be mass-produced and obtain a useful substance with high activity.

［一般式（１）中、Ｒ¹は水素原子又は炭素数１〜５０の一価の炭化水素基であって、炭化水素基のいずれかの位置に、水酸基、エーテル基、アミド基、アミノ基、アクリロイル基、メタクリロイル基、カルボニル基、カルボキシル基及びエステル基からなる群より選ばれる少なくとも１種の置換基を有していてもよい炭化水素基を表し、Ｒ²は炭素数１〜５０の一価の炭化水素基であって、炭化水素基のいずれかの位置に、水酸基、エーテル基、アミド基、アミノ基、アンモニウム基、アクリロイル基、メタクリロイル基、カルボニル基、カルボキシル基及びエステル基からなる群より選ばれる少なくとも１種の置換基を有していてもよい炭化水素基を表し、ＸはＯ^-又はヒドロキシル基を表し、Ｒ²において、炭化水素基のいずれかの位置にアンモニウム基を有している場合はＯ^-を表す。］ As a result of intensive studies to solve the above problems, the present inventors have reached the present invention.
That is, the present invention is a method of secreting and producing a useful substance into a culture solution by a microorganism contained in the culture solution, and the phosphate compound (A) represented by the following general formula (1) is added to the culture solution. This is a method for producing useful substances.

[In General Formula (1), R ¹ is a hydrogen atom or a monovalent hydrocarbon group having 1 to 50 carbon atoms, and a hydroxyl group, an ether group, an amide group, an amino group at any position of the hydrocarbon group Represents an optionally substituted hydrocarbon group selected from the group consisting of an acryloyl group, a methacryloyl group, a carbonyl group, a carboxyl group and an ester group, and R ² is a group having 1 to 50 carbon atoms. A group consisting of a hydroxyl group, an ether group, an amide group, an amino group, an ammonium group, an acryloyl group, a methacryloyl group, a carbonyl group, a carboxyl group and an ester group at any position of the hydrocarbon group represents at least one substituent hydrocarbon group which may have a are more selected, X is O ^- or a hydroxyl group, in R ^2, any position of the hydrocarbon group If having an ammonium group O ^- represents a. ]

  By using the useful substance production method of the present invention, a highly active useful substance can be mass-produced.

  The present invention is a method for producing a useful substance using a microorganism contained in a culture solution, which is a method for producing a useful substance containing a phosphate ester compound (A) represented by the following general formula (1) in the culture solution. It is a production method.

［一般式（１）中、Ｒ¹は水素原子又は炭素数１〜５０の一価の炭化水素基であって、炭化水素基のいずれかの位置に、水酸基、エーテル基、アミド基、アミノ基、アクリロイル基、メタクリロイル基、カルボニル基、カルボキシル基及びエステル基からなる群より選ばれる少なくとも１種の置換基を有していてもよい炭化水素基を表し、Ｒ²は炭素数１〜５０の一価の炭化水素基であって、炭化水素基のいずれかの位置に、水酸基、エーテル基、アミド基、アミノ基、アンモニウム基、アクリロイル基、メタクリロイル基、カルボニル基、カルボキシル基及びエステル基からなる群より選ばれる少なくとも１種の置換基を有していてもよい炭化水素基を表し、ＸはＯ^-又はヒドロキシル基を表し、Ｒ²において、炭化水素基のいずれかの位置にアンモニウム基を有している場合はＯ^-を表す。］

[In General Formula (1), R ¹ is a hydrogen atom or a monovalent hydrocarbon group having 1 to 50 carbon atoms, and a hydroxyl group, an ether group, an amide group, an amino group at any position of the hydrocarbon group Represents an optionally substituted hydrocarbon group selected from the group consisting of an acryloyl group, a methacryloyl group, a carbonyl group, a carboxyl group and an ester group, and R ² is a group having 1 to 50 carbon atoms. A group consisting of a hydroxyl group, an ether group, an amide group, an amino group, an ammonium group, an acryloyl group, a methacryloyl group, a carbonyl group, a carboxyl group and an ester group at any position of the hydrocarbon group represents at least one substituent hydrocarbon group which may have a are more selected, X is O ^- or a hydroxyl group, in R ^2, any position of the hydrocarbon group If having an ammonium group O ^- represents a. ]

  In the present invention, microorganisms include gram-negative bacteria and gram-positive bacteria.

Examples of gram-negative bacteria in the present invention are shown below, but are not limited thereto. Gram-negative bacteria include Escherichia, Thermus, Rhizobium, Pseudomonas, Shewanella, Vibromo, Vibromo, Vibromo, Vibromo Examples include Salmonella, Acetobacter genus, Synechocystis genus, and the like.
Of these, Escherichia is preferred from the viewpoint of productivity of useful substances, and Escherichia coli is more preferred.

Examples of Gram-positive bacteria include the genus Bacillus (genus Bacillus), the genus Saccharomyces (genus Saccharomyces), the genus Candida (genus Candida), the genus Corynebacterium (genus Corynebacterium), the genus Brevibacillus, and the genus Bifidobacterium ( Bifidobacterium genus), Lactococcus genus (Lactococcus genus), Enterococcus genus (Enterococcus genus), Pediococcus genus (Pediococcus genus), Leuconostoc genus (Leuconostoc genus), Streptomyces genus (Streptomyces genus etc.
Of these, Saccharomyces is preferable from the viewpoint of productivity of useful substances.
Examples of the genus Saccharomyces include Saccharomyces cerevisiae, Saccharomyces pastorianus;
The gram-positive bacterium is preferably Saccharomyces genus, more preferably Saccharomyces cerevisiae, from the viewpoint of productivity of useful substances.
In the present invention, “productivity of useful substances” means that useful substances can be obtained in large quantities.

  As the microorganism, from the viewpoint of productivity of useful substances, Gram-negative bacteria are preferable, Escherichia bacteria are more preferable, and Escherichia coli is particularly preferable.

  Useful substances produced in the present invention include proteins, oligosaccharides and nucleic acids.

Proteins include enzymes (oxidoreductases (cholesterol oxidase, glucose oxidase, ascorbate oxidase, peroxidase, etc.), hydrolases (lysozyme, protease, serine protease, amylase, lipase, cellulase, glucoamylase, etc.), isomerase ( Glucose isomerase, etc.), transferases (acyltransferase, sulfotransferase, etc.), synthetic enzymes (fatty acid synthase, phosphate synthase, citrate synthase, etc.) and elimination enzymes (pectin lyase, etc.)}, hormone proteins {bone morphogenetic factor ( BMP), interferon α, interferon β, interleukin 1-12, growth hormone, erythropoietin, insulin, granular colony stimulating factor (G-CSF) Tissue plasminogen activator (TPA), natriuretic peptide, blood coagulation factor VIII, somatomedin, glucagon, growth hormone releasing factor, serum albumin and calcitonin}, antibody {single chain antibody, IgG large subunit, IgG small subunit, etc.}, antigen protein {hepatitis B surface antigen, etc.}, functional protein {pronectin (registered trademark), antifreeze peptide, antibacterial peptide, etc.}, fluorescent protein (GFP, etc.), photoprotein (lucerase, etc.) And peptides (in particular, the amino acid composition is not limited but oligopeptides, dipeptides, tripeptides, etc.).
Of these proteins, enzymes and hormone proteins are preferred from the viewpoint of ease of protein production.

  Examples of the oligosaccharide include sucrose, lactose, maltose, trehalose, panose, raffinose, cyclodextrin, galactooligosaccharide and fructooligosaccharide.

  Examples of nucleic acids include inosine monophosphate, adenosine monophosphate, guanosine monophosphate, and the like.

  The useful substance is preferably a protein, more preferably an enzyme and a hormone protein, from the viewpoint of easy preparation of a useful substance for microorganisms.

When the useful substance is a protein and the microorganism is a gram-negative bacterium, it is preferable that part or all of the protein has a property of transferring to the periplasm after being expressed in the gram-negative bacterium. More preferably, it is a protein encoding a signal sequence necessary for the transition to the periplasm in the Open Reading Frame (ORF).
The periplasm is the space from the cytoplasmic membrane of Gram-negative bacteria to the outermost surface of Gram-negative bacteria.
Examples of signal sequences necessary for transition to the periplasm include Sec secretion signal sequences, TAT secretion signals, α-factor-derived secretion signals, and the like.

一般式（１）中、Ｒ¹は水素原子又は炭素数１〜５０の一価の炭化水素基であって、炭化水素基のいずれかの位置に、水酸基、エーテル基、アミド基、アミノ基、アクリロイル基、メタクリロイル基、カルボニル基、カルボキシル基及びエステル基からなる群より選ばれる少なくとも１種の置換基を有していてもよい炭化水素基を表し、Ｒ²は炭素数１〜５０の一価の炭化水素基であって、炭化水素基のいずれかの位置に、水酸基、エーテル基、アミド基、アミノ基、アンモニウム基、アクリロイル基、メタクリロイル基、カルボニル基、カルボキシル基及びエステル基からなる群より選ばれる少なくとも１種の置換基を有していてもよい炭化水素基を表し、ＸはＯ^-又はヒドロキシル基を表し、Ｒ²において、炭化水素基のいずれかの位置にアンモニウム基を有している場合はＯ^-を表す。なお、炭化水素基の炭素数について、炭化水素基のいずれかの位置にアミド基、アクリロイル基、メタクリロイル基、カルボニル基、カルボキシル基及びエステル基のように炭素原子を有する置換基を有する場合は、置換基中の炭素についても炭化水素基の炭素として炭化水素基の炭素数とする。以下において同じである。 The production method of the present invention includes a phosphate ester compound (A) represented by the following general formula (1) in a culture solution.

In general formula (1), R ¹ is a hydrogen atom or a monovalent hydrocarbon group having 1 to 50 carbon atoms, and at any position of the hydrocarbon group, a hydroxyl group, an ether group, an amide group, an amino group, Represents a hydrocarbon group optionally having at least one substituent selected from the group consisting of an acryloyl group, a methacryloyl group, a carbonyl group, a carboxyl group and an ester group, and R ² is a monovalent group having 1 to 50 carbon atoms. From any group consisting of a hydroxyl group, an ether group, an amide group, an amino group, an ammonium group, an acryloyl group, a methacryloyl group, a carbonyl group, a carboxyl group, and an ester group at any position of the hydrocarbon group. represents at least one substituent which may have a hydrocarbon group selected, X is O ^- represents or hydroxyl group, in R ^2, in any position of the hydrocarbon group If a ammonium group O ^- represents a. As for the carbon number of the hydrocarbon group, when it has a substituent having a carbon atom such as an amide group, acryloyl group, methacryloyl group, carbonyl group, carboxyl group and ester group at any position of the hydrocarbon group, The carbon in the substituent is also the carbon number of the hydrocarbon group as the carbon of the hydrocarbon group. The same applies to the following.

In R ¹ in the general formula (1), the monovalent hydrocarbon group having 1 to 50 carbon atoms may be a methyl group or a saturated or unsaturated chain or branched hydrocarbon group having 2 to 50 carbon atoms (ethyl Group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, lauryl group, stearyl group, linole group and oleyl group), alicyclic carbonization having 3 to 50 carbon atoms A hydrogen group (such as a cyclopentyl group and a cyclohexyl group), an aromatic hydrocarbon group having 6 to 50 carbon atoms (such as a phenyl group, a methylphenyl group, a biphenyl group and a naphthyl group) and an araliphatic group having 7 to 50 carbon atoms (benzyl) Group) and the like.
The hydrocarbon group is preferably a saturated or unsaturated chain or branched hydrocarbon group from the viewpoint of the stability of (A) when dissolved in water and the stability of useful substances.
As the R ^1, from the viewpoint of the stability of solubility and useful substances in water (A), preferably from 1 to 45 carbon atoms, more preferably from 2 to 40.
In the present invention, the stability of the useful substance means that the modification and / or aggregation of the useful substance is suppressed.

R ¹ is at least one selected from the group consisting of a hydroxyl group, an ether group, an amide group, an amino group, an acryloyl group, a methacryloyl group, a carbonyl group, a carboxyl group, and an ester group at any position of the hydrocarbon group. It may have a substituent.
Examples of the hydrocarbon group having a substituent include those having a hydroxyl group (hydroxyalkyl group, phenol group, etc.), those having an ether group (hydroxyl groups of those having a hydroxyl group). Those having an alkylene oxide having 2 to 4 carbon atoms), those having an amino group (aminoalkyl group and aromatic amine, etc.), those having an amide group (having an amino group) Amino acids having an amide bond with an amino group having 1 to 49 carbon atoms), those having an acryloyl group (such as those having a hydroxyl group esterified with acrylic acid), having a methacryloyl group (E.g., those having a hydroxyl group esterified with methacrylic acid), those having a carboxyl group (e.g., carboxyalkyl group and aromatic carboxylic acid), Those having an ester group (those aliphatic alcohol 1-49 carbon atoms in the carboxyl group of those having a carboxyl group is esterified, etc.) and the like.

In R ¹ , the hydrocarbon group has at least one substituent selected from the group consisting of a hydroxyl group, an ether group, an amide group, an amino group, an acryloyl group, a methacryloyl group, a carbonyl group, a carboxyl group, and an ester group. However, from the viewpoint of the stability of (A) when dissolved in water and the stability of useful substances, it comprises a hydroxyl group, an ether group, an amide group, an amino group, an acryloyl group, a methacryloyl group, a carbonyl group, and an ester group. At least one selected from the group is preferred, more preferably at least one selected from the group consisting of a hydroxyl group, an ether group, an amide group, an amino group and an ester group, and most preferably an ester group.

In R ² in the general formula (1), the monovalent hydrocarbon group having 1 to 50 carbon atoms is the same as R ¹ described above.

In R ² in the general formula (1), the hydrocarbon group includes a saturated or unsaturated chain or branched fat from the viewpoint of the stability of (A) when dissolved in water and the stability of useful substances. An aromatic hydrocarbon group is preferable, and a saturated or unsaturated linear aliphatic hydrocarbon group is more preferable.
R ² is preferably 1 to 28 carbon atoms, more preferably 1 to 20 carbon atoms, and particularly preferably 1 to 15 carbon atoms from the viewpoint of the solubility of (A) in water and the stability of useful substances. Most preferably, it is 1-5.

R ² is selected from the group consisting of a hydroxyl group, an ether group, an amide group, an amino group, an ammonium group, an acryloyl group, a methacryloyl group, a carbonyl group, a carboxyl group, and an ester group at any position of the hydrocarbon group. It may have at least one substituent.
Examples of the hydrocarbon group having a substituent include those having a hydroxyl group (hydroxyalkyl group, phenol group, etc.), those having an ether group (hydroxyl groups of those having a hydroxyl group). Those having an alkylene oxide having 2 to 4 carbon atoms), those having an amino group (aminoalkyl group and aromatic amine, etc.), those having an ammonium group (having an amino group) In which the amino group is alkylated to form quaternary ammonium, etc.), in which an amide group is present (in which the amino group has an amino group, a fatty acid having 1 to 49 carbon atoms is amide-bonded, etc.) ), Having an acryloyl group (such as those having a hydroxyl group esterified with acrylic acid), having a methacryloyl group (having a hydroxyl group) Those having a carboxyl group (such as carboxyalkyl groups and aromatic carboxylic acids), those having an ester group (such as those having a carboxyl group) And the like, which are esterified from an aliphatic alcohol having 1 to 49 carbon atoms).

R ² is selected from the group consisting of a hydroxyl group, an ether group, an amide group, an amino group, an ammonium group, an acryloyl group, a methacryloyl group, a carbonyl group, a carboxyl group, and an ester group at any position of the hydrocarbon group. Although it may have at least one type of substituent, from the viewpoint of the stability of (A) when dissolved in water and the stability of useful substances, a hydroxyl group, an ether group, an amide group, an amino group, an acryloyl group And preferably at least one substituent selected from the group consisting of a methacryloyl group and an ammonium group, more preferably at least one substituent selected from the group consisting of a hydroxyl group, an ether group, an amide group, an amino group and an ammonium group. Particularly preferably at least one selected from the group consisting of a hydroxyl group, an amino group and an ammonium group, Preferably an ammonium group.

  Specific examples of the compound represented by the general formula (1) include methacryloyloxyethyl phosphorylethanolamine, 1,2-dilaurolylglycero-3-phosphoethanolamine, and 1,2-diercoylglycero-3-phosphoethanol. Amine, phosphatidylcholine, dibutyrylphosphatidylcholine, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, monoministoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, distearoylphosphatidylcholine, 1-palmitoylglycero-3-phosphocholine, dipalmitoylphosphatidylcholine Proylphosphatidylcholine, 1-palmitoyl-2-arachidonylphosphatidylcholine, dioctanoyl-L-α-glyce Phosphorylcholine, didecanoylphosphatidylcholine, 1-stearoyl-2-arachidonoylphosphatidylcholine, 1,2-diachiladylphosphatidylcholine, 1-palmitoyl-2- (9-hydroxy-9-hydroperoxynonanoyl) -sn-glycero-3 -Phosphatidylcholine, diphytanoylphosphatidylcholine, 1-palmitoyl-2- (5-hydroxy-8-oxo-6-octenoyl) -sn-glycero-3-phosphatidylcholine, glycerophosphocholine, decylphosphorylcholine, hexadecylphosphorylcholine, lysophosphatidylcholine, Sphingomyelin, 2-methacryloyloxyethyl phosphorylcholine, phosphorylethanolamine, phosphoethanolamine-N-methoxy (polyethylene Lenglycol) 350] 1,2-dioleoyl-glycero-3-ammonium salt, phosphoethanolamine-N- [methoxy (polyethylene glycol) 1000] 1,2-dioleoyl-glycero-3-ammonium salt, dioleoylphosphatidic acid , Phosphorylglycerol, phosphatidylglycerol ethylene oxide 14 mol adduct, phosphocholine and the like.

一般式（２）〜（５）中、Ｒ³及びＲ⁷はそれぞれ炭素数１〜５０の一価の炭化水素基であって、炭化水素基のいずれかの位置に、水酸基、エーテル基、アミド基、アミノ基、アクリロイル基、メタクリロイル基、カルボニル基、カルボキシル基及びエステル基からなる群より選ばれる少なくとも１種の置換基を有していてもよい炭化水素基を表し、Ｒ⁴、Ｒ⁸、Ｒ¹²及びＲ¹⁵はそれぞれ炭素数１〜１２の二価の炭化水素基であって、炭化水素基のいずれかの位置に、水酸基、エーテル基、アミド基、アミノ基、アクリロイル基、メタクリロイル基、カルボニル基、カルボキシル基及びエステル基からなる群より選ばれる少なくとも１種の置換基を有していてもよい炭化水素基を表し、Ｒ⁵、Ｒ⁶、Ｒ⁹、Ｒ¹⁰、Ｒ¹¹、Ｒ¹³、Ｒ¹⁴、Ｒ¹⁶、Ｒ¹⁷及びＲ¹⁸はそれぞれ水素原子又は炭素数１〜４９の脂肪族炭化水素基であって、炭化水素基のいずれかの位置に水酸基及び／又はエーテル基を有していてもよい炭化水素基を表す。 In the present invention, from the viewpoint of productivity of useful substances and stability of useful substances, the phosphate ester compound (A) is a phosphate ester compound represented by the following general formulas (2) to (5). preferable.

In the general formulas (2) to (5), R ³ and R ⁷ are each a monovalent hydrocarbon group having 1 to 50 carbon atoms, and at any position of the hydrocarbon group, a hydroxyl group, an ether group, an amide Represents a hydrocarbon group which may have at least one substituent selected from the group consisting of a group, an amino group, an acryloyl group, a methacryloyl group, a carbonyl group, a carboxyl group and an ester group, R ⁴ , R ⁸ , R ¹² and R ¹⁵ are each a divalent hydrocarbon group having 1 to 12 carbon atoms, and at any position of the hydrocarbon group, a hydroxyl group, an ether group, an amide group, an amino group, an acryloyl group, a methacryloyl group, It represents a hydrocarbon group which may have at least one substituent selected from the group consisting of a carbonyl group, a carboxyl group and an ester group, and R ⁵ , R ⁶ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹³ , R ^14, R ^{16, 17} and R ¹⁸ are each an aliphatic hydrocarbon group of a hydrogen atom or a C 1 to 49, any of which may have a hydroxyl group and / or ether group in position hydrocarbon group hydrocarbon group Represent.

In general formulas (2) to (5), in R ³ and R ⁷ , preferred hydrocarbon groups are the same as those for R ¹ .

In the general formulas (2) to (5), in R ⁴ , R ⁸ , R ¹² and R ¹⁵ , the divalent hydrocarbon group having 1 to 12 carbon atoms is a methylene group, saturated or having 2 to 12 carbon atoms, Unsaturated chain or branched aliphatic hydrocarbon group (ethylene group, propylene group, butylene group, pentylene group, hexylene group, heptylene group, octylene group, nonylene group, decylene group, etc.), C3-C12 fat A cyclic hydrocarbon group (such as a cyclopentylene group and a cyclohexylene group), an aromatic hydrocarbon group having 6 to 12 carbon atoms (such as a phenyl group, a methylphenyl group, a biphenyl group, and a naphthyl group) and a 7 to 12 carbon atoms. An araliphatic group (benzyl group etc.) etc. are mentioned.

In R ⁴ , R ⁸ , R ¹² and R ¹⁵ , the divalent hydrocarbon group having 1 to 12 carbon atoms is a saturated or unsaturated chain from the viewpoint of the productivity of useful substances and the stability of useful substances. Or a branched aliphatic hydrocarbon group is preferable, a saturated or unsaturated chain aliphatic hydrocarbon group is more preferable, and an alkylene group is particularly preferable.
Further, in R ⁴ , R ⁸ , R ¹² and R ¹⁵ , the number of carbon atoms is preferably 1 to 10 and more preferably 1 to 5 from the viewpoint of the productivity of useful substances and the stability of useful substances.

R ⁴ , R ⁸ , R ¹² and R ¹⁵ are each composed of a hydroxyl group, an ether group, an amide group, an amino group, an acryloyl group, a methacryloyl group, a carbonyl group, a carboxyl group and an ester group at any position of the hydrocarbon group. It may have at least one substituent selected from the group.
Examples of the hydrocarbon group having a substituent include those having a hydroxyl group (such as a hydroxyalkylene group) and those having an ether group (having a hydroxyl group but having 2 to 4 carbon atoms in the hydroxyl group). Having an amino group (such as an aminoalkylene group), having an amide group (having an amino group, the amino group has 1 to 11 carbon atoms) Fatty acids of which amide bonds are present), those having an acryloyl group (such as those having a hydroxyl group esterified with acrylic acid), those having a methacryloyl group (having a hydroxyl group) Those having esterified methacrylic acid), those having a carboxyl group (carboxyalkylene group, etc.), those having an ester group (having a carboxyl group) Aliphatic alcohol 1-11 carbon atoms in the carboxyl group of the can be mentioned, etc.) or the like esterified.

R ⁴ , R ⁸ , R ^12, and R ¹⁵ are a hydroxyl group, an ether group, an amide group, an amino group, an acryloyl group, a methacryloyl group, a carbonyl group, a carboxyl group, and an ester group at any position of the hydrocarbon group. May have at least one substituent selected from the group consisting of: from the viewpoint of the stability of (A) when dissolved in water and the stability of useful substances, a hydroxyl group, an ether group, an amide group , At least one substituent selected from the group consisting of an amino group, a carboxyl group and an ester group is preferred, and a hydroxyl group and / or a carboxyl group is more preferred.

R ⁵ , R ⁶ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹³ , R ¹⁴ , R ¹⁶ , R ¹⁷ and R ¹⁸ each represent a hydrogen atom or an aliphatic hydrocarbon group having 1 to 49 carbon atoms.

In R ⁵ , R ⁶ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹³ , R ¹⁴ , R ¹⁶ , R ¹⁷ and R ¹⁸ , the aliphatic hydrocarbon group having 1 to 49 carbon atoms includes a methyl group and a carbon number 2 to 49 saturated or unsaturated chain or branched hydrocarbon groups (ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, lauryl group, etc.), etc. Is mentioned.
In R ⁵ , R ⁶ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹³ , R ¹⁴ , R ¹⁶ , R ¹⁷ and R ¹⁸ , from the viewpoints of productivity of useful substances and stability of useful substances, It is preferably a chain hydrocarbon group having 1 to 4 carbon atoms, more preferably a hydrogen atom or a methyl group.

In the general formula (2), the total number of carbon atoms of R ^4, R ⁵ and R ⁶ are 50, from the viewpoint of stability of the solubility and useful substances in water (A), a carbon number 1 -28 are preferable, More preferably, it is 1-20, Especially preferably, it is 1-15, Most preferably, it is 1-5.
In the general formula (3), the total number of carbon atoms of R ^8, R ^9, R ¹⁰ and R ¹¹ is 1 to 50, in view of the stability of solubility and useful substances in water (A), C1-C28 is preferable, More preferably, it is 1-20, Most preferably, it is 1-15, Most preferably, it is 1-5.
Moreover, in General formula (4), the sum total of carbon number of R < ¹² >, R < ¹³ > and R < ¹⁴ > is 1-50, and from a viewpoint of the solubility to the water of (A) and the stability of a useful substance, carbon number 1 -28 are preferable, More preferably, it is 1-20, Especially preferably, it is 1-15, Most preferably, it is 1-5.
Moreover, in General formula (5), the sum total of carbon number of R <^15> , R <^16> , R < ¹⁷ > and R < ¹⁸ > is 1-50, From a viewpoint of the solubility to the water of (A) and a useful substance, C1-C28 is preferable, More preferably, it is 1-20, Most preferably, it is 1-15, Most preferably, it is 1-5.

Specific examples of the compound represented by the general formula (2) include methacryloyloxyethyl phosphorylethanolamine, 1,2-dilaurolylglycero-3-phosphoethanolamine, and 1,2-dielcoylglycero-3-phosphoethanol. Amine, glycerophosphorylserine, phosphoethanolamine-N- [methoxy (polyethylene glycol) 350] -1,2-dioleyl-glycero-3-ammonium salt, glycerophosphorylserine, phosphoethanolamine-N- [methoxy (polyethylene glycol) 1000] -1,2-dioleoyl-glycero-3-ammonium salt and the like.
Specifically, as represented by the general formula (3), phosphatidylcholine, dibutyrylphosphatidylcholine, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, monoministoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, distearoylphosphatidylcholine, 1-palmitoylglycero-3-phosphocholine, dipalmitoylphosphatidylcholine, dicaproylphosphatidylcholine, 1-palmitoyl-2-arachidonylphosphatidylcholine, dioctanoyl-L-α-glycerophosphorylcholine, didecanoylphosphatidylcholine, 1-stearoyl-2-arachidonoyl Phosphatidylcholine, 1,2-diaarachidonylphosphatidylcholine, 1-palmitoy -2- (9-hydroxy-9-hydroperoxynonanoyl) -sn-glycero-3-phosphatidylcholine, diphytanoylphosphatidylcholine, 1-palmitoyl-2- (5-hydroxy-8-oxo-6-octenoyl) -sn -Glycero-3-phosphatidylcholine, glycerophosphocholine, decylphosphorylcholine, hexadecylphosphorylcholine, lysophosphatidylcholine, sphingomyelin, 2-methacryloyloxyethyl phosphorylcholine and the like.
Specific examples of the compound represented by the general formula (4) include phosphorylethanolamine.
Specific examples of the compound represented by the general formula (5) include phosphocholine.

  Among those represented by the general formulas (2) to (5), the phosphate ester compound (A) is represented by the general formula (3) from the viewpoint of the productivity of useful substances and the stability of useful substances. Those are preferred.

一般式（６）中、Ｒ¹⁹は水素原子又は炭素数１〜５０の一価の炭化水素基であって、炭化水素基としては、いずれかの位置に、水酸基、エーテル基、アミド基、アミノ基、アクリロイル基、メタクリロイル基、カルボニル基、カルボキシル基及びエステル基からなる群より選ばれる少なくとも１種の置換基を有していてもよい炭化水素基を表す。 In the present invention, from the viewpoint of productivity of useful substances and stability of useful substances, the phosphate ester compound (A) is preferably a phosphate ester compound represented by the following general formula (6).

In the general formula (6), R ¹⁹ is a hydrogen atom or a monovalent hydrocarbon group having 1 to 50 carbon atoms, and the hydrocarbon group may be a hydroxyl group, an ether group, an amide group, an amino group at any position. Represents a hydrocarbon group which may have at least one substituent selected from the group consisting of a group, an acryloyl group, a methacryloyl group, a carbonyl group, a carboxyl group and an ester group.

In general formula (6), in R ¹⁹ , preferred hydrocarbon groups are the same as those in R ¹ .

  Specific examples of the compound represented by the general formula (6) include phosphatidylcholine, dibutyrylphosphatidylcholine, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, monoministoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, distearoylphosphatidylcholine. 1-palmitoylglycero-3-phosphocholine, dipalmitoylphosphatidylcholine, dicaproylphosphatidylcholine, 1-palmitoyl-2-arachidonylphosphatidylcholine, dioctanoyl-L-α-glycerophosphorylcholine, didecanoylphosphatidylcholine, 1-stearoyl-2-arachid Noyl phosphatidylcholine, 1,2-dialachidonyl phosphatidylcholine, 1-palmi Ile-2- (9-hydroxy-9-hydroperoxynonanoyl) -sn-glycero-3-phosphatidylcholine, diphytanoylphosphatidylcholine, 1-palmitoyl-2- (5-hydroxy-8-oxo-6-octenoyl)- Examples include sn-glycero-3-phosphatidylcholine, glycerophosphocholine, decylphosphorylcholine, hexadecylphosphorylcholine, lysophosphatidylcholine, sphingomyelin, 2-methacryloyloxyethylphosphorylcholine.

一般式（７）中、Ｒ²⁰及びＲ²¹はそれぞれ水素原子又は炭素数２〜２２の｛Ｒ²²−ＣＯ−｝で表されるアシル基を表し、Ｒ²²はいずれかの位置に、水酸基、エーテル基、アミド基、アミノ基、アクリロイル基、メタクリロイル基、カルボニル基、カルボキシル基及びエステル基からなる群より選ばれる少なくとも１種の置換基を有していてもよい炭化水素基を表す。
Ｒ²²としては、メチル基、炭素数２〜２１の飽和又は不飽和の鎖状又は分岐の炭化水素基（エチル基、プロピル基、ブチル基、ペンチル基、ヘキシル基、ヘプチル基、オクチル基、ノニル基、デシル基及びラウリル基等）等が挙げられる。
Ｒ²²において、有用物質の生産性及び有用物質の安定性の観点から、炭素数５〜２１が好ましく、さらに好ましくは炭素数１０〜２１である。また、有用物質の生産性及び有用物質の安定性の観点から、飽和又は不飽和の鎖状又は分岐の炭化水素基が好ましく、さらに好ましくは飽和又は不飽和の鎖状の炭化水素基である。 In the present invention, from the viewpoint of productivity of useful substances and stability of useful substances, the phosphate ester compound (A) is preferably a phosphate ester compound represented by the following general formula (7).

In General Formula (7), R ²⁰ and R ²¹ each represent a hydrogen atom or an acyl group represented by {R ²² —CO—} having 2 to ²² carbon atoms, and R ²² is a hydroxyl group at any position, It represents a hydrocarbon group which may have at least one substituent selected from the group consisting of an ether group, an amide group, an amino group, an acryloyl group, a methacryloyl group, a carbonyl group, a carboxyl group and an ester group.
R ²² includes a methyl group, a saturated or unsaturated chain or branched hydrocarbon group having 2 to 21 carbon atoms (ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group). Group, decyl group, lauryl group, etc.).
R ²² preferably has 5 to 21 carbon atoms, more preferably 10 to 21 carbon atoms, from the viewpoint of productivity of useful substances and stability of useful substances. Further, from the viewpoint of productivity of useful substances and stability of useful substances, saturated or unsaturated chain or branched hydrocarbon groups are preferable, and saturated or unsaturated chain hydrocarbon groups are more preferable.

As the compound represented by the general formula (7), specifically, phosphatidylcholine, lysophosphatidylcholine, dibutyrylphosphatidylcholine, dimyristoylphosphatidylcholine, monoministoylphosphatidylcholine, dioleoylphosphatidylcholine, 1-palmitoyl-2 -Oleoylphosphatidylcholine, distearoylphosphatidylcholine, 1-palmitoylglycero-3-phosphocholine, dipalmitoylphosphatidylcholine, dicaproylphosphatidylcholine, 1-palmitoyl-2-arachidonylphosphatidylcholine, dioctanoylphosphatidylcholine, didecanoylphosphatidylcholine, 1-stearoyl 2-Arachidonoyl phosphatidylcholine, 1,2-dialachidonyl phospha Zylcholine, 1-palmitoyl-2- (9-hydroxy-9-hydroperoxynonanoyl) -sn-glycero-3-phosphatidylcholine, diphytanoylphosphatidylcholine, 1-palmitoyl-2- (5-hydroxy-8-oxo-6) -Octenoyl) -sn-glycero-3-phosphatidylcholine and the like.
A phosphate ester compound (A) may be used individually by 1 type, or may use 2 or more types together.

  The content of the phosphate ester compound (A) in the culture solution is the weight of the culture solution [medium, E. coli and other additives added during culture [phosphate ester compound (A), amino acid (B), saccharide (C ), Peptide (D), spikrispolic acid (E), surfactant (F), additive used for inductive expression described later (Isopropyl β-D-1-thiogalactopyranoside etc.) and fed-batch culture It means the total weight of the nutrient source. Same in the following. ] From the viewpoint of the productivity of useful substances and the stability of useful substances, the content is preferably 0.0003 to 3% by weight, more preferably 0.0003 to 1% by weight.

  In the method for producing a useful substance of the present invention, at least selected from the group consisting of an amino acid (B), a saccharide (C), a peptide (D), spikrispolic acid (E) and a surfactant (F) in the culture solution. One compound may be included. In the surfactant (F), the phosphate ester compound (A) is not included.

  Examples of the amino acid (B) used in the method for producing a useful substance of the present invention include a protein-constituting amino acid (B-1) and a non-protein-constituting amino acid (B-2).

  Examples of the protein-constituting amino acid (B-1) include α-alanine, arginine, asparagine, glycine, histidine, leucine, lysine, serine, threonine, and valine.

  Non-proteinogenic amino acids (B-2) include β-alanine, canavamin, citrulline, H-homoarginine, isoserine, glycocyanine, 4-amino entangled acid, homoserine, aminoadipic acid, diaminopimelic acid, diaminobutyric acid, diaminopropion Examples include acids and isoserine.

Amino acid (B) may be used individually by 1 type, or may use 2 or more types together.
The amino acid (B) used in the present invention is preferably a protein-constituting amino acid from the viewpoint of secretion efficiency, and more preferably α-alanine, arginine, glycine, lysine and serine.

  Preferred examples of the saccharide (C) used in the method for producing a useful substance of the present invention include disaccharide (C-1), glycolipid (C-2), and cyclodextrin (C-3). .

  Examples of the disaccharide (C-1) include sucrose, lactulose, lactose, maltose, trehalose, cellobiose, isotrehalose, neotrehalose, gentiobiose, mannobiose, galactosucrose, xylobiose and primeverose.

Examples of the glycolipid (C-2) include glyceroglycolipid, sphingoglycolipid, glycolipid biosurfactant and the like.
Examples of the glycolipid biosurfactant include rhamnolipid, sophorolipid, trehalolipid, and cellolipid.

  As cyclodextrin (C-3), α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, derivatives thereof (methylation, ethylation, hydroxyethylation, hydroxypropylation, maltose conjugation) Cationization, quaternary ammonium formation, anionization, amphotericization, etc.).

Sugar (C) may be used individually by 1 type, or may use 2 or more types together.
The saccharide (C) used in the present invention is preferably sucrose, lactose, maltose and glycolipid biosurfactant from the viewpoint of secretion efficiency, and more preferably sucrose, lactose and glycolipid biosurfactant.

  Examples of the peptide (D) used in the method for producing a useful substance of the present invention include an oligopeptide (D-1), a polypeptide (D-2), and an acyl peptide biosurfactant (D-3).

Examples of the oligopeptide (D-1) include oligopeptides composed of protein-constituting amino acids and oligopeptides composed of non-protein-constituting amino acids.
Among oligopeptides (D-1), examples of oligopeptides composed of protein-constituting amino acids include alanyl glycyl glycine, triglycine, tetraglycine, alanyl histidine, glycyl serine, leucyl glycine, and leucyl tyrosine. .

Examples of the polypeptide (D-2) include a polypeptide composed of protein-constituting amino acids and a polypeptide composed of non-protein-constituting amino acids.
Among the polypeptides (D-2), examples of the polypeptide composed of protein-constituting amino acids include glutamic acid lysine copolymer, leucine lysine copolymer, polylysine, polyglutamic acid, polygamma glutamic acid, nisin, microcin, lactoferrin and depsipeptide. It is done.

  The number of amino acids of the oligopeptide (D-1) and polypeptide (D-2) is preferably 3 to 200, more preferably 3 to 100, from the viewpoint of secretion of useful substances.

Surfactin etc. are mentioned as an acyl peptide type | system | group biosurfactant (D-3).
Surfactin can be obtained from the market as Kaneka Surfactin [manufactured by Kaneka Corporation].

The peptide (D) used in the production method of the useful substance of the present invention preferably has a solubility in water of 25 ° C. of 1.0 to 500.0 g / L or less, and has a mass number of less than 5000. It is preferable.
Note that the mass number of the peptide (D) can be determined by the SDS-PAGE method.

A peptide (D) may be used individually by 1 type, or may use 2 or more types together.
The peptide (D) used in the present invention is preferably an oligopeptide composed of protein-constituting amino acids and a polypeptide composed of protein-constituting amino acids from the viewpoint of secretion efficiency.

  The surfactant (F) used in the production method of the useful substance of the present invention includes an alkylene oxide adduct (F-1) of a chain aliphatic alcohol, an alkylene oxide adduct of a chain aliphatic alcohol, a fatty acid, Ester (F-2), fatty acid amide betaine (F-3), alkyl ether carboxylate (F-4), alkyl ether sulfate (F-5) and imidazolinium betaine (F-6) It is.

The chain aliphatic alcohol used for the alkylene oxide adduct (F-1) of a chain aliphatic alcohol is a chain aliphatic alcohol having 1 to 24 carbon atoms, and is a chain aliphatic alcohol having 1 to 24 carbon atoms. Is a C1-C24 chain aliphatic monohydric alcohol [methanol, ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, 1-heptanol, 1-octanol, 1-nonanol, 1 -Decanol];
C2-C24 chain aliphatic dihydric alcohol [ethylene glycol, 1,2- or 1,3-propylene glycol, 1,4-butanediol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol, lauryl glycol, tetradecanediol, neopentyl glycol and 2,2-diethyl-1,3-propanediol, etc.];
C3-C24 trihydric alcohol [glycerin, trimethylolpropane, etc.];
C5-C24 chain aliphatic 3-8 valent alcohol [pentaerythritol, sorbitol, mannitol, sorbitan, diglycerin, dipentaerythritol, etc.];
Saccharides [sucrose, glucose, mannose, fructose, methyl glucoside and derivatives thereof]; and the like.

  The number of carbon atoms of the chain aliphatic alcohol is preferably 6-18, more preferably 8-14, from the viewpoint of secretion of useful substances.

  Examples of the alkylene oxide added to the chain aliphatic alcohol include ethylene oxide, 1,2- or 1,3-propylene oxide and 1,2-, 1,3-, 1,4- or 2,3-butylene oxide. Is mentioned.

  The number of moles of alkylene oxide added to the chain aliphatic alcohol is preferably 1-30, more preferably 1-20, from the viewpoint of solubility in the culture medium.

  Specific examples of the alkylene oxide adduct of chain aliphatic alcohol include 1 to 20 mol of ethylene oxide adduct of lauryl alcohol.

For the chain aliphatic alcohol, an alkylene oxide added in a block form and / or a random form can be used.
Among these, what was added to the block shape can be obtained from the market as Pluronic F-127NF (manufactured by BASF).

  In the present invention, the fatty acid used for the alkylene oxide adduct of chain aliphatic alcohol and the ester of fatty acid (F-2) is a fatty acid having 2 to 22 carbon atoms, and the fatty acid having 2 to 22 carbon atoms is butanoic acid. , Pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, lauric acid and oleic acid.

  Specific examples of esters of chain aliphatic alcohol alkylene oxide adducts and fatty acids include lauric acid 1 molar condensates of pluronic F-127NF.

  Examples of the fatty acid amide betaine (F-3) include coconut oil fatty acid amide betaine and the like (coconut oil fatty acid amidopropyldimethylaminoacetic acid betaine and the like) and lauric acid amidopropyl betaine and the like.

As alkyl ether carboxylate (F-4), the salt of ether carboxylic acid which has a hydrocarbon group (C8-C24) is contained.
Specific examples of alkyl ether carboxylates include polyoxyethylene lauryl ether acetic acid, polyoxyethylene lauryl ether acetate sodium salt, polyoxyethylene tridecyl ether acetate sodium salt, polyoxyethylene octyl ether acetate sodium salt and sodium lauryl glycol acetate Salt etc.

The alkyl ether sulfate (F-5) includes a salt of an ether sulfate having a hydrocarbon group (8 to 24 carbon atoms).
Specific examples of the alkyl ether sulfate include polyoxyethylene lauryl ether sulfate sodium salt and polyoxyethylene lauryl ether sulfate triethanolamine salt.

  Examples of imidazolinium betaine (F-6) include 2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazolinium betaine.

In the present invention, as the surfactant (F), these surfactants may be used as they are, or may be used as an aqueous diluent (aqueous solution or aqueous dispersion) by mixing with water if necessary.
The total concentration of these surfactants (F) in the aqueous dilution is appropriately selected depending on the target microorganism, the type of physiologically active substance, and the type of extraction method. From the viewpoint of secretion and handling properties of useful substances From 0.1 to 99% by weight, preferably 1 to 50% by weight, based on the weight of the aqueous diluent.

The amino acid (B), saccharide (C), peptide (D) and spikrispolic acid (E) used in the production method of the useful substance of the present invention may be used as they are, or mixed with water if necessary. Alternatively, it may be used as an aqueous diluent (aqueous solution or aqueous dispersion).
The weight ratio of amino acids (B), saccharides (C), peptides (D) and splicrispolic acid (E) used in the method for producing useful substances in the aqueous dilution is the type of the target microorganism or physiologically active substance. And, depending on the type of extraction method, from the viewpoint of secretion of useful substances and handling properties, 0.1 to 99% by weight is preferable, more preferably 1 to 50% by weight based on the weight of the aqueous diluent. %.

  The culture solution used in the present invention can be obtained by culturing a medium (TB medium or the like) containing a carbon source, a nitrogen source and other essential nutrients that can be assimilated by a host such as a microorganism.

  The content of the surfactant (F) is preferably 0.1 to 10.0% by weight, more preferably 0.3 to 3.3% by weight based on the weight of the culture solution, from the viewpoint of cell growth. 8% by weight.

  Moreover, each weight ratio of the at least 1 sort (s) of compound chosen from the group which consists of an amino acid (B), saccharide | sugar (C), a peptide (D), spikrispolic acid (E), and surfactant (F) is secretion efficiency. In view of the above, it is preferably 0.05 to 5.0% by weight, more preferably 0.1 to 3.0% by weight, based on the weight of the culture solution.

  In the present invention, the culture solution is composed of the above-described phosphate ester compound (A), amino acid (B), saccharide (C), peptide (D), spikrispolic acid (E), and surfactant (F). In addition to at least one compound selected from the group, other surfactants (G) and the like may be included.

  Examples of other surfactants (G) include amphoteric surfactants (G-) except the amino acid (B), saccharide (C), peptide (D), spikrispolic acid (E) and surfactant (F). 1), anionic surfactant (G-2), nonionic surfactant (G-3) and cationic surfactant (G-4).

Examples of the amphoteric surfactant (G-1) include carboxylate-type amphoteric surfactants [betaine-type amphoteric surfactants [alkyldimethylbetaines (such as stearyldimethylaminoacetic acid betaine and lauryldimethylaminoacetic acid betaine)] and lauric acid Amidopropyl betaine), alkyldihydroxyalkyl betaines (such as lauryl dihydroxyethyl betaine) and hydrogenated coconut oil fatty acid amidopropyldimethylaminoacetic acid betaines]]];
Sulfobetaine-type amphoteric surfactants (pentadecyl sulfotaurine, etc.);
And alkylamine oxide type amphoteric surfactants (such as lauryl dimethylamine oxide).

Examples of the anionic surfactant (G-2) include ether carboxylic acids (polyoxyethylene lauryl ether acetic acid, polyoxyethylene tridecyl ether acetic acid, polyoxyethylene octyl ether acetic acid, lauryl glycol acetic acid and the like);
Ether sulfate (polyoxyethylene lauryl ether sulfate, etc.);
Sulfonates (such as sodium dodecyl diphenyl ether disulfonate, sodium dodecylbenzene sulfonate and sodium naphthalene sulfonate);
Sulfosuccinates (such as polyoxyethylene lauryl sulfosuccinic acid disodium salt, sulfosuccinic acid lauryl disodium salt and sulfosuccinic acid polyoxyethylene lauroylethanolamide disodium salt);
Fatty acid salts (such as sodium octylate, sodium laurate and sodium stearate);
Acylated amino acid salts;
And naturally-occurring carboxylic acids and salts thereof (such as chenodeoxycholic acid, cholic acid, and deoxycholic acid).

Examples of the nonionic surfactant (G-3) include an alkylphenol alkylene oxide adduct [1 to 20 mol of ethylene oxide of octylphenol and / or 1 to 20 mol of adduct of propylene oxide and 1 to 20 mol of ethylene oxide of nonylphenol and / Or 1-20 mol adduct of propylene oxide, etc.];
Fatty acid alkylene oxide adduct [oleic acid ethylene oxide 9 mol adduct (HLB = 11.8), dioleic acid ethylene oxide 12 mol adduct (HLB = 10.4), dioleic acid ethylene oxide 20 mol adduct ( HLB = 12.9) and an ethylene oxide 9 mol adduct of stearic acid (HLB = 11.9) etc.] and the like.
In addition, HLB in this invention is a numerical value calculated by the following formula (Takehiko Fujimoto, Introduction to Surfactant, page 142, published by Sanyo Chemical Industries, Ltd.).
HLB = 20 × {molecular weight of hydrophilic group / molecular weight of surfactant}

Examples of the cationic surfactant (G-4) include amine salt type cationic surfactants (stearylamine acetate and the like);
And quaternary ammonium salt type cationic surfactants (such as distearyldimethylammonium chloride and alkylbenzyldimethylammonium chloride).

The useful substance production method of the present invention preferably includes a step (a) of producing a useful substance by secretion and a step (b) of separating the useful substance from the culture solution.
Step (a): A step of causing a useful substance to be secreted extracellularly (in the culture solution) by simultaneously existing a culture solution for culturing a microorganism that produces a useful substance and the phosphate ester compound (A).
Step (b): A step of separating useful substances from the culture solution after the step (a).

  Below, an example [(i)-(iii)] of the method of producing the useful substance (for example, protein) secreted with the method of this invention in microorganisms (for example, E. coli) is shown.

(I) Preparation of expression vector (i-1) Isolation of messenger RNA (mRNA) from cells expressing the target protein, synthesis of single-stranded cDNA and then double-stranded DNA from the mRNA, Double-stranded DNA is incorporated into phage DNA or plasmid. The resulting recombinant phage or plasmid is transformed into host E. coli to prepare a cDNA library.
(I-2) Examples of a method for screening phage DNA or plasmid containing the target DNA include a hybridization method of the phage DNA or plasmid and a DNA probe encoding a part of the target protein gene or complementary sequence. .
(I-3) The target cloned DNA or a part thereof is cut out from the screened phage or plasmid, and the cloned DNA or a part thereof is ligated downstream of the promoter in the expression vector. Expression vectors can be prepared. DNAs encoding a signal sequence for translocating the inner membrane (a signal sequence for expressing the target substance in the periplasm) can be linked simultaneously.

(Ii) Culture [corresponding to the above step (a)]
(Ii-1) The host E. coli is transformed with the expression vector prepared in (i-3) to prepare recombinant E. coli, and the recombinant E. coli is pre-cultured. Pre-culture is performed on a medium (LB medium or the like) at 15 to 43 ° C. for 3 to 72 hours.
(Ii-2) A culture solution used for protein production is autoclaved at 121 ° C. for 20 minutes, and a medium (a carbon source capable of assimilating a host such as a microorganism such as a TB medium, a nitrogen source, and other essential nutrients) Incubate the recombinant Escherichia coli pre-cultured in the medium containing). The culture is performed at 15 to 43 ° C. for 12 to 72 hours.
To this culture broth, Isopropyl β-D-1-thiogalactopylanoside and the like are added for inducible expression, followed by the phosphoric ester compound (A), amino acid (B), saccharide (C), peptide (D), When using at least one compound selected from the group consisting of spicrispolic acid (E) and surfactant (F), the above compound and culture medium are mixed and homogenized, and the same is used. Perform the operation.
In addition, when the above compound is added 6 to 72 hours after the addition of the above-mentioned Isopropyl β-D-1-thiogalactopylanoside or the like to the culture solution, the culture is continued for 1 to 1000 hours after the addition of the above compound.
Note that fed-batch culture in which a nutrient source of E. coli (such as a glycerin / protein solution containing an antifoaming agent and ampicillin) is supplied during the above-described culturing step.

(Iii) Purification [corresponding to the above step (b)]
(Iii-1) Proteins secreted into the culture solution are separated from microorganisms and microbial residues by centrifugation, hollow fiber separation, filtration, and the like.
(Iii-2) The culture solution containing protein is subjected to precipitation treatment such as ethanol precipitation, ammonium sulfate precipitation and polyethylene glycol precipitation by repeating column treatments such as ion exchange column, gel filtration column, hydrophobic column, affinity column and ultra-column. Separation and purification are carried out as necessary.

The host E. coli isolated in (iii-1) can then be further cultured by supplying a new culture solution. The culture solution and the like are further subjected to the step (iii), and purification and culture are repeated, whereby protein can be continuously produced.

  Examples of the packing material used in the column chromatography in the above-mentioned (iii) protein separation / removal step include silica, dextran, agarose, cellulose, acrylamide, vinyl polymer, etc., and commercially available products such as Sephadex series and Sephacryl series. And Sepharose series (manufactured by Pharmacia) and Bio-Gel series (manufactured by Bio-Rad).

The useful substance production method of the present invention includes the step of causing the phosphate compound (A) and the microorganism to simultaneously exist in the culture solution and secreting the useful substance into the culture solution.
In this step, as long as the microorganism is alive, the microorganism can make a useful substance and secrete it into the culture solution.
Moreover, if the microorganism has the ability to create a useful substance, the production method of the present invention can be used regardless of the kind of useful substance to be created.
The useful substance production method of the present invention is particularly effective when the useful substance prepared in the microorganism is transferred to the periplasm of the microorganism. By transferring the useful substance to the periplasm, the useful substance is easily secreted into the culture medium.

  Evaluation of the production amount of the useful substance in the useful substance production method of the present invention can be expressed by a secretion efficiency ratio.

Secretion efficiency ratio is an index indicating the ratio of useful substances in microorganisms secreted out of microorganisms by this technology, and is an index that varies depending on useful substances to be secreted, microorganisms to be used, and compounds described in this patent to be used. . In the present invention, it is defined by the following formula.
Secretion efficiency ratio = (Xp / Xs) / (Yp / Ys)
Xp: amount of useful substance in the culture supernatant obtained by centrifuging the culture solution that produced the useful substance using the present invention Xs: dry cell density of the culture solution produced the useful substance using the present invention Yp : Phosphate ester compound (A), amino acid (B), saccharide (C), peptide (D), spicrispolic acid (E) and surfactant (F) are the same as in the method of the present invention except that the surfactant is not added. The amount of useful substance in the culture supernatant obtained by centrifuging the culture solution that produced the useful substance Ys: phosphate compound (A), amino acid (B), saccharide (C), peptide (D), spikli The dry cell density of the culture solution in which useful substances were produced in the same manner as in the method of the present invention, except that the spuric acid (E) and the surfactant (F) were not added

In the present invention, the dry cell density represents the weight of microorganisms in a state where the microorganisms contained in 1 L of the culture solution are dried. The dry cell density is determined by the following procedures (1) to (5).
Procedure (1): The weight of the container (centrifugal tube) is measured in advance.
Procedure (2): 100 ml of the culture solution is placed in the container of Procedure (1), centrifuged (4,000 G, 15 minutes, 4 ° C.), the supernatant is extracted, and microorganisms are collected.
Procedure (3): The collected microorganisms in the container are washed with 0.9 wt% NaCl aqueous solution [the same volume as the culture solution used in Procedure (2)], and centrifuged again (4,000 G, 15 minutes, 4 ℃), extract the supernatant and collect the microorganisms.
Procedure (4): The microorganisms obtained in the procedure (3) are dried for 10 hours at 105 ° C. with the microorganisms kept in the container, and then the total weight of the container and the microorganisms is measured.
Procedure (5): After the procedure (4), after further drying at 105 ° C. for 2 hours, the total weight of the container and the microorganism was measured, and the change in the weight was the weight of the microorganism measured in the procedure (4) [the container and Subtract the weight of the container measured in the procedure (1) from the total weight of the microorganisms] to confirm that it is 2% by weight or less. When the weight change is larger than 2% by weight, the drying is continued at 105 ° C. until the weight change is eliminated.
The dry cell density is determined by applying the measured value of procedure (5) and procedure (1) and the volume (L) of the culture solution used in procedure (2) to the following equation.
Dry cell density (g / L) = ([measured value of procedure (5)] − [measured value of procedure (1)]) / [volume of culture medium used in procedure (2) (L)]

The dry cell density in the production method of the useful substance of the present invention is preferably 1.5 to 500 g / L, more preferably 3 to 200 g / L based on the volume of the culture solution, from the viewpoint of the useful substance production amount. L, particularly preferably 4 to 100 g / L.
The dry cell density when the microorganism is Escherichia coli is preferably 1.5 to 500 g / L, more preferably 3 to 500 g / L based on the volume of the culture solution, from the viewpoint of enabling the production of useful substances. 100 g / L, particularly preferably 10 to 50 g / L, and most preferably 12 to 27 g / L.

Moreover, the evaluation of the activity of the useful substance produced using the useful substance production method of the present invention can be expressed by the activity value ratio.
The activity value ratio is an index indicating the activity of a useful substance secreted outside the cell by the present technology, and is an index that varies depending on the useful substance to be secreted, the microorganism to be used, and the compound described in this patent to be used. In the present invention, it is defined by the following formula.
Activity value ratio = (Xa / Xp) / (Ya / Yp)
Xa: Activity of the protein in the culture supernatant obtained by centrifuging the culture solution that produced the useful substance using the present invention (activity as an enzyme, etc.)
Ya: Same as the method of the present invention except that the phosphate ester compound (A), amino acid (B), saccharide (C), peptide (D), spicrisporic acid (E) and surfactant (F) are not added. Activity of useful substances in the culture supernatant obtained by centrifuging the culture medium that produced useful substances in this way (activity as an enzyme, etc.)
Xp: Same as Xp explained in secretory efficiency ratio Yp: Same as Yp explained in secretory efficiency ratio

The reason why the present invention exerts a high production amount of useful substances is not clear in detail, but by using the above-mentioned phosphate ester compound (A), it is produced during cultivation that causes a decrease in the production amount of useful substances. It is conceivable that the useful substance is denatured and / or aggregation of the secreted useful substance is suppressed.
Even when the phosphate ester compound (A) is not used, if a compound having the property of suppressing the denaturation of the useful substance and / or the aggregation of the secreted useful substance is used, the production amount of the useful substance is also improved. It is considered effective.

  The useful substance obtained by the method of the present invention exhibits high activity as an enzyme or the like, and in particular, the phosphate ester compound (A) and the amino acid (B), saccharide (C), peptide (D), spikyl spall. When used in combination with at least one selected from the group consisting of an acid (E) and a surfactant (F), when an alanine, arginine, glycine or lysine is used as the amino acid (B), the saccharide (C) When sucrose, 2-hydroxypropyl-β-cyclodextrin or rhamnolipid is used, when polyglutamic acid or glutamic acid lysine copolymer is used as peptide (D), when splicrisporic acid (E) is used, or these compounds Higher activity when used in combination. Also in this regard, as described above, the useful substance produced during the culture by using the phosphate ester compound (A), the amino acid (B), the saccharide (C), the peptide (D), and spikrispolic acid (E). This is presumed to be due to the suppression of denaturation and / or aggregation of secreted useful substances.

  The present invention will be further described with reference to the following examples and comparative examples, but the present invention is not limited thereto. Unless otherwise specified, parts means parts by weight.

<Production Example 1: Preparation of E. coli (α)>
The alkaline phosphatase (phoA) gene of E. coli strain W3110 was amplified by PCR using primers 1 and 2 (Table 1). The PCR fragment was treated with restriction enzymes NdeI and BamHI and then bound to the NdeI restriction enzyme site and the BamHI restriction enzyme site of the pET-22b plasmid (Novagen). Thereafter, using λDE3 Lysogenization Kit (Novagen), this plasmid was transformed into AG1 (DE3) E. coli strain prepared by modifying E. coli strain AG1 (ToYoBo) to obtain an alkaline phosphatase expression strain [E. coli (α)]. Created. It was analyzed and confirmed based on the method of METHODS IN ENZYMOLOGY 353 2002 p. 121 that the expressed alkaline phosphatase was localized in the periplasm fraction.

<Production Example 2: Preparation of Escherichia coli (β)>
Bacillus licheni by PCR using primers 3 and 4 (Table 1)
The formis bglC gene was amplified. The PCR fragment was treated with restriction enzymes NdeI and BamHI and then bound to the NdeI restriction enzyme site and the BamHI restriction enzyme site of the pET-22b plasmid (Novagen). Thereafter, this plasmid was transformed into BL21 (DE3) E. coli strain (Novagen) to prepare a cellulase expression strain [E. coli (β)]. Based on the method of METHODS IN ENZYMOLOGY 353 2002 page 121, it was confirmed that the expressed cellulase was localized in the periplasmic fraction.

<Production Example 3: Preparation of methacryloyloxyethyl phosphorylethanolamine>
19 g (0.08 mol) of 2- (methacryloyloxy) ethyl-2 ′-(trimethylammonium) ethyl phosphoric acid (manufactured by Tokyo Chemical Industry Co., Ltd.), 23.5 g (0.4 mol) of trimethylamine and 200 ml of acetonitrile in a pressure resistant reaction tube The mixture was shaken at 50 ° C. for 10 hours. Immediately after the reaction, about 150 ml of acetonitrile was removed by an evaporator and stored in a cold place to obtain crude crystals of white crystals (crude yield about 100%).
The crude crystals were dissolved in dehydrated chloroform and reprecipitated with dehydrated acetone to obtain purified methacryloyloxyethyl phosphorylethanolamine (A-2).

<Production Example 4: Production of Pluronic F-127NF 1 Mole Condensate of Lauric Acid>
30 parts of Pluronic F-127NF (manufactured by BASF) and 1 part of lauric acid are esterified in 10 parts of dimethylformamide at a reaction temperature of 10 ° C. for 10 hours, and then dimethylformamide is distilled under reduced pressure. To give 31 parts of a lauric acid 1 molar condensate of Pluronic F-127NF.

<Examples 1 to 25 and Comparative Examples 1 to 8: Cultivation of E. coli (α) and evaluation of phosphatase produced by E. coli (α)>
1 ml of the culture solution of E. coli (α) prepared in Production Example 1 was inoculated into 10 ml of LB medium [manufactured by LB Broth, Miller, Difco Laboratories] and cultured with shaking at 37 ° C. for 12 hours to prepare a preculture solution.
The above pre-cultured solution is centrifuged (7000 rpm × 15 minutes) using a centrifuge [Suprema 21, manufactured by Tommy Seiko Co., Ltd.], and the cells after removing the supernatant are placed in 10 ml of TB medium (Difco). After resuspending, Isopropyl β-D-1-thiogalactopylanoside was added to a final concentration of 1 mM, and the phosphate compound (A) described in Tables 2 and 3 and, if necessary, the amino acid (B), saccharide (C ) And surfactant (F) are added to the weight of the culture solution [medium, preculture solution, Isopropyl β-D-1-thiogalactopylanoside, and phosphate ester compound (A), amino acid (B), saccharide (C) to be added. , Total weight of peptide (D), spikrispolic acid (E) and surfactant (F)] 3 was added such that the weight percent described, starts shaking culture at 37 ° C., in E. coli (alpha), to produce phosphatase is a protein and secreted production by phosphatase in the culture solution.
After 21 hours from the start of the shaking culture, the culture solution was sampled to evaluate the secretion efficiency (relative ratio) and activity value ratio (relative ratio). The results are shown in Tables 2 and 3.

<Examples 26 to 30 and Comparative Examples 9 to 16: Cultivation of E. coli (β) and evaluation of cellulase produced by E. coli (β)>
Inoculate 0.5 ml of the overnight culture solution of E. coli (β) prepared in Production Example 2 into 5 ml of LB medium [LB Broth, Miller, manufactured by Difco Laboratories], and perform shaking culture at 30 ° C. for 3 hours. It was created.
5 mL of the pre-culture solution was added to 50 mL of a medium [yeast extract [manufactured by Nippon Pharmaceutical Co., Ltd.] 1.2 g, polypeptone [manufactured by Nippon Pharmaceutical Co., Ltd.] 0.6 g, dipotassium phosphate 0.47 g, and potassium phosphate 1. 11g, ammonium sulfate 0.35g, disodium phosphate dodecahydrate 0.66g, sodium citrate dihydrate 0.02g, glycerol 0.2g, lactalbumin hydrolyzate 1.5g, antifoam [Shin-Etsu Chemical “KM-70” manufactured by Co., Ltd. 0.3 g, 1 mM magnesium sulfate, trace metal solution [calcium chloride 18.9 μg, iron (III) chloride 500 μg, zinc sulfate heptahydrate 9.0 μg, copper sulfate 5. 1 μg, manganese chloride tetrahydrate 6.7 μg, cobalt chloride 4.9 μg, ethylenediaminetetraacetic acid tetrasodium 200 μg], 100 mg / L ampicillin] Culture apparatus [Able Co., Ltd., product name "BioJr.8"] was used to start the aeration-agitation culture while maintaining the pH6.8,30 ℃.
After 3 hours from the start of the culture, Isopropyl β-D-1-thiogalactopylanoside was added to a final concentration of 1 mM, and the phosphate compound (A) described in Tables 4 and 5 and, if necessary, the amino acid (B), The saccharide (C) and the surfactant (F) are added to the weight of the culture solution [medium, preculture solution, Isopropyl β-D-1-thiogalactopyroside, glycerin / protein solution added later, and phosphate ester compound (A ), Amino acids (B), saccharides (C), peptides (D), spiculisporic acid (E) and surfactant (F)] based on the total weight] so that the weight percentages are as shown in Tables 4 and 5. 14 hours after the start of culture, glycerin / protein solution [50% glycerin, 50 g / L Lactalbumin hydrolyzate, 33 g / L Antifoaming agent [manufactured by Shin-Etsu Chemical Co., Ltd., “KM-70”], 100 mg / L ampicillin] quantitative liquid feed pump [MP-1000, Tokyo Science Instruments Co., Ltd.] The drop was started at a rate of 2 ml / hr.
The culture solution was sampled 48 hours after the start of the culture, the secretion efficiency (relative ratio) and the activity value ratio (relative ratio) were evaluated, and the results are shown in Tables 4 and 5.

In addition, the thing of the following Table 6 was used for the phosphate ester compound (A) in Tables 2-5.

In addition, the following were used for the amino acid (B), polysaccharide (C), and surfactant (F) in Tables 2-5.
B1: Glycine [Wako Pure Chemical Industries, Ltd.]
C1: 2-hydroxypropyl-β-cyclodextrin [Wako Pure Chemical Industries, Ltd.]
F1: Ethylene oxide 1 mol adduct of lauryl alcohol [New Pole DDE-10, manufactured by Sanyo Chemical Industries, Ltd.]
F2: Palm oil fatty acid amide betaine [Levon 2000, manufactured by Sanyo Chemical Industries, Ltd.]
F3: 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine [Levon 105, manufactured by Sanyo Chemical Industries, Ltd.]
F4: Sodium polyoxyethylene tridecyl ether acetate [Burelite ECA, manufactured by Sanyo Chemical Industries, Ltd.]
F5: Sodium polyoxyethylene lauryl ether sulfate [Sun Dead EN, manufactured by Sanyo Chemical Industries, Ltd.]
F6: Pluronic F-127NF [manufactured by BASF]
F7: Pluronic F-127NF 1 mol lauric acid adduct (obtained in Production Example 4)

[Method for evaluating secretion efficiency ratio]
Secretion efficiency ratio was defined by the following formula. Secretion efficiency ratio is an index indicating the ratio of protein in Gram-negative bacteria secreted out of Gram-negative bacteria by this technology.
Secretion efficiency ratio = (Xp / Xs) / (Yp / Ys)
Xp: The amount of protein in the culture supernatant obtained by centrifuging (13000 G × 15 min) each culture solution obtained in Examples 1 to 50 and Comparative Examples 1 to 16 (detailed measurement method for protein weight is as follows. Described in)
Xs: dry cell density of each culture solution obtained in Examples 1-50 and Comparative Examples 1-16 Yp: In Examples 1-25 and Comparative Examples 1-8, the amount of protein in Comparative Example 1, Example 26 ~ 50 and Comparative Examples 9-16, the amount of protein Ys of Comparative Example 9: In Examples 1-25 and Comparative Examples 1-8, the dry cell density of Comparative Example 1, Examples 26-50 and In Comparative Examples 9 to 16, the dry cell density of Comparative Example 9

<Method for quantifying protein content>
The weight of the protein after recovering the culture solution was analyzed by analyzing the total protein in the culture supernatant by SDS-PAGE, and the density of the protein band was quantified by ImageJ (National Institutes of Health, USA), and the produced recombinant protein The quantity was quantified. (Based on standard methods performed by those skilled in the art.)

<Dry cell density measurement method>
The dry cell density in Examples 1 to 50 and Comparative Examples 1 to 16 was determined by the following procedures (1) to (5).
Procedure (1): The weight of the container (centrifugal tube) is measured in advance.
Procedure (2): 100 ml of each culture solution obtained in Examples 1 to 50 and Comparative Examples 1 to 16 is placed in the container of Procedure (1), centrifuged (4,000 G, 15 minutes, 4 ° C.), and the supernatant is obtained. Extract and collect Gram-negative bacteria.
Procedure (3): The collected Gram-negative bacteria in the container are washed with 0.9 wt% NaCl aqueous solution [the same volume as the culture solution used in Procedure (2)], and centrifuged again (4,000 G, 15 minutes) 4 ° C.), the supernatant is extracted, and Gram-negative bacteria are collected.
Procedure (4): The gram-negative bacterium obtained in the procedure (3) is dried at 105 ° C. for 10 hours in a container, and then the total weight of the container and the gram-negative bacterium is measured.
Step (5): After drying at 105 ° C. for 2 hours after step (4), the total weight of the container and the gram-negative bacterium was measured, and the change in weight was determined for the gram-negative bacterium measured in step (4). Confirm that the weight is 2% by weight or less based on the weight [subtracting the weight of the container measured in the procedure (1) from the total weight of the container and Gram-negative bacteria]. When the weight change is larger than 2% by weight, the drying is continued at 105 ° C. until the weight change is eliminated.
The dry cell density is determined by applying the measured value of procedure (5) and procedure (1) and the volume (L) of the culture solution used in procedure (2) to the following equation.
Dry cell density (g / L) = ([measured value of procedure (5)] − [measured value of procedure (1)]) / [volume of culture medium used in procedure (2) (L)]

[Evaluation method of activity value ratio]
The activity value ratio is an index indicating the activity of the protein secreted outside the cell by the present technology, and is an index that varies depending on the protein to be secreted, the Gram-negative bacterium to be used, and the compound described in this patent to be used. In the present invention, it is defined by the following formula.
Activity value ratio = (Xa / Xp) / (Ya / Yp)
Xa: Activity of protein in supernatant obtained by centrifugation of each culture solution obtained in Examples 1-50 and Comparative Examples 1-16 (detailed measurement method for protein activity is described below)
Ya: In Examples 1 to 25 and Comparative Examples 1 to 8, the activity of the protein in the supernatant of Comparative Example 1, and in Examples 26 to 50 and Comparative Examples 9 to 16, above Comparative Example 9 Protein activity in clear liquid Xp: Same as Xp explained in secretory efficiency ratio Yp: Same as Yp explained in secretory efficiency ratio

<In Examples 1-25 and Comparative Examples 1-8, activity measurement of proteins produced using E. coli (α): measurement of alkaline phosphatase activity>
12.4 g of diethanolamine (MW = 105.14, purity 85%) is diluted with 80 ml of distilled water and 0.5 ml of 1M MgCl 2 is added. Furthermore, the solution A [1M diethanolamine buffer (pH 9.8)] was prepared by adjusting the pH to 9.8 with 2N hydrochloric acid while keeping the temperature at 37 ° C., and adjusting the final solution volume to 100 ml.
P-Nitrophenylphosphoric acid was dissolved in solution A to 0.67M to prepare solution B.
2.9 ml of the solution A and 0.1 ml of the solution B were put into an Eppendorf tube, and then the temperature was adjusted to 37 ° C. Subsequently, each culture solution obtained in Examples 1 to 25 and Comparative Examples 1 to 8 was centrifuged (13000 G × 15 min). The culture supernatant (0.1 ml) thus obtained was put into an Eppendorf tube and mixed by inversion for 3 seconds. The liquid after inversion mixing was transferred to a cell (optical path length = 1.0 cm), and the absorbance at 405 nm was measured with a spectrophotometer [PharmacSpec UV-1700, manufactured by Shimadzu Corporation] controlled at 37 ° C. with water as a control. Read after 3, 4 and 5 minutes after transferring to the cell, plot XY coordinates with time (minutes) on the X axis and absorbance on the Y axis, and 1 minute from the slope when a straight line is drawn by the least squares method Absorbance change per unit is obtained (ΔODTEST). In the blank, 0.1 ml of water is added instead of 0.1 ml of the culture solution, and the change in absorbance per minute is similarly determined (ΔODBLANK). Using these values, alkaline phosphatase activity was determined from the following formula.
Alkaline phosphatase activity (U / ml) = {(ΔODTEST−ΔODBLANK) × 3.1} / {18.2 × 1.0 × 0.1}
3.1: Amount of reaction solution after addition of culture supernatant (ml)
18.2: Molar molecular extinction coefficient of p-nitrophenol (cm2 / μmol) under the above measurement conditions
1.0: Optical path length (cm)
0.1: Amount of enzyme solution added (ml)

<Activity measurement of proteins produced using E. coli (β) in Examples 26-50 and Comparative Examples 9-16: Cellulase activity measurement>
Endo-1,4-β-glucanase activity was measured as cellulase activity.
Carboxymethyl cellulose [manufactured by Wako Pure Chemical Industries, Ltd.] was dissolved in a 100 mM phosphate buffer aqueous solution of pH 7.5 so as to be 17.5 g / L to prepare a substrate solution.
5 μL of the culture supernatant obtained by centrifuging each culture solution obtained in Examples 25 to 50 and Comparative Examples 9 to 16 (13000 G × 15 min) and 3 mL of the substrate solution were mixed, and this mixture was used as a viscometer. Transfer to a temperature-controlled cup (TVE-22L viscometer, 2 rpm) at 40 ° C.
Read the viscosity immediately after mixing, read the viscosity after 1, 2, 3, 4, 5 and 6 minutes, and plot the XY coordinate diagram with the time (minutes) as the X axis and the viscosity (mPa · s) as the Y axis. The absolute value of the slope when a straight line was drawn by the least square method was defined as cellulase activity (endo-1,4-β-glucanase activity).

  Examples 1-50 using the production method of the present invention include the phosphate ester compound (A) in the culture medium, thereby comparing with Comparative Example 1 or 9 not including the phosphate ester compound (A). From the fact that the secretion efficiency is large, the production amount of useful substances (proteins) is large, and the activity value ratio is large, indicating that the activity is high. Moreover, when Examples 18-25 and 43-50 using surfactant (F) were compared with Comparative Examples 2-8 and 10-16, although the production amount of a useful substance (protein) was comparable, It turns out that the Example of this invention using a phosphate ester compound has a large activity value ratio, and can obtain a large amount of what has high activity.

The production method of a useful substance of the present invention can be used when secreting production of a useful substance from a microorganism. Examples of useful substances to be produced include enzymes, hormone proteins, antibodies and peptides. Among them, when useful substances to be produced are enzymes (proteases, cellulases, lipases, amylases, etc.), It can be suitably used for cleaning agents, fiber treatment, papermaking applications, enzyme conversion applications, and the like.

<Production Example 1: Preparation of E. coli (α)>
The alkaline phosphatase (phoA) gene of E. coli strain W3110 was amplified by PCR using primers 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2) (Table 1). The PCR fragment was treated with restriction enzymes NdeI and BamHI and then bound to the NdeI restriction enzyme site and the BamHI restriction enzyme site of the pET-22b plasmid (Novagen). Thereafter, using λDE3 Lysogenization Kit (Novagen), this plasmid was transformed into AG1 (DE3) E. coli strain prepared by modifying E. coli strain AG1 (ToYoBo) to obtain an alkaline phosphatase expression strain [E. coli (α)]. Created. It was analyzed and confirmed based on the method of METHODS IN ENZYMOLOGY 353 2002 p. 121 that the expressed alkaline phosphatase was localized in the periplasm fraction.

<Production Example 2: Preparation of Escherichia coli (β)>
The bglC gene of Bacillus licheni formis was amplified by PCR using primers 3 (SEQ ID NO: 3) and 4 (SEQ ID NO: 4) (Table 1). The PCR fragment was treated with restriction enzymes NdeI and BamHI and then bound to the NdeI restriction enzyme site and the BamHI restriction enzyme site of the pET-22b plasmid (Novagen). Thereafter, this plasmid was transformed into BL21 (DE3) E. coli strain (Novagen) to prepare a cellulase expression strain [E. coli (β)]. Based on the method of METHODS IN ENZYMOLOGY 353 2002 page 121, it was confirmed that the expressed cellulase was localized in the periplasmic fraction.

Claims (6)

A method for producing a useful substance containing a phosphate ester compound (A) represented by the following general formula (1) in the culture solution, wherein the useful substance is secreted and produced in the culture solution by a microorganism contained in the culture solution. .

[In General Formula (1), R ¹ is a hydrogen atom or a monovalent hydrocarbon group having 1 to 50 carbon atoms, and a hydroxyl group, an ether group, an amide group, an amino group at any position of the hydrocarbon group Represents an optionally substituted hydrocarbon group selected from the group consisting of an acryloyl group, a methacryloyl group, a carbonyl group, a carboxyl group and an ester group, and R ² is a group having 1 to 50 carbon atoms. A group consisting of a hydroxyl group, an ether group, an amide group, an amino group, an ammonium group, an acryloyl group, a methacryloyl group, a carbonyl group, a carboxyl group and an ester group at any position of the hydrocarbon group represents at least one substituent hydrocarbon group which may have a are more selected, X is O ^- or a hydroxyl group, in R ^2, any position of the hydrocarbon group If having an ammonium group O ^- represents a. ] The method for producing a useful substance according to claim 1, wherein the phosphate ester compound (A) is a phosphate ester compound represented by the following general formulas (2) to (5).

[In General Formulas (2) to (5), R ³ and R ⁷ are each a monovalent hydrocarbon group having 1 to 50 carbon atoms, and at any position of the hydrocarbon group, a hydroxyl group, an ether group, Represents a hydrocarbon group which may have at least one substituent selected from the group consisting of an amide group, an amino group, an acryloyl group, a methacryloyl group, a carbonyl group, a carboxyl group and an ester group, R ⁴ , R ⁸ , R ¹² and R ¹⁵ are each a divalent hydrocarbon group having 1 to 12 carbon atoms, and at any position of the hydrocarbon group, a hydroxyl group, an ether group, an amide group, an amino group, an acryloyl group, a methacryloyl group Represents a hydrocarbon group optionally having at least one substituent selected from the group consisting of a carbonyl group, a carboxyl group and an ester group, and R ⁵ , R ⁶ , R ⁹ , R ¹⁰ , R ¹¹ , R ^13, R ^14, R ¹⁶ An aliphatic hydrocarbon group of R ¹⁷ and R ¹⁸ are each a hydrogen atom or a C 1 to 49, or a hydroxyl group and / or may have an ether hydrocarbon group in the position of the hydrocarbon group Represents. ] The method for producing a useful substance according to claim 1 or 2, wherein the phosphate ester compound (A) is a phosphate ester compound represented by the following general formula (6).

[In the general formula (6), R ¹⁹ is a hydrogen atom or a monovalent hydrocarbon group having 1 to 50 carbon atoms, and at any position of the hydrocarbon group, a hydroxyl group, an ether group, an amide group, an amino group Represents a hydrocarbon group which may have at least one substituent selected from the group consisting of acryloyl group, methacryloyl group, carbonyl group, carboxyl group and ester group. ]   At least selected from the group consisting of a surfactant (F) other than the amino acid (B), saccharide (C), peptide (D), spikrispolic acid (E) and phosphate ester compound (A) in the culture solution. The method for producing a useful substance according to any one of claims 1 to 3, comprising one kind of compound.   The method for producing a useful substance according to any one of claims 1 to 4, wherein the content of the phosphate ester compound (A) is 0.0003 to 3 wt% based on the weight of the culture solution.   The method for producing a useful substance according to any one of claims 1 to 5, wherein the microorganism is a gram-negative bacterium.