JP7050528B2 - Production method of useful substances - Google Patents

Description

The present invention relates to a method for producing a useful substance.

Technology for producing useful substances such as amino acids and proteins by microorganisms is widely used in industrial fields such as medical treatment.
As a microorganism that produces useful substances such as amino acids and proteins, a method of producing by Escherichia coli is known. However, there are problems that useful substances to be produced are limited and secretory production is difficult. Therefore, by using fungi such as Gram-positive bacteria, yeast and basidiomycete, it is possible to secrete and produce a wide range of useful substances (for example, Non-Patent Documents 1 and 2).

However, such fungi such as Gram-positive bacteria, yeasts and basidiomycetes have a cell wall made of thick polysaccharide in order to withstand stress from the outside world. This cell wall made of polysaccharide causes adsorption of useful substances and suppression of permeation of useful substances, and the secretory efficiency deteriorates, which is a big problem in secretory production.

Therefore, in yeast, for the purpose of thinning the cell wall due to polysaccharides, S. There is a technique (Non-Patent Document 3) for increasing the amount of protein secretion by using a sugar synthesis-related gene-disrupted strain of S. cerevisiae or Yarrowia lipolytica. However, it has been pointed out that the glycoprotein obtained by this method is limited to proteins that do not have glycosylated chains, and no useful solution has been found yet.

Bioindustry Association, Fermentation Handbook, Kyoritsu Shuppan, July 2001 New Biochemical Engineering (2nd Edition), Sankyo Publishing, March 2013, p17-21 J. Bacteriol, 1998, vol. 180, no. 24, p6736-6742

An object of the present invention is to provide a method for producing a useful substance capable of efficiently secreting a useful substance produced by yeast into a culture solution.

The present inventors have arrived at the present invention as a result of diligent studies to solve these problems.
That is, the present invention is a method for producing a useful substance by secreting and producing a useful substance in the culture solution by yeast contained in the culture solution, and the yeast is at least one selected from the group consisting of the genus Saccharomyces and the genus Pichia. The nonionic surfactant (A) is contained in the culture broth, (A) is the fatty acid ester (A1 ) of the aliphatic polyhydric alcohol, and the nonionic surfactant (A) is contained in the culture broth. This is a method for producing a useful substance in which the amount is 0.003 to 5% by weight based on the weight of the culture solution at the start of culture .

By using the method for producing a useful substance of the present invention, the useful substance produced by a microorganism can be secreted into a culture solution with high efficiency.

The method for producing a useful substance of the present invention is a method for producing a useful substance by secreting and producing a useful substance in a culture solution by yeast contained in the culture solution, wherein the yeast belongs to the genus Ogataea, the genus Saccharomyces, the genus Kluyveromyces, and the genus Hansenula. , Pichia, Yarrowia, and Candida, which is at least one selected from the group, contains a nonionic surfactant (A) in a culture solution, and (A) is a fatty acid ester of an aliphatic polyhydric alcohol (A1). ), Fatty acid ester of alkylene oxide adduct of aliphatic polyhydric alcohol (A2), polyalkylene glycol fatty acid ester (A3), aliphatic monovalent alcohol alkylene oxide adduct (A4), alkylene oxide adduct of fatty acid (A5). It is a method for producing a useful substance which is at least one nonionic surfactant selected from the group consisting of an alkylene oxide adduct (A6) of an aromatic monovalent alcohol having an aliphatic hydrocarbon group.

The culture medium used in the production method of the present invention can be used without particular limitation as long as it is a cell culture medium generally used in the art, and is a natural medium containing a carbon source, a nitrogen source and other essential nutrients, and synthetic. Any medium may be used.

Examples of the carbon source include carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol.

Examples of the nitrogen source include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate or other nitrogen-containing compounds, as well as peptone, meat extract and corn steep liquor.

Examples of other essential nutrients include inorganic salts, and examples of the inorganic salts include primary potassium phosphate, ferric potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, and copper sulfate. , Calcium carbonate and the like are used.

The yeast in the present invention is at least one selected from the group consisting of the genera Ogataea, Saccharomyces, Kluyveromyces, Hansenula, Pichia, Yarrowia and Candida.
It is preferably at least one selected from the group consisting of the genera Saccharomyces, Pichia and Candida.

The useful substance in the present invention is not particularly limited, and includes proteins (enzymes, hormone proteins, antibodies, peptides, etc.), oligosaccharides, nucleic acids, and the like.

Examples of proteins include enzymes {oxidation-reducing enzymes (cholesterol oxidase, glucose oxidase, ascorbic acid oxidase, peroxidase, etc.), transferases (lysoteam, protease, serine protease, amylases, lipase, cellulase, glucoamylase, etc.), isomerizing enzymes (, etc.). Glucose isomerase, etc.), transferases (acyltransferase, sulfotransferase, etc.), synthases (fatty acid synthase, phosphate synthase, citrate synthase, etc.) and desorption enzymes (pectinriase, etc.)}; BMP), interferon α, interferon β, interleukins 1-12, growth hormone, erythropoetin, insulin, granular colony stimulator (G-CSF), tissue plasminogen activator (TPA), sodium diuretic peptide, blood coagulation Factor VIII, somatomedin, glucogon, growth hormone releasing factor, serum albumin and calcitonin, etc.};
Antibodies {single-stranded antibody, IgG large subunit, IgG small subunit, etc.}, antigen protein {hepatitis B surface antigen, etc.};
Functional proteins {Pronectin®, antifreeze peptides, antibacterial peptides, etc.};
Examples thereof include fluorescent proteins (GFP and the like), luminescent proteins (lucerase and the like) and peptides (not particularly limited in amino acid composition, oligopeptides, dipeptides and tripeptides and the like).

Examples of oligosaccharides include hyaluronic acid, chondroitin, xanthane, cellulose, sucrose, lactose, trehalose, maltose, raffinose, panose, cyclodextrin, galactooligosaccharides and fructooligosaccharides.

Examples of the nucleic acid include DNA, RNA, inosinic acid, adenosine monophosphate, guanosine monophosphate and the like.

Among these useful substances, proteins are preferable, and enzymes, hormonal proteins and antibodies are more preferable from the viewpoint of easiness of producing useful substances.

When the useful substance is a protein, it is preferable that the protein has a property that a part or all of the protein is transferred to periplasm after being expressed in the microorganism. More preferably, it is a protein that encodes the signal sequence required for the transition to periplasm in the ORF.
Periplasm is the space outside the cytoplasmic membrane of microorganisms and the cell wall layer containing polysaccharides.
Examples of the signal sequence required for the transition to periplasm include an α-signal sequence, a signal sequence of the PHO1 gene, and the like.

The nonionic surfactant (A) used in the method for producing a useful substance of the present invention includes a fatty acid ester of an aliphatic polyhydric alcohol (A1), a fatty acid ester of an alkylene oxide adduct of an aliphatic polyhydric alcohol (A2), and the like. Polyalkylene glycol fatty acid ester (A3), aliphatic monohydric alcohol alkylene oxide adduct (A4), fatty acid alkylene oxide adduct (A5) and aromatic monohydric alcohol alkylene oxide adduct having an aliphatic hydrocarbon group (A5). It is at least one nonionic surfactant selected from the group consisting of A6).

The aliphatic hydrocarbon group includes a monovalent or divalent aliphatic hydrocarbon group, and specifically, a monovalent aliphatic hydrocarbon group {a linear or branched alkyl group, a linear or branched alkyl group. An alkenyl group, an alicyclic hydrocarbon group, etc.}, a divalent aliphatic hydrocarbon group {a linear or branched alkylene group, etc.} and the like can be mentioned.
As the aliphatic hydrocarbon group, a monovalent aliphatic hydrocarbon group is preferable from the viewpoint of secretion efficiency, and a linear or branched alkyl group and a linear or branched alkenyl group are more preferable.
The carbon number of the aliphatic hydrocarbon group is preferably 8 to 22, more preferably 8 to 18, from the viewpoint of cytotoxicity and secretory efficiency.

The fatty acid ester (A1) of the aliphatic polyhydric alcohol of the present invention is an ester compound of the aliphatic polyhydric alcohol and the fatty acid, and is a specific fatty acid as long as one or more hydroxyl groups of the polyhydric alcohol are ester-bonded. Octanoic acid, nonanoic acid, decanoic acid, lauric acid, myristic acid, pentadecylic acid, palmitic acid, palmitreic acid, margaric acid, stearic acid, oleic acid, baxenoic acid, linoleic acid, linolenic acid, arachidic acid, behen Acids and the like can be mentioned.
Of these, fatty acids having 8 to 22 carbon atoms are preferable, and 8 to 18 are more preferable, from the viewpoint of cytotoxicity and secretory efficiency.

The aliphatic polyhydric alcohol is a dihydric aliphatic alcohol {alkylene glycol having 2 to 20 carbon atoms (ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, 1,4-butanediol, neopentyl). Glycer, 1,6-hexanediol, 1,9-nonanediol, 1,10-decanediol, etc.), trihydric or higher aliphatic alcohols {including those having 3 to 20 carbon atoms, for example, alkane polyols and theirs. Intramolecular or intermolecular dehydrations (glycerin, trimethylolethane, trimethylolpropane, pentaerythritol, sorbitol, sorbitan, polyglycerin, and dipentaerythritol), saccharides and derivatives thereof (sucrose and methyl glucoside), etc. Be done.

The fatty acid ester (A2) of the aliphatic polyhydric alcohol of the present invention is an ester compound of an alkylene oxide of an aliphatic polyhydric alcohol (hereinafter, the alkylene oxide may be abbreviated as AO) adduct and a fatty acid. It suffices if one or more hydroxyl groups of the A adduct of the polyhydric alcohol are ester-bonded.
Examples of the AO adduct of the aliphatic polyhydric alcohol include AO (ethylene oxide, 1,2-propylene oxide, 1,3-propylene oxide and the like) adduct having 2 to 4 carbon atoms to the aliphatic polyhydric alcohol. Can be mentioned.
The addition (polymerization) of AO may be monopolymerization or copolymerization. In the case of copolymerization, block copolymerization, random copolymerization, combined use of block and random, etc. may be used, but a block copolymer is preferable from the viewpoint of secretion efficiency.
From the viewpoint of secretory efficiency, the degree of polymerization of AO is preferably 1 to 100, more preferably 1 to 50.
Examples of the fatty acid include those exemplified in (A1), and among these, fatty acids having 8 to 22 carbon atoms are preferable, and 8 to 18 are more preferable, from the viewpoint of cytotoxicity and secretory efficiency.

The polyalkylene glycol fatty acid ester (A3) of the present invention is an ester compound of polyalkylene glycol and a fatty acid, and it is sufficient that one or more hydroxyl groups of the polyalkylene glycol are ester-bonded.
As the polyalkylene glycol, AO (ethylene oxide, 1,2-propylene oxide and 1,3-) having 2 to 4 carbon atoms to an alkylene glycol having 2 to 4 carbon atoms (ethylene glycol, propylene glycol, butylene glycol, etc.) (Propylene oxide, etc.) Additives and the like can be mentioned.
The addition (polymerization) of AO may be monopolymerization or copolymerization. In the case of copolymerization, block copolymerization, random copolymerization, combined use of block and random, etc. may be used, but a block copolymer is preferable from the viewpoint of secretion efficiency.
From the viewpoint of secretory efficiency, the degree of polymerization of AO is preferably 1 to 100, more preferably 1 to 50.
Examples of the fatty acid include those exemplified in (A1), and among these, fatty acids having 8 to 22 carbon atoms are preferable, and 8 to 18 are more preferable, from the viewpoint of cytotoxicity and secretory efficiency.

The aliphatic monohydric alcohol alkylene oxide adduct (A4) is an alkylene oxide adduct of the aliphatic monohydric alcohol.
The aliphatic monohydric alcohols include capryl alcohol, 2-ethylhexanol, pelargone alcohol, caprin alcohol, undecyl alcohol, lauryl alcohol, tridecyl alcohol, myristyl alcohol, pentadecyl alcohol, palmityl alcohol, cetostearyl alcohol, and palmitrail. Alcohol, 1-Heptadecanol, Stearyl Alcohol, Elysyl Alcohol, Oleyl Alcohol, Linoleil Alcohol, Elide Linoleil Alcohol, Linolenyl Alcohol, Nonadesyl Alcohol, Arakidyl Alcohol, Heneikosanol, Behenyl Alcohol and Elsyl Alcohol and the like can be mentioned.
The addition (polymerization) of AO may be monopolymerization or copolymerization. In the case of copolymerization, block copolymerization, random copolymerization, combined use of block and random, etc. may be used, but a block copolymer is preferable from the viewpoint of secretion efficiency.
From the viewpoint of secretory efficiency, the degree of polymerization of AO is preferably 1 to 100, more preferably 1 to 50.

Examples of the fatty acid alkylene oxide adduct (A5) include those exemplified in (A1), and preferred ones are also the same.
The addition (polymerization) of AO may be monopolymerization or copolymerization. In the case of copolymerization, block copolymerization, random copolymerization, combined use of block and random, etc. may be used, but a block copolymer is preferable from the viewpoint of secretion efficiency.
From the viewpoint of secretory efficiency, the degree of polymerization of AO is preferably 1 to 100, more preferably 1 to 50.
Of these, fatty acids having 8 to 22 carbon atoms are preferable, and 8 to 18 are more preferable, from the viewpoint of cytotoxicity and secretory efficiency.

The aromatic monohydric alcohol having an aliphatic hydrocarbon group of the alkylene oxide adduct (A6) of the aromatic monohydric alcohol having an aliphatic hydrocarbon group includes an alkylphenol having an alkyl group having 8 to 22 carbon atoms. Specific examples thereof include 4-octylphenol, 4-t-octylphenol, nonylphenol, dodecylphenol and the like.
The addition (polymerization) of AO may be monopolymerization or copolymerization. In the case of copolymerization, block copolymerization, random copolymerization, combined use of block and random, etc. may be used, but a block copolymer is preferable from the viewpoint of secretion efficiency.
From the viewpoint of secretory efficiency, the degree of polymerization of AO is preferably 1 to 100, more preferably 1 to 50.

HLB is known as a measure of hydrophilicity and hydrophobicity of nonionic surfactants. The higher the HLB value, the higher the hydrophilicity. The HLB in the present invention is a numerical value calculated by the following formula (1) (Takehiko Fujimoto, Introduction to Surfactants, p. 212, published by Sanyo Chemical Industries, Ltd.).
HLB = 10 × (inorganic / organic) (1)

The HLB of the nonionic surfactant (A) is preferably 6 to 20, particularly preferably 6 to 17, from the viewpoint of dispersibility in the culture medium and secretory efficiency.

Specific examples of the fatty acid ester (A1) of the aliphatic polyhydric alcohol include sorbitan palm oil fatty acid ester (HLB = 8.5).

Specific examples of the fatty acid ester (A2) of the alkylene oxide adduct of the aliphatic polyhydric alcohol include monococonut oil fatty acid ester (HLB = 16.3) of sorbitan EO 20 mol adduct and monoolein of sorbitan EO 20 mol adduct. Acid ester (HLB = 15.4, Tween80), monolauric acid ester with 20 mol of sorbitan EO (HLB = 16.7, Tween20), dioleic acid ester with 12 mol of EO (HLB = 10.4), 20 mol of EO Examples thereof include dioleic acid ester (HLB = 12.9) of the substance.

Specific examples of the polyalkylene glycol fatty acid ester (A3) include a stearic acid ester of polyethylene glycol with 6 mol of EO (HLB = 7.3) and a stearic acid ester of polyethylene glycol with 65 mol of EO (HLB = 17.0). ), Oleic acid ester of polyethylene glycol with 9 mol of EO (HLB = 9.4), Oleic acid ester of polyethylene glycol with 12 mol of EO (HLB = 10.4), Olein of PO15 mol of polyethylene glycol with 30 mol of EO. Examples thereof include an acid ester (HLB = 7.0), a lauryl acid ester of polyethylene glycol with 290 mol of EO (HLB = 18.4), and the like.

Specific examples of the aliphatic monohydric alcohol alkylene oxide adduct (A4) include lauryl alcohol EO 9 mol adduct (HLB = 13.6), lauryl alcohol EO 11 mol adduct (HLB = 14.4), and lauryl alcohol. And EO 8 mol adduct (HLB = 13.6) of myristyl alcohol mixture, EO 10 mol adduct (HLB = 13.3) and oleyl alcohol EO 10 mol adduct (HLB = 12.4) of hexadecanol and stearyl alcohol mixture. And so on.

Specific examples of the fatty acid alkylene oxide adduct (A5) include oleic acid EO 9 mol adduct (HLB = 11.8), stearic acid EO 23 mol adduct (HLB = 15.7), and stearic acid EO 9 mol adduct. An object (HLB = 11.9) and the like can be mentioned.
In the polyalkylene glycol fatty acid ester (A3) and the alkylene oxide adduct (A5) of the fatty acid, the polyalkylene glycol portion of (A3), the portion to which the alkylene oxide of (A5) is added, and the fatty acid portion of (A3). If the fatty acid portion of (A5) is the same, it is presumed that the same effect is obtained.

Specific examples of the alkylene oxide adduct (A6) of the aromatic monohydric alcohol having an aliphatic hydrocarbon group include 4- (1,1,3,3-tetramethylbutyl) phenyl-polyethylene glycol (HLB =). 13.5, TritonX-100), octylphenyl-polyethylene glycol (HLB = 13.0, NonidetP-40) and the like.

The nonionic surfactant (A) includes a fatty acid ester of an aliphatic polyhydric alcohol (A1), a fatty acid ester of an alkylene oxide adduct of the aliphatic polyhydric alcohol (A2), and a polyalkylene glycol fatty acid ester from the viewpoint of secretion efficiency. At least one selected from the group consisting of (A3), an alkylene oxide adduct of a fatty acid (A5) and an alkylene oxide adduct of an aromatic monohydric alcohol having an aliphatic hydrocarbon group (A6) is preferable, and more preferably fat. Of group polyhydric alcohol fatty acid esters (A1), aliphatic polyhydric alcohol alkylene oxide adduct fatty acid esters (A2), polyalkylene glycol fatty acid esters (A3) and aromatic monohydric alcohols having an aliphatic hydrocarbon group. It is at least one selected from the group consisting of the alkylene oxide adduct (A6).

Further, as the nonionic surfactant (A), from the viewpoint of cytotoxicity, a fatty acid ester of an aliphatic polyhydric alcohol (A1), a fatty acid ester of an alkylene oxide adduct of the aliphatic polyhydric alcohol (A2), and a polyalkylene glycol fatty acid. At least one of the fatty acids constituting the ester (A3) or the alkylene oxide adduct (A5) of the fatty acid has 8 to 22 carbon atoms.

The amount (% by weight) of the nonionic surfactant (A) used in the method for producing a useful substance of the present invention is appropriately selected depending on the target microorganism, the type of useful substance produced, and the type of extraction method. However, from the viewpoint of secretion efficiency, 0.0001 to 20% by weight is preferable, more preferably 0.005 to 10% by weight, and then 0.005 to 10% by weight, based on the weight of the culture solution at the start of culture (at the time of inoculation). It is more preferably 0.007 to 7% by weight, and particularly preferably 0.01 to 5% by weight.

In the method for producing a useful substance of the present invention, the secretory efficiency (%) of the surfactant in the culture solution is preferably 55 to 100%, more preferably 60 to 100%, and further preferably further, from the viewpoint of productivity. Is 65 to 100%, particularly preferably 70 to 100%.

The secretory efficiency indicates that the useful substance in the microorganism is secreted to the outside of the microorganism (in the culture solution) by the surfactant.
In the present invention, it is defined by the following formula.
Secretion efficiency (%) = 100 × (X / OD) / {(X / OD) + (Y / OD)}
X (culture supernatant): Concentration of useful substances in the culture solution remaining after removal of cells by centrifugation Y (surface of cells): Cell suspension collected by centrifugation and resuspended. Concentration of useful substances in OD: Concentration of cells during centrifugation

As for the secretory efficiency, for example, if the protein produced in the microorganism is more periplasmically transferred, the secretory efficiency is increased, and if the protein produced in the microorganism is not transferred to the periplasm, the secretory efficiency is decreased. In addition, the secretory efficiency can be increased by selecting a surfactant having a high secretory efficiency by screening.

The nonionic surfactant (A) may be added later to the culture solution in which the microorganism is suspended, in addition to being used by mixing with the culture solution in advance. Mixing with the culture solution can be performed by adding a surfactant to the culture solution and stirring with a stirring blade, a stirrer or the like. When mixing later, it can be added while stirring with a stirring blade or the like.

<Measurement method of turbidity of culture solution>
Using a culture solution containing microorganisms recovered by sampling, turbidity is measured using a turbidity meter [UV-1700, manufactured by Shimadzu Corporation] and a quartz cell having an optical path length of 1 cm.
Centrifuge the culture at 1500 rpm, 5 minutes, 4 ° C and discard the supernatant. The precipitate is resuspended in the same amount of physiological saline as the sample solution, diluted with physiological saline so as to have an appropriate absorbance (0.1 to 0.8), and the absorbance at 600 nm is measured. The turbidity of the culture solution is calculated by the following formula (1).
Turbidity of culture solution (OD) = (absorbance of diluted culture solution at 600 nm) × dilution ratio of culture solution (Formula 1)

The turbidity of the culture solution in the method for producing a useful substance of the present invention is preferably 1 to 300 OD, more preferably 10 to 300 OD, and particularly preferably 20 to 300 OD.
When the turbidity of the culture solution is 1 OD or more, the amount of useful substances produced increases, and when the turbidity is 300 OD or less, the production of useful substances is easy, which is preferable.
In the method for producing a useful substance of the present invention, within the above range, the greater the turbidity of the culture solution, the greater the amount of the useful substance produced.

In the method for producing a useful substance of the present invention, from the viewpoint of the production amount of the useful substance, the time during which the turbidity of the culture solution is within the above range is 10% or more of the time required for the step of secreting the useful substance. It is preferable, more preferably 50% or more.

The turbidity of the culture can be increased, for example, by feeding at an appropriate rate using a semi-batch culture method under sufficient aeration conditions and reduced by performing batch cultures under restricted aeration conditions. be able to.
Further, it can be increased by lengthening the time from the start of culture (at the time of inoculation) to the addition of the surfactant, and can be decreased by shortening the time from the start of culture to the addition of the surfactant. Further, it can be increased by slowing the charging rate of the surfactant and decreasing by increasing the charging rate.

In the method for producing a useful substance of the present invention, the production method for secretory production of a useful substance includes a microbial exocrine production method including the following steps (a) and (b). In the following steps, the step of secreting and producing useful substances is step (a).
Step (a): A step of simultaneously presenting a culture solution for culturing a microorganism (yeast or the like) that produces a useful substance and a surfactant to secrete the useful substance to the outside of the microorganism (in the culture solution).
Step (b): After step (a), a step of separating useful substances from the culture broth.

The following is an example of a method for producing a useful substance using the nonionic surfactant (A) of the present invention.
(I) Genetic recombination (i-1) A messenger RNA (mRNA) is isolated from cells expressing the target protein, a single-stranded cDNA is synthesized from the mRNA, and then a double-stranded DNA is synthesized, and the double-stranded DNA is synthesized. Incorporate strand DNA into phage DNA or plasmid. The obtained recombinant phage or plasmid is transformed into a microorganism to prepare a cDNA library.
(I-2) As a method for screening a phage DNA or a plasmid containing the target DNA, a hybridization method between the phage DNA or the plasmid and a DNA probe encoding a part of the target protein gene or complementary sequence can be mentioned. ..
(I-3) The target gene by cutting out the target cloned DNA or a part thereof from the phage or plasmid after screening and ligating the cloned DNA or a part thereof downstream of the promoter in the expression vector. Expression vector can be prepared. DNA encoding a signal sequence that translocates the intima (a signal sequence that expresses the target substance in the periplasm) can also be ligated at the same time.
(Ii) Culture (ii-1) The host microorganism is transformed with an expression vector to prepare a recombinant microorganism, and the recombinant microorganism is precultured. Preculture is usually carried out on an agar medium at 15-43 ° C. for 3-72 hours.
(Ii-2) The culture medium used for the production of useful substances is autoclaved at 121 ° C. for 20 minutes, and the recombinant microorganisms precultured on an agar medium are cultured here. Culturing is usually carried out at 15 to 43 ° C. for 12 to 72 hours. When the surfactant (A) is used at the same time as the start of culturing, the same operation is performed by using a homogenized mixture of the surfactant (A) and the culture broth as the culture broth. When the surfactant (A) is added 6 to 72 hours after the culture, the culture is continued for 1 to 1000 hours after the surfactant is added.
(Iii) Purification (iii-1) The protein secreted in the culture medium is separated from microorganisms and microbial residues by centrifugation, hollow fiber separation, filtration and the like.
(Iii-2) The culture solution containing the protein is repeatedly subjected to column treatments such as an ion exchange column, a gel filtration column, a hydrophobic column, an affinity column and an extraordinary column, and is subjected to a precipitation treatment such as ethanol precipitation, ammonium sulfate precipitation and polyethylene glycol precipitation. It is separated and purified by performing it as needed.

The host microorganism isolated in (iii-1) can then be further cultured by supplying a new culture solution. By further subjecting the culture solution or the like to the step (iii) and repeating purification and culturing, continuous production of useful substances can be performed.

When column treatment is performed in the above purification step (iii), examples of the filler used for column chromatography include silica, dextran, agarose, cellulose, acrylamide, vinyl polymer and the like, and commercially available products include the Capto series. Examples thereof include Sephadex series, Sephadex series, Sepharose series (hereinafter referred to as GE Healthcare), Bio-Gel series (Bio-Rad) and the like.

By using the method for producing a useful substance of the present invention, the useful substance produced by yeast can be secreted into the culture medium with high efficiency, and a high yield can be obtained in a short time. Can be achieved. Further, in the method for producing a useful substance of the present invention, since the useful substance is secreted into the culture broth, the purification of the useful substance is easy.

Since the useful substance obtained by the production method of the present invention is obtained by the above method, it has a higher specific activity than the conventional one.

The method for producing a useful substance of the present invention comprises a step of simultaneously presenting a surfactant and a microorganism to secrete the useful substance into a culture solution. In this step, it is presumed that as long as the microorganism is alive, the microorganism can produce a useful substance and secrete it into the culture medium. Further, if the microorganism has the ability to produce a useful substance, it is presumed that the production method of the present invention can be used regardless of the type of the useful substance to be produced.
The method for producing a useful substance of the present invention is particularly effective when the useful substance produced in the microorganism is transferred to the periplasm of the microorganism. The transition of useful substances to periplasm facilitates the secretion of useful substances into the culture medium.

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 mean parts by weight. Examples 5 to 10 and 15 to 30 are reference examples.

<Examples 1 to 10>
Luciferase-expressing yeast (Saccharomyces cerevisiae) was transformed using Saccharomyces cerevisiae distributed by (Biotechnology Center, Product Evaluation Technology Infrastructure Organization). The pYES2 vector (manufactured by Thermo) was cleaved with HindIII and XbaI, and an α-factor-luciferase gene having a HindIII and XbaI cleavage site was incorporated into both ends of the artificially synthesized (consigned synthesis by Thermo). Transformation of the α-factor-luciferase gene-incorporated pYES2 vector into a microorganism was carried out by the method described in the pYES2 vector instruction manual. Inoculate a luciferase-expressing microorganism into a YPD culture medium (Yeast extract 1 wt% manufactured by Difco, Bacto pepper 2 wt% manufactured by Difco, 2 wt% glucose) using a platinum loop and cultured at 30 ° C. for 15 hours at 200 rpm. The culture broth prepared in the above was resuspended in 125 ml YPD culture broth (pH 6 containing 100 mM potassium hydrogen phosphate buffer) so that the final turbidity (hereinafter sometimes abbreviated as OD) was 0.1 OD / ml.
The turbidity of the culture solution was measured under the same conditions as in the "method for measuring the turbidity of the culture solution" described later.
Further, the resuspension was continuously stirred and cultured at 30 ° C. for about 17 hours at 1100 rpm until the OD reached 20 to 30. When the OD reaches 20 to 30, the culture medium is centrifuged (1500 g, 10 minutes), and 125 ml of the galactose-induced culture solution (the above-mentioned YPD culture solution contains 100 mM potassium phosphate buffer, 2% instead of glucose). It was resuspended in pH 6) containing galactose and cultured at 30 ° C. and 1100 rpm for 2 hours. Then, the surfactants shown in Table 1 were added so as to have the weight% shown in Table 1, and the cells were cultured at 30 ° C. and 1100 rpm for 8 hours. Then, sampling was performed for measuring turbidity (turbidity at the end of culture), cells and culture supernatant were separated from the culture solution by a centrifuge, and the culture supernatant and cells were collected.

<Comparative Example 1>
In Example 1, the same procedure was carried out except that pure water was added instead of the surfactant, and the culture supernatant and the cells were collected.

<Examples 11 to 20>
Luciferase-expressing yeast (Pichia pastoris) was transformed using Pichia pastoris distributed by (Biotechnology Center, Product Evaluation Technology Infrastructure Organization). The pPICZα vector (manufactured by Thermo) was cleaved with XhoI and XbaI, and an artificially synthesized (consigned synthesis by Thermo) luciferase gene having XhoI and XbaI cleavage sites was incorporated into both ends. Transformation of the luciferase gene-incorporated pPICZα vector into a microorganism was carried out by the method described in the pPICZα vector instruction manual. A 125 ml BMGY culture was prepared by inoculating a luciferase-expressing microorganism into a BMGY culture solution using a loop loop and shaking the culture solution at 30 ° C. for 15 hours at 200 rpm so that the final OD was 0.1 OD / ml. Liquid (1 wt% Yeast extract manufactured by Difco, 2 wt% Bacto peptone manufactured by Difco, 1.34 wt% Yeast microbial base w / o amino acid 1.34 wt% manufactured by Difco, re-potassium hydrogen phosphate, pH 6.0). Suspended. Further, the resuspension was continuously stirred and cultured at 30 ° C. for about 17 hours at 1100 rpm until the OD reached 20 to 30. When the OD reached 20 to 30, the culture broth was centrifuged (1500 × g, 10 minutes), resuspended in 125 ml BMMY culture broth, and cultured at 30 ° C. and 1100 rpm for 2 hours. After 2 hours, the surfactants shown in Table 2 were added to the weight% shown in Table 2 and cultured at 30 ° C. and 1100 rpm for 8 hours. Then, sampling was performed for measuring turbidity (turbidity at the end of culture), and the cells and the culture supernatant were separated from the culture solution by a centrifuge, and the culture supernatant and the cells were collected.

<Comparative Example 2>
In Example 11, the same procedure was carried out except that pure water was added instead of the surfactant, and the culture supernatant and the cells were collected.

<Examples 21 to 30>
Acid phosphatase-expressing yeast (Candida boidinii, known in the literature (Regulation and evaluation of five methanol-inducible promoters in the methylotrophic yeast Candida boidinii, H. Yurimoto et al., Biochimica et Biophysica Acta, 1493, 2000, according to 56-63) The culture solution prepared by inoculating BMGY medium with platinum ears and shaking at 30 ° C. for 15 hours at 200 rpm was prepared so that the final OD was 0.1 OD / ml. It was resuspended in 125 ml BMGY medium. Further, the resuspension was continuously stirred and cultured at 30 ° C. for about 17 hours at 1100 rpm until the OD became 2 to 5. When the OD reached 2 to 5, methanol was added so that the OD was 2 v / v%, and the cells were cultured at 30 ° C. and 1100 rpm for 2 hours. After 2 hours, the surfactants listed in Table 3 were added by weight as shown in Table 3 and cultured at 30 ° C. and 1100 rpm for 8 hours. After 8 hours, sampling was performed for measuring turbidity (turbidity at the end of culture), cells and culture supernatant were separated from the culture solution by a centrifuge, and the culture supernatant and cells were collected.
The cells were washed by adding 50 mM NaBf acetate (pH 4.0) to the cells and centrifuging. The washed cells were resuspended with 50 mM NaBf acetate (pH 4.0) to prepare a sample for measuring acid phosphatase activity on the surface of the cells.

<Comparative Example 3>
In Example 21, the same procedure was carried out except that pure water was added instead of the surfactant, and the culture supernatant and the cells were collected.

In Tables 1 to 3, the following nonionic surfactants were used.
Nonionic Surfactant (A1-1): Sorbitan Palm Oil Fatty Acid Ester, HLB = 8.5
Nonion Surfactant (A2-1): Monococonut oil fatty acid ester of sorbitan EO 20 mol adduct, oleic acid esterified product of HLB = 16.3 mol adduct), HLB = 7.0
Nonionic Surfactant (A2-2): Monooleic Acid Ester of 20 mol Adduct of Sorbitan EO, HLB = 15.4 (Tween80)
Nonionic Surfactant (A2-3): Monolauric Acid Ester with 20 Mole Adduct of Sorbitan EO, HLB = 16.7 (Tween 20)
Nonionic Surfactant (A3-1): Polyalkylene Glycol (oleic acid esterified product of EO30 mol PO15, HLB = 7.0)
Nonionic Surfactant (A6-1): 4- (1,1,3,3-Tetramethylbutyl) Phenyl-Polyethylene Glycol, HLB = 13.5 (TritonX-100)
Nonionic Surfactant (A6-2): Octylphenyl-Polyethylene Glycol, HLB = 13.0 (NonidetP-40)

With respect to the culture supernatants obtained in Examples 1 to 30 and Comparative Examples 1 to 3, the turbidity of the culture solution, the amount of secreted production of useful substances, the secretory efficiency and the like were evaluated as described below. The results are shown in Tables 1-3.

<Measurement method of turbidity of culture solution>
The turbidity was measured using a turbidity meter [UV-1700, manufactured by Shimadzu Corporation] using a culture solution containing microorganisms recovered by sampling, using a quartz cell having an optical path length of 1 cm.
The culture broth was centrifuged at 1500 rpm, 5 minutes and 4 ° C., and the supernatant was discarded. The precipitate was resuspended in the same amount of physiological saline as the sample solution, diluted with physiological saline so as to have an appropriate absorbance (0.1 to 0.8), and the absorbance at 600 nm was measured. The turbidity of the culture solution was calculated by the following formula (1).
Turbidity of culture solution (OD) = (absorbance of diluted culture solution at 600 nm) × dilution ratio of culture solution (Formula 1)

<Evaluation of secretory production of useful substances: Measurement of luciferase activity of secreted and produced useful substances>
60 μl of the following luciferin solution was added to 20 μl of each culture supernatant obtained in Examples 1 to 20 and Comparative Examples 1 to 2 diluted 10 to 1000 times with 0.2 M Tris-HCl buffer (pH 7.4). Using the resulting mixed solution as a sample, the luminescence intensity measured under the following conditions using a luminometer was used as the value obtained by applying the following formula to the luciferase activity of the useful substance secreted and produced.
Luminometer: "Glomax Navigator" manufactured by Promega Co., Ltd.
Luciferin solution: New England Biolab Japan Co., Ltd. Product measurement temperature: 25 ° C
Detection device: Luminescent detector Detection wavelength: 350-700nm
[Luciferase activity of secreted and produced useful substances] = [Emission intensity] x Dilution ratio / [Turbidity at the end of culture]
In Tables 1 and 2, the luciferase activity was represented by a relative value when Comparative Example 1 or 2 was set to 1.
The higher the luciferase activity of the secreted and produced useful substance, the larger the amount of secreted and produced useful substance.

<Measurement of luciferase activity of useful substances remaining on the cell surface>
To the cells obtained in Examples 1 to 20 and Comparative Examples 1 and 2, 200 μl of 0.2 M Tris-HCl buffer (pH 7.4) in the same amount as the removed supernatant was added, and the cells were centrifuged to centrifuge. Was washed. The washed cells were resuspended in 200 μl of 0.2 M Tris-HCl buffer (pH 7.4) in the same amount as the washed supernatant, and 10 in 0.2 M Tris-HCl buffer (pH 7.4). A sample diluted ~ 1000 times was used as a sample for measuring the luciferase activity on the cell surface.
Using a mixed solution prepared by adding 60 μl of the above luciferin solution to 20 μl of the sample for measuring luciferase activity on the surface of the cells as a sample, the luminescence intensity was measured under the same conditions as in "Measurement of luciferase activity of secreted and produced useful substances", and the measured luminescence was measured. The intensity was applied to the following formula, and the value obtained was taken as the luciferase activity of the useful substance remaining on the cell surface.
[Luciferase activity of useful substances remaining on the cell surface] = [Luminescent value] x Dilution ratio / [Turbidity at the end of culture]
In Tables 1 and 2, the luciferase activity was represented by a relative value when Comparative Example 1 or 2 was set to 1.

<Evaluation of secretory efficiency>
Using the culture supernatants and cells obtained in Examples 1 to 20 and Comparative Examples 1 and 2, the secretory efficiency in each example was calculated by the following formula.
Secretory efficiency (%) = 100 x "Luciferase activity of secreted and produced useful substances" / "Total luciferase activity"
The total luciferase activity means "luciferase activity of secreted and produced useful substance" + "luciferase activity of useful substance remaining on the cell surface".

<Measurement of protein amount: Acid phosphatase activity measurement>
To each of the culture supernatants obtained in Examples 21 to 30 and Comparative Example 3, 1/40 amount of 2M sodium acetate buffer (pH 4.0) was added to 5 μl. A substrate solution (p-nitrophenylphosphate 0.64 mg / ml-containing 50 mM sodium acetate buffer pH 4.0) was added to the culture supernatant to which the buffer was added or each cell surface measurement sample obtained in Examples 21 to 30 by volume ratio = 1. The mixture was mixed at 1: 1 and reacted at 35 ° C. for 10 minutes.
After the reaction, an equal amount of 10% trichloroacetic acid solution as the reaction solution was added to terminate the reaction. To the solution in which the reaction was stopped, an equal amount of a saturated solution of sodium carbonate was added to the whole reaction solution to develop a color. The insoluble fraction was removed from the color-developed liquid at 1500 g in 5 minutes, and the absorbance at 420 nm was measured using (Biotek microplate reader PowerWave). The reference wavelength was 630 nm.
The acid phosphatase activity was defined as the value calculated from the following formula for each of the culture supernatant and the cell surface measurement sample. In the result of the acid phosphatase activity of the obtained culture supernatant, the relative value when the value of Comparative Example 3 was set to 1 was defined as the "acid phosphatase activity of the secreted and produced useful substance", and the acid phosphatase activity of the cell surface measurement sample. In the results of, the relative value when the value of Comparative Example 3 was set to 1 is shown in Table 3 as "acid phosphatase activity of useful substances remaining on the cell surface".
Acid phosphatase activity = {(Abs420nm)-(Abs630nm)} / (turbidity at the end of culture)

<Evaluation of secretory efficiency>
Using the values of "acid phosphatase activity of the useful substance secreted and produced" and "acid phosphatase activity of the useful substance remaining on the cell surface" obtained in Examples 21 to 30 and Comparative Example 3, the secretory efficiency was calculated from the following formula. Calculated. The results are shown in Table 3.
Secretory efficiency (%) = 100 x "acid phosphatase activity of secreted and produced useful substances" / "total acid phosphatase activity"
The total acid phosphatase activity means "acid phosphatase activity of the useful substance secreted and produced" + "acid phosphatase activity of the useful substance remaining on the cell surface".

From Tables 1 to 3, by containing the nonionic surfactant (A) in the culture medium, the luciferase activity or acid phosphatase activity of the secreted and produced useful substance is increased, and the secretory efficiency of the useful substance to the outside of the microorganism is increased. Can be seen to be rising.
Further, in Examples 1 to 30, by using the nonionic surfactant (A), the turbidity is equal to or higher than that of Comparative Examples 1 to 3, and therefore the bacterial growth is high. You can see that. Further, since the total luciferase activity or the total acid phosphatase activity is equal to or higher than that, it can be seen that the production amount of the useful substance per microbial bacterium is equal to or higher than that, and the obtained useful substance is not easily denatured.
Therefore, it can be seen that a large amount of useful substances can be obtained by using the method for producing useful substances of the present invention.

The method for producing a useful substance of the present invention can be used when producing a useful substance such as protein in yeast. Examples of proteins include enzymes, hormonal proteins, antibodies and peptides. When the protein produced is an enzyme (protease, cellulase, lipase, amylase, etc.), it can be suitably used for food processing, cleaning agents, fiber processing, papermaking, enzyme conversion, etc., especially biopharmaceuticals. It is useful for use in the production of.

Claims (4)

A method for producing a useful substance by secreting and producing a useful substance in the culture solution by yeast contained in the culture solution, wherein the yeast is at least one selected from the group consisting of the genus Saccharomyces and the genus Pichia, and the culture solution. The nonionic surfactant (A) is contained therein, (A) is a fatty acid ester of an aliphatic polyhydric alcohol (A1 ), and the content of the nonionic surfactant (A) in the culture broth starts culturing. A method for producing a useful substance, which is 0.003 to 5% by weight based on the weight of the culture solution at the time . The method for producing a useful substance according to claim 1 , wherein the nonionic surfactant (A) has an HLB of 6 to 20. The method for producing a useful substance according to claim 1 or 2, wherein at least one of the fatty acids constituting the fatty acid ester (A1 ) of the aliphatic polyhydric alcohol has 8 to 22 carbon atoms. The method for producing a useful substance according to any one of claims 1 to 3 , wherein the useful substance is a protein.