How proteins are made

Abstract

The present invention provides a method for producing protein in a microbiological manner inexpensively and efficiently. A method for producing protein comprises: culturing a microorganism in the presence of a glycosylamine.

Description

Technical Field

[0001] The present invention relates to a method for producing a protein using a microorganism. Background Art

[0002] Since filamentous fungi produce a variety of cellulases and xylanases, they have attracted much attention as plant polysaccharide degrading bacteria. Among them, Trichoderma can produce cellulases and xylanases simultaneously and in large quantities, and therefore has been studied as a microorganism for producing cellulase-based biomass degrading enzymes.

[0003] In the cultivation of microorganisms, glucose is currently used as a carbon source. However, in the presence of glucose, a control mechanism known as catabolite repression is used to produce a reduction or saturation in the material productivity of the microorganism. It is reported that wide-area control transcription factors CreA, or CreB, CreC, CreD, etc. are involved in the catabolite repression of filamentous fungi such as Aspergillus (Aspergillus) (patent document 1). It is possible to regulate the catabolite repression of Aspergillus by the control of these transcription factors, but sufficient results have not yet been obtained. Regarding Trichoderma, the mechanism analysis of catabolite repression is also being carried out (patent document 2, non-patent document 1). However, there are still many unclear places in the mechanism of catabolite repression of Trichoderma, and repression has not been avoided.

[0004] In addition, in the production of proteins such as enzymes using microorganisms, in addition to carbon sources, inducing substances are sometimes required. For example, the expression of the α-amylase gene of Aspergillus oryzae is induced by starch or maltose. In addition, the expression of the main cellulase genes cbh1, cbh2, egl1 and egl2 of Trichoderma is induced by cellulose, cellobiose, etc. (Non-patent Document 2). Starch or maltose, cellulose, cellobiose can also be used as a carbon source for culture. In the production of cellulase using microorganisms, microcrystalline cellulose such as Avicel is generally used.

[0005] Patent Document 3 discloses a method for producing cellulase using microorganisms, which uses lignocellulose as an inducing substance instead of pure cellulose. In Patent Document 3, in a culture medium containing a cellulose-based material, glucose as a carbon source and phosphoric acid or ammonia water as a pH regulator are added separately, while Trichoderma is cultured to produce cellulase. Non-Patent Document 3 discloses that while repeatedly or continuously adding sugars to the culture medium, coryneform bacteria are made to produce amino acids. Patent Document 4 and Non-Patent Document 4 disclose that a mixed solution of glucose and ammonia is added to the culture, and lysine is fermented and produced by controlling the pH value while adding a carbon source and a nitrogen source. By this method, the productivity of lysine is increased.

[0006] Glycosylamine is a monosaccharide derivative in which the hydroxyl group at the C-1 position is substituted by an amino group. In many cases, it is synthesized and used as an intermediate of various sugar derivatives. It is reported that β-D-glycosylamine acts as an inhibitor of β-glycosidase (Non-Patent Document 5). Non-Patent Document 6 records that β-D-glycosylamine is generated by the reaction of glucose and ammonia. Non-Patent Document 7 records that various D-glucose derivatives containing β-D-pyranose glucoamine can be used as a carbon source for the hyphae growth of Candida albicans.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent Application Publication No. 2015-39349

[0010] Patent Document 2: Japanese Patent Publication No. 11-512930

[0011] Patent Document 3: U.S. Patent Publication No. 2008 / 0199908

[0012] Patent Document 4: China Patent Grant No. 101967501

[0013] Non-patent literature

[0014] Non-patent document 1: Appl. Environ. Microbiol., 63: 1298-1306 (1997)

[0015] Non-patent document 2: Curr. Genomics, 14: 230-249 (2013)

[0016] Non-patent document 3: Journal of Biotechnology, 104: 155-172 (2003)

[0017] Non-patent document 4: The Chinese Journal of Process Engineering, 11(3):492-496(2011)

[0018] Non-patent document 5: Adv. Carbohydr. Chem. Biochem., 48: 319-384 (1990)

[0019] Non-patent document 6: Journal of the Japanese Society of Agricultural Chemistry, 36: 305-310 (1962)

[0020] Non-patent document 7: Journal of Basic Microbiology, 23(5):303-312 (1983) Summary of the invention

[0021] The present invention provides a method for producing a protein, comprising: a step of culturing a microorganism in the presence of a glycosylamine. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The values ​​represent the relative values ​​on the second, third, or fourth day of culture, when the protein production on the third day of culture in Comparative Example 1 is set as 100%.

[0023] Figure 2 The values ​​are relative values ​​when the protein production on the third day of culture in Comparative Example 1 is set as 100%. DETAILED DESCRIPTION

[0024] All patent documents, non-patent documents and other publications cited in this specification are incorporated herein by reference in their entirety.

[0025] In this specification, "upstream" and "downstream" related to a gene refer to the upstream and downstream of the transcription direction of the gene. For example, "a gene arranged downstream of a promoter" means that the gene exists on the 3' side of the promoter in the sense strand of DNA, and the upstream of a gene refers to the region on the 5' side of the gene in the sense strand of DNA.

[0026] In this specification, "operably linked" a promoter and a gene means that the promoter and the gene are linked in such a way that the promoter can induce transcription of the gene. The steps of "operably linking" a promoter and a gene are well known to those skilled in the art.

[0027] In the present specification, "promoter activity" refers to an activity that promotes the expression of a gene located downstream thereof, and more specifically, refers to an activity that promotes transcription from DNA of a gene located downstream to mRNA.

[0028] In this specification, the term "originally" used for the function, property, or morphology of a cell is used to indicate that the function, property, or morphology originally exists in the cell. In contrast, the term "exogenous" is used to indicate that the function, property, or morphology does not originally exist in the cell, but is introduced from the outside. For example, an "exogenous" gene or polynucleotide refers to a gene or polynucleotide that is introduced into a cell from the outside. An exogenous gene or polynucleotide may be derived from an organism of the same species as the cell into which it is introduced, or may be derived from an organism of a different species (i.e., a gene or polynucleotide of a different species).

[0029] In the present specification, "at least 90% identity" with respect to an amino acid sequence or a nucleotide sequence means: 90% or more, preferably 95% or more, more preferably 96% or more, further preferably 97% or more, further more preferably 98% or more, and further preferably 99% or more identity.

[0030] In the present specification, the identity of nucleotide sequences and amino acid sequences was calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, the identity was calculated by setting the unit size to compare (ktup) to 2 using the homology analysis (Search homology) program of the genetic information processing software Genetyx-Win.

[0031] There is a demand for developing a culture technology for producing proteins such as enzymes cheaply and efficiently using microorganisms. By repeatedly or continuously adding glucose to a culture in a fed-batch culture, catabolite repression is suppressed, and there is a possibility that the protein productivity of the microorganism can be improved. However, when there is little glucose, the protein synthesis process does not proceed sufficiently, and on the other hand, the addition of a large amount of glucose causes catabolite repression. Therefore, in the production of proteins using glucose fed-batch culture, precise control of the glucose concentration in the culture is required.

[0032] The inventors of the present invention have found that the productivity of proteins such as enzymes using microorganisms is improved by culturing microorganisms in the presence of glycosylamines.

[0033] According to the present invention, the productivity of protein using microorganisms can be improved through simple steps.

[0034] The method for producing the protein of the present invention comprises the step of culturing a microorganism in the presence of a glycosylamine. Examples of the glycosylamine used in the present invention include β-D-glucose pyranosamine (CAS No. 7284-37-9), β-D-mannopyranosamine (CAS No. 7388-99-0), β-D-galactopyranosamine (CAS No. 74867-91-7), and the like, and any one of them or a combination of two or more thereof can be used. Preferably, the glycosylamine used in the present invention is β-D-glucose pyranosamine, which is a compound in which the hydroxyl group at the C-1 position of β-D-glucose is substituted with an amino group. Glycosylamines are commercially available, or can be produced by referring to the steps described in J. Org. Chem, 1958, 23 (9): 1309-1319.

[0035] In one embodiment of the method of the present invention, a microorganism is cultured with a culture medium containing glycosylamine. In one example, glycosylamine is added to the initial culture medium for culture, and the microorganism is cultured using the culture medium. The amount of glycosylamine added to the initial culture medium is preferably 50 g or less per 1L of initial culture medium, more preferably 30 g or less, more preferably 0.5 to 20 g, and more preferably 1 to 10 g. In another example, glycosylamine is added to the culture of the microorganism. The amount of glycosylamine added relative to the culture is preferably 5 g / hr or less per 1L of initial culture medium, more preferably 4 g / hr or less, more preferably 0.005 to 4 g / hr, and more preferably 0.01 to 3 g / hr. Alternatively, the above-mentioned addition of glycosylamine to the initial culture medium and the addition of glycosylamine to the mixture can also be combined. At this time, the amount of glycosylamine added to the initial culture medium and the amount of glycosylamine fed are preferably adjusted so that the content of glycosylamine in the culture is preferably 0.5 to 50 g, more preferably 0.5 to 20 g, and even more preferably 1 to 10 g per 1 L of the culture. The fed feed may be intermittent, and the flow rate may be changed. The conditions of the fed feed are set based on consideration of the productivity of the protein.

[0036] In the method of the present invention, when the glycosylamine is added to the culture of the microorganism, it is preferred that an aqueous solution containing the glycosylamine is added to the culture. The content of the glycosylamine in the aqueous solution is preferably 2 to 90% by mass, more preferably 2 to 50% by mass. When the amount of the glycosylamine in the aqueous solution is too small, a large amount of aqueous solution will be added to the mixed flow, thereby placing a burden on the culture equipment. On the other hand, when the amount of the glycosylamine in the aqueous solution is too much, it becomes difficult to control the amount of the aqueous solution added to the mixture.

[0037] The aqueous solution containing glycosylamine can be an aqueous solution obtained by directly adding glycosylamine to water and dissolving it, or it can be a mixture of glucose and ammonia (containing glycosylamine generated by the reaction of glucose and ammonia, refer to non-patent document 6). Therefore, as an embodiment, the method of the present invention may include the step of culturing microorganisms while flowing the mixture of glucose and ammonia. The mixture of glucose and ammonia is a liquid mixture prepared by mixing glucose and ammonia. For example, the mixture is a mixture of glucose, ammonia and their solvents (such as water). Preferably, the mixture is a mixture prepared by mixing glucose or its aqueous solution with ammonia, its salt or their aqueous solution, and water as needed. More preferably, the mixture is a mixture obtained by mixing glucose and an aqueous ammonia solution and water as needed. When preparing the mixture of glucose and ammonia, glucose and ammonia are preferably mixed at a mass ratio of 0.5 to 10:1, more preferably 2 to 8:1. When the ratio of glucose is too high, the cells change from the enzyme production state to the proliferation state, and there is a tendency for the enzyme productivity to decrease. On the other hand, when the ratio of glucose is too low, the carbon source becomes insufficient, and as a result, the improvement of the enzyme productivity cannot be expected. In addition, when preparing the mixture of glucose and ammonia, the amount of glucose added is adjusted to preferably 2 to 90 g, more preferably 5 to 80 g per 100 mL of the obtained mixture. When the amount of glucose added to the mixture is too little, in order to achieve the above-mentioned flow addition amount of glycosylamine, a large amount of mixture will be added to the mixture flow, therefore, causing a burden on the culture equipment. On the other hand, when the amount of glucose added to the mixture is too much, it becomes difficult to control the flow addition amount of the mixture of the culture. For example, the mixture can be prepared by mixing an appropriate amount of glucose and an aqueous ammonia solution in a manner that can achieve the above-mentioned glucose: ammonia ratio and / or the amount of glucose added, or, it can also be prepared by mixing an arbitrary amount of glucose and an aqueous ammonia solution, and then diluting the obtained aqueous solution with appropriate water in a manner that can achieve the above-mentioned glucose: ammonia ratio and / or the amount of glucose added.

[0038] When the mixture of glucose and ammonia is added to the culture of the microorganism, the amount of the mixture added relative to the culture can be an amount within the range of the amount of glycosylamine added as described above. For example, the amount of glucose added to the mixture per 1L of the initial culture medium (the culture medium not containing the mixture added), is preferably 8 g / hr or less, more preferably 6 g / hr or less, further preferably 0.05 to 8 g / hr, and further preferably 0.1 to 6 g / hr. When the amount of glucose added is large, catabolite repression may sometimes be caused. On the other hand, the amount of the mixture (whole) added relative to the culture can be appropriately adjusted according to the content of glucose or ammonia, or the pH of the culture described later, etc., and is not particularly limited. From the viewpoint of economic efficiency, the amount of the mixture (whole) added per 1L of the initial culture medium is adjusted to about 0.1 to 10 g / hr, preferably about 0.3 to 8 g / hr.

[0039] The pH of the culture is preferably adjusted by adding the mixture of glucose and ammonia. At this time, the amount and time of the addition of the mixture depend on the pH of the culture to which the mixture is added. It is preferred to prepare an initial culture medium of a specified pH using the usual steps, and then, the culture is cultured while adding the mixture of glucose and ammonia in such a manner that the culture after the addition maintains the specified pH value. It is preferred to use only the mixture of glucose and ammonia in the adjustment of the pH of the culture during the culture, but other pH regulators may also be used in combination. The determination of the pH of the culture and the control of the amount of addition based on the pH value can be carried out using a commercially available fermenter (jar fermenter) or the like. The pH of the culture can be set to an appropriate value depending on the type of microorganism or the type of protein produced. For example, when the microorganism is a filamentous fungus, the pH of the culture is preferably maintained at pH 3 to 7, more preferably pH 3.5 to 6. The pH of the culture can usually be measured using an electrode installed in the fermenter. Preferably, the pH of the culture in the present invention refers to a value measured at a culture temperature of 28° C. using a pH sensor such as F-635 Autoclavable pH Electrode (Broadley-James Corp) or 405-DPAS-SC-K8S pH sensor (METTLER TOLEDO).

[0040] From the viewpoint of the stability of the glycosylamine, the pH of the aqueous solution containing the glycosylamine is preferably alkaline at 25°C, more preferably pH 8 or more, further preferably pH 9 or more, further preferably pH 10 or more, and preferably pH 13 or less. When the aqueous solution containing the glycosylamine is a mixture of glucose and ammonia, the pH of the mixture of glucose and ammonia is preferably alkaline at 25°C, more preferably pH 8 or more, further preferably pH 9 or more, further preferably pH 10 or more, and preferably pH 13 or less. It is preferred to adjust the pH of the aqueous solution containing the glycosylamine to the above range using a pH adjuster such as an alkali agent (ammonia, etc.) as needed. The pH of the aqueous solution containing the glycosylamine can be measured using a general pH meter. For example, a pH meter (HORIBALTd. pH / ion meter F-52, etc.) connected to a pH measurement composite electrode (HORIBALTd. glass sliding sleeve type, etc.) is used. As the internal liquid of the pH electrode, a saturated potassium chloride aqueous solution (3.33 mol / L) is used. The measurements were performed at 25°C.

[0041] The aqueous solution containing the glycosylamine may further contain other substances that can be usually added to the culture medium of microorganisms. Examples of the other substances include organic salts, inorganic salts, pH regulators, carbon sources or nitrogen sources other than glycosylamine, surfactants, defoamers, etc. Preferably, glucose is used as a carbon source, and ammonia is preferably used as a nitrogen source. Ammonia can also be used as a pH regulator. One or more selected from glucose and ammonia can be contained in part or all of the initial culture medium, or can also be fed. In the case of feeding one or more of glucose and ammonia, the flow acceleration of the aqueous solution containing them is preferably a speed at which the combined flow addition amount of glycosylamine, glucose and ammonia does not exceed the flow addition amount of the above-mentioned glycosylamine. In addition, the total amount of glucose, ammonia and glycosylamine in the initial culture medium and the culture is also preferably not more than the content of the above-mentioned glycosylamine in the initial culture medium and the culture.

[0042] The mixture of glucose and ammonia may also further contain other substances that can be usually added to the culture medium of microorganisms. As examples of these other substances, preferably, substances that do not damage the pH regulating function of the mixture, such as organic salts, inorganic salts, other pH adjusting agents, carbon sources or nitrogen sources other than glucose and ammonia, surfactants, defoamers, etc. are enumerated.

[0043] In the method of the present invention, the initial culture medium used in the cultivation of the microorganism can be any culture medium commonly used in the cultivation of the microorganism. For example, the initial culture medium can contain various components commonly contained in the culture medium of the microorganism such as carbon source, nitrogen source, magnesium salt, zinc salt and other metal salts, sulfate, phosphate, pH regulator, surfactant, defoamer, etc. The composition of the components in the culture medium can be appropriately selected. The initial culture medium can be any one of a synthetic culture medium, a natural culture medium, a semi-synthetic culture medium, or a commercially available culture medium. The initial culture medium is preferably a liquid culture medium.

[0044] It is preferred that the cultivation of the microorganism in the method of the present invention is carried out in the presence of an inducing substance for the expression of the gene encoding the target protein. Thus, the productivity of the target protein can be further improved. In more detail, the culture of the microorganism only needs to contain the inducing substance. For example, the inducing substance can be contained in the initial culture medium, can be added to the culture, or can be both. As preferred examples of the inducing substance, cellulose and cellobiose can be cited, and either or both of them can be used. The cellulose or cellobiose in the culture not only acts as an inducing substance, but also can be consumed as a carbon source. The concentration of cellulose or cellobiose in the culture is preferably 1 to 15 mass / volume% as the total concentration of cellulose and cellobiose. For example, when cellulose or cellobiose is added to the initial culture medium, the concentration of cellulose or cellobiose in the initial culture medium is preferably 1 to 15 mass / volume% as the total concentration of cellulose and cellobiose. It is known that the expression of cellulase genes of filamentous fungi such as Trichoderma is induced by cellulose, cellobiose, etc., and is repressed by catabolites through glucose (Patent Document 2, Non-Patent Documents 1 to 2). Therefore, the method of the present invention using the inducing substance can be preferably used for the production of cellulase using filamentous fungi.

[0045] Alternatively, by linking the gene encoding the target protein to a promoter induced by cellulose or cellobiose, such as a promoter of a gene encoding a cellulase possessed by filamentous fungi (so-called filamentous fungus cellulase gene promoter), the expression of the gene encoding the target protein can be induced by cellulose or cellobiose. Therefore, in a preferred embodiment of the method of the present invention, the microorganism is cultured in the presence of at least one selected from cellulose and cellobiose, and the microorganism contains a promoter induced by cellulose or cellobiose, and the gene encoding the target protein is operably linked downstream of the promoter.

[0046] As examples of promoters induced by cellulose or cellobiose, promoters of filamentous fungal cellulase genes or xylanase genes can be cited. As examples of promoters of filamentous fungal cellulase genes, promoters of cellulase genes of Trichoderma can be cited, and as preferred examples, promoters of cellulase genes of Trichoderma reesei consisting of the nucleotide sequence of sequence number 1 or 2 can be cited. As examples of promoters of filamentous fungal xylanase genes, promoters of xylanase genes of Trichoderma can be cited, and as preferred examples, promoters of xylanase genes of Trichoderma reesei consisting of the nucleotide sequence of sequence number 3 can be cited. As further examples of promoters induced by cellulose or cellobiose, polynucleotides consisting of a nucleotide sequence having at least 90% identity with any one of the nucleotide sequences of sequence numbers 1 to 3 and having promoter activity induced by cellulose or cellobiose can be cited.

[0047] The promoter can be a promoter originally possessed by the microorganism cultivated by the method of the present invention, or can be an external promoter introduced into the microorganism. The connection of these promoters and the gene encoding the target protein can be carried out by known steps such as endonuclease method and homologous recombination method. For example, a vector or DNA fragment having a polynucleotide containing the gene encoding the target protein is introduced into a microbial cell, and the polynucleotide is inserted downstream of the target promoter in the genome, so that the promoter and the gene encoding the target protein are operably linked on the genome. Alternatively, a vector or DNA fragment having a promoter sequence and a polynucleotide containing the gene encoding the target protein linked to its downstream can also be constructed, and the vector or fragment is introduced into a microbial cell. As required, the vector or DNA fragment can also have selection markers such as antibiotic resistance genes and nutrient requirement-related genes.

[0048] The vector may be a plasmid or other vector that can self-propagate and replicate outside the chromosome, or may be a vector that is inserted into the chromosome. The preferred type of vector depends on the type of microorganism into which it is introduced. For example, as a preferred example of a vector for filamentous fungi, a plasmid containing AMA1 that acts as a self-replication factor in Aspergillus microorganisms may be cited, but is not limited to these.

[0049] In order to introduce a vector or a DNA fragment into a microorganism, a general transformation method such as electroporation, transformation, transfection, conjugation, protoplast method, particle gun method, Agrobacterium-mediated method and the like can be used.

[0050] The microorganisms introduced with the target vector or DNA fragment can be selected using a selection marker. For example, when the selection marker is an antibiotic resistance gene, the microorganisms introduced with the target vector or DNA fragment can be selected by culturing the microorganism in a culture medium to which the antibiotic has been added. In addition, for example, when the selection marker is a nutrient requirement-related gene such as an amino acid synthesis-related gene or a base synthesis-related gene, after gene introduction in the host of the nutrient requirement, the microorganisms introduced with the target vector or DNA fragment can be selected with the presence or absence of the nutrient requirement as an indicator. Alternatively, the introduction of the target vector or DNA fragment can be confirmed by investigating the DNA sequence of the microorganism using PCR, etc.

[0051] The gene encoding the target protein linked to the promoter can be linked to a secretion signal peptide. By linking to the secretion signal peptide, the expressed target protein is secreted outside the cell, so the target protein can be separated from the culture supernatant without destroying the microbial cells. Examples of secretion signal peptides include cellobiohydrolase 1 of Trichoderma reesei, α-amylase of Aspergillus oryzae, glucoamylase of Rhizopus oryzae, or a signal peptide derived from α-factor of Saccharomyces cerevisiae.

[0052] Examples of target proteins produced by the method of the present invention include oxidoreductase, transferase, hydrolase, lyase, isomerase, synthetase, glycolytic enzyme, lactate synthase (LDH, etc.), enzyme of TCA cycle, etc. The target protein may be a protein originally possessed by the microorganism cultured by the method of the present invention, or an external protein. Preferred examples of target proteins include enzymes related to biomass decomposition or biomass saccharification. Examples of enzymes related to biomass decomposition or biomass saccharification include cellulases (e.g., β-endoglucanase, cellobiohydrolase, β-glucosidase, etc.), hemicellulases (e.g., endoxylanase, β-xylosidase, arabinofuranosidase, glucuronidase, acetylxylanesterase, mannanase, β-mannosidase, ferulic acid esterase, etc.), xylanases, etc., among which cellulases are preferred. The enzymes related to biomass decomposition or biomass saccharification are preferably derived from filamentous fungi, more preferably from Trichoderma.

[0053] Examples of microorganisms that can be cultured by the method of the present invention include bacteria, yeast, and filamentous fungi. Among them, filamentous fungi are preferred. Examples of filamentous fungi include Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromycetes, and others. s), Pleurotus, Rhizopus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes and Trichoderma, among which Acremonium, Aspergillus , Chrysosporium, Fusarium, Humicola, Myceliophthora, Neurospora, Penicillium, Piromyces, Talaromyces, Thermoascus, Thielavia and Trichoderma, more preferably Trichoderma. As Trichoderma, preferably Trichoderma reesei and its variants. As examples of Trichoderma reesei and its variants, Trichoderma reesei QM9414 strain and its variants can be listed.

[0054] The various conditions for culturing the microorganism in the method of the present invention are suitable for the type of the microorganism or the scale of the culture, and can be appropriately set according to conventional methods. For example, when the microorganism is a filamentous fungus, the pH of the culture is preferably maintained at pH 3 to 7, more preferably pH 3.5 to 6. The method for measuring the pH of the culture is as described above.

[0055] Next, the target protein is recovered from the culture. When the protein is secreted in the culture supernatant, the protein can be recovered from the culture supernatant. When the protein is contained in cells, the cells are destroyed and the components containing the protein are taken out for protein recovery. Protein recovery can be carried out by methods commonly used in the field, such as decantation, membrane separation, centrifugation, electrodialysis, use of ion exchange resins, distillation, salting out, etc., or a combination thereof. The recovered target protein can also be further separated or purified.

[0056] When the target protein is secreted in the culture supernatant, the microorganism used in the production of the protein in the present invention can be reused. That is, the microbial cells separated from the culture supernatant are recovered and cultured again in the presence of glycosylamine as described above, thereby producing the target protein again.

[0057] The method for producing a protein of the present invention may be a batch method in which the cultivation of microorganisms, the recovery of proteins accumulated in the culture, and the replacement of the culture medium are performed alternately, or may be a semi-batch or continuous method in which the cultivation of microorganisms and the recovery of proteins are performed simultaneously while a part of the microorganisms and the culture medium are replaced intermittently or continuously.

[0058] As exemplary embodiments of the present invention, the following substances, production methods, uses, methods, etc. are further disclosed in this specification. However, the present invention is not limited to these embodiments.

[0059] [1] A method for producing a protein, comprising: culturing a microorganism in the presence of a glycosylamine.

[0060] [2] The production method according to [1], wherein the microorganism is preferably cultured in the presence of at least one selected from cellulose and cellobiose.

[0061] [3] The production method according to [2], wherein the microorganism preferably contains a cellulose-inducible promoter or a cellobiose-inducible promoter,

[0062] The promoter is preferably a promoter of a filamentous fungus cellulase gene or a promoter of a filamentous fungus xylanase gene.

[0063] More preferably, it is a promoter of a cellulase gene of Trichoderma or a promoter of a xylanase gene of Trichoderma.

[0064] [4] The production method according to [3], wherein a gene encoding the protein is preferably linked downstream of the promoter.

[0065] [5] The production method according to [4], wherein the promoter is preferably a filamentous fungal cellulase gene promoter, and the protein is an enzyme.

[0066] [6] The production method according to [4] or [5], wherein the protein is preferably an enzyme, preferably an enzyme involved in biomass decomposition or biomass saccharification, more preferably cellulase,

[0067] The enzyme involved in the biomass decomposition or biomass saccharification is preferably an enzyme derived from filamentous fungi, more preferably an enzyme derived from Trichoderma, further preferably a cellulase derived from filamentous fungi, further preferably a cellulase derived from Trichoderma.

[0068] [7] The production method according to any one of [1] to [6], wherein the glycosylamine is preferably added to the initial culture medium.

[0069] [8] The production method according to [7], wherein the amount of the glycosylamine added is preferably 50 g or less, more preferably 30 g or less, further preferably 0.5 to 50 g, further preferably 0.5 to 30 g, further preferably 0.5 to 20 g, further preferably 1 to 10 g per 1 L of the initial culture medium.

[0070] [9] The production method according to any one of [1] to [8], wherein the glycosylamine is preferably fed to the culture.

[0071]

[10] The production method according to [9], wherein the flow rate of the above-mentioned glycosylamine is preferably 5 g / hr or less, more preferably 4 g / hr or less, further preferably 0.005 to 4 g / hr, and further preferably 0.01 to 3 g / hr per 1 L of the initial culture medium.

[0072]

[11] The production method according to any one of [1] to

[10] , wherein the glycosylamine is preferably added to an initial culture medium and fed to the culture, and the content of the glycosylamine in the culture is preferably 0.5 to 50 g, more preferably 0.5 to 20 g, and even more preferably 1 to 10 g per 1 L of the culture.

[0073]

[12] The production method according to any one of [9] to

[11] , wherein an aqueous solution containing the above-mentioned glycosylamine is preferably fed, and the content of the glycosylamine in the aqueous solution is preferably 2 to 90% by mass, more preferably 2 to 50% by mass.

[0074]

[13] The production method according to

[12] preferably includes the step of culturing the microorganism while feeding the mixture of glucose and ammonia.

[0075]

[14] The production method according to

[13] , wherein the mixture is converted into the amount of glucose to be added to the mixture, and the mixture is added at a rate of preferably 8 g / hr or less, more preferably 6 g / hr or less, further preferably 0.05 to 8 g / hr, and further preferably 0.1 to 6 g / hr per 1 L of initial culture medium.

[0076]

[15] The production method according to

[13] or

[14] , wherein the pH of the culture is adjusted to preferably pH 3 to 7, more preferably pH 3.5 to 6, by feeding the mixture.

[0077]

[16] The production method according to any one of

[13] to

[15] , wherein the mixture is a mixture containing glucose and ammonia at a mass ratio of preferably 0.5 to 10:1, more preferably 2 to 8:1.

[0078]

[17] The production method according to any one of

[13] to

[16] , wherein the mixture preferably contains 2 to 90 g of glucose per 100 mL.

[0079]

[18] The production method according to any one of

[12] to

[17] , wherein the pH of the aqueous solution containing the above-mentioned glycosylamine or the above-mentioned mixture of glucose and ammonia at 25°C is preferably alkaline, more preferably above pH 8, further preferably above pH 9, further preferably above pH 10, and preferably below pH 13.

[0080]

[19] The production method according to any one of [1] to

[18] , wherein the glycosylamine is preferably at least one selected from β-D-pyranoglucopyranosamine, β-D-mannopyranosamine and β-D-galactopyranosamine, and more preferably β-D-pyranoglucopyranosamine.

[0081]

[20] The production method according to any one of [1] to

[19] , wherein during the culture, the pH of the culture is adjusted to preferably pH 3 to 7, more preferably pH 3.5 to 6.

[0082]

[21] The production method according to any one of [1] to

[20] , wherein the microorganism is a filamentous fungus.

[0083]

[22] The production method according to

[21] , wherein the filamentous fungus is a Trichoderma fungus.

[0084]

[23] The production method according to

[22] , wherein the Trichoderma fungus is Trichoderma reesei.

[0085]

[24] The production method according to any one of [2] to

[23] , wherein the microorganism is preferably a filamentous fungus, and the protein is preferably an enzyme related to biomass decomposition or biomass saccharification, and more preferably cellulase.

[0086]

[25] The production method according to

[24] , wherein the filamentous fungus is preferably a Trichoderma fungus.

[0087]

[26] The production method according to

[25] , wherein the Trichoderma fungus is Trichoderma reesei.

[0088]

[27] The production method according to any one of [2] to

[26] , wherein the concentration of cellulose or cellobiose in the culture is preferably 1 to 15 mass / volume % as the total concentration of cellulose and cellobiose.

[0089]

[28] The production method according to any one of [1] to

[27] , preferably further comprising: a step of recovering the protein from the culture of the above-mentioned microorganism.

[0090] Example

[0091] The present invention is further described in detail below based on examples, but the present invention is not limited to the examples. It should be noted that, in the following examples, "%" means "w / v%" unless otherwise specified. The pH of the culture was measured using a pH sensor such as a F-635 Autoclavable pH Electrode (Broadley-James Corp) or a 405-DPAS-SC-K8S pH sensor (METTLER TOLEDO) installed in a fermenter.

[0092] Reference Example 1 Synthesis of β-D-glucopyranosamine

[0093] 80 g of D-glucose and 2 g of ammonium chloride were added to 200 mL of methanol, and ammonia gas was bubbled in until dissolved at 13°C. After dissolution, crystals were obtained by standing at 4°C. The obtained crystals were recovered by filtration, washed with methanol, and dried to obtain β-D-pyranose glucopyranosamine. The obtained β-D-pyranose glucopyranosamine was used in the following examples.

[0094] Comparative Example 1

[0095] The protein containing cellulase was produced by culturing the Trichoderma reesei X3AB1 strain (J. Ind. Microbiol. Biotechnol., 2012, 174: 1-9; hereinafter referred to as the X3AB1 strain) in a medium containing cellulose. As a pre-culture, 50 mL of the medium was added to a 500 mL flask to a concentration of 1×10 5 Spores of the X3AB1 strain were inoculated at 100 μg / mL and cultured with shaking at 28°C and 220 rpm (PRECI, PRXYg-98R). The medium composition was as follows: 1% glucose, 0.14% (NH 4 ) 2 SO 4 , 0.2% KH 2 PO 4 , 0.03% CaCl 2 ·2H 2 O, 0.03% MgSO 4 7H 2 O, 0.1% Bacto Peptone (BD Difco), 0.05% Bacto Yeast extract (BD Difco), 0.1% Tween 80, 0.1% trace element, 50 mM tartaric acid buffer (pH 4.0). The composition of trace elements is as follows: 6 mg H 3 BO 3 、26mg(NH 4 ) 6 Mo 7 O 24 ·4H 2 O, 100 mg FeCl 3 6H 2 O, 40 mg CuSO 4 ·5H 2 O, 8mgMnCl 2 ·4H 2 O, 200 mg ZnCl 2 Make up to 100 mL with distilled water.

[0096] After 2 days of pre-culture, main culture was performed. The initial medium for main culture contained 10% powdered cellulose (KC FlockW100; Nippon Paper Corporation) and the following other medium components: 0.42% (NH 4 ) 2 SO 4 , 0.2% KH 2 PO 4 , 0.03% CaCl 2 ·2H2 O, 0.03% MgSO 4 7H 2 O, 0.1% bacterial peptone, 0.05% Bacto yeast extract, 0.1% Tween80, 0.1% trace elements, 0.2% defoaming agent PE-L. In a 1L volume fermenter BMZ-01KP2 (Biott) containing 500mL of the culture medium, 10% (v / v%) pre-culture solution was inoculated and cultured for 4 days. The fermenter was set as follows: temperature 28°C, ventilation 0.5vvm, pH: 4.5±0.1, and the stirring number was changed to maintain DO=3.0ppm. During the culture, the pH of the culture solution was maintained within the above-specified range by adding 10% ammonia solution. The culture solution was sampled on the 2nd, 3rd, and 4th day of culture.

[0097] Example 1

[0098] The microorganisms were cultured in the same manner as in Comparative Example 1 except that 0.5% of β-D-glucopyranosamine was added to the initial medium of the main culture, and the culture solution was sampled.

[0099] Example 2

[0100] In the main culture, an 8% aqueous solution of β-D-pyranoglucopyranosamine was added to the culture medium under the conditions shown in Table 1 (a total of 0.2% of β-D-pyranoglucopyranosamine was added per unit amount of the culture medium). Otherwise, the microorganisms were cultured in the same manner as in Comparative Example 1 and the culture fluid was sampled.

[0101] [Table 1]

[0102]

[0103] Experiment 1 Protein production

[0104] The culture solution obtained in Comparative Example 1 and Example 1-2 was centrifuged, and then the cells and the supernatant were separated using a membrane filter 25CS020AN (Advantech). The protein concentration of the supernatant was determined by the Bradford method. The protein concentration of the supernatant was calculated as the protein production amount using a QuickStart protein assay (BioRad) based on the Bradford method based on a standard curve using bovine gamma globulin as a standard protein. Figure 1The relative protein production on the second, third, and fourth days of culture in Comparative Example 1 and Examples 1-2 is shown in the figure, when the protein production on the third day of culture in Comparative Example 1 is set as 100%. In Example 1, the maximum productivity was shown on the third day of culture, and in Example 2, the maximum productivity was shown on the fourth day of culture. In all Examples, the productivity was 20% or more higher than that in Comparative Example 1. From the above, it can be seen that by using glycosylamine as a medium raw material, protein production is greatly improved regardless of whether it is pre-added to the medium components or fed during culture.

[0105] Comparative Example 2

[0106] In the main culture, except that a 40% aqueous glucose solution was fed to the culture under the conditions shown in Table 2, the same procedure as in Comparative Example 1 was followed to culture the microorganisms, and the culture solution was sampled.

[0107] Example 3

[0108] In the main culture, the microorganisms were cultured in the same manner as in Comparative Example 1 except that a 38% glucose / 2% β-D-glucopyranosamine aqueous solution was fed to the culture under the conditions shown in Table 2, and the culture solution was sampled.

[0109] Example 4

[0110] In the main culture, microorganisms were cultured in the same manner as in Comparative Example 1 except that a 35% glucose / 5% β-D-glucopyranosamine aqueous solution was fed to the culture under the conditions shown in Table 2, and the culture solution was sampled.

[0111] [Table 2]

[0112]

[0113] Comparative Example 3

[0114] In the main culture, except that a 40% aqueous glucose solution was fed to the culture under the conditions shown in Table 3, the same procedure as in Comparative Example 1 was followed to culture the microorganisms, and the culture solution was sampled.

[0115] Example 5

[0116] In the main culture, microorganisms were cultured in the same manner as in Comparative Example 1 except that a 38% glucose / 2% β-D-glucopyranosamine aqueous solution was fed to the culture under the conditions shown in Table 3, and the culture solution was sampled.

[0117] Example 6

[0118] In the main culture, microorganisms were cultured in the same manner as in Comparative Example 1 except that a 35% glucose / 5% β-D-glucopyranosamine aqueous solution was fed to the culture under the conditions shown in Table 3, and the culture solution was sampled.

[0119] [Table 3]

[0120]

[0121] Experiment 2 Protein production

[0122] The protein production amount was measured in the same manner as in Experiment 1 for the culture solutions obtained in Comparative Example 2-3 and Example 3-6. Figure 2 The relative protein production at the time of maximum production in Comparative Examples 2-3 and Examples 3-6 is shown when the protein production on the third day of culture in Comparative Example 1 is set as 100%. The following trend was confirmed: the protein productivity increases with the increase in the amount of β-D-pyranose glucopyranosamine added to the culture. In addition, when Comparative Example 2 and Examples 3-4, which have different times for starting the flow addition and flow acceleration of the flow addition liquid, are compared with Comparative Example 3 and Examples 5-6, it was confirmed that the situation where the flow addition is started little by little from the beginning shows a trend of higher productivity. From the above, it can be seen that the addition of glycosylamine to the culture will increase the protein productivity even when it is added together with other carbon sources such as glucose. Sequence Listing <110> Kao Co., Ltd. <120> How proteins are made <130> KS1629 <150> JP 2018-127519 <151> 2018-07-04 <150> JP 2018-211690 <151> 2018-11-09 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 1640 <212> DNA <213> Trichoderma reesei <220> <223> cbh1 promoter <400> 1 tgcaaagttt tgtttcggct acggtgaaga actggatact tgttgtgtct tctgtgtatt 60 tttgtggcaa caagaggcca gagacaatct attcaaacac caagcttgct cttttgagct 120 acaagaacct gtggggtata tatctagagt tgtgaagtcg gtaatcccgc tgtatatagtaa 180 tacgagtcgc atctaaatac tccgaagctg ctgcgaaccc ggagaatcga gatgtgctgg 240 aaagcttcta gcgagcggct aaattagcat gaaaggctat gagaaattct ggagacggct 300 tgttgaatca tggcgttcca ttcttgca agcaaagcgt tccgtcgcag tagcaggcac 360 tcattcccga aaaaactcgg agattcctaa gtagcgatgg aaccggata atataatagg 420 caatacattg agttgcctcg acggttgcaa tgcaggggta ctgagcttgg acataactgt 480 tccgtacccc acctcttctc aacctttggc gtttccctga ttcagcgtac ccgtacaagt 540 cgtaatcact attaacccag actgaccgga cgtgttttgc ccttcatttg gagaataat 600 gtcattgcga tgtgtaattt gcctgcttga ccgactgggg ctgttcgaag cccgaatgta 660 ggattgttat ccgaactctg ctcgtagagg catgttgtga atctgtgtcg ggcaggacac 720 gcctcgaagg ttcacggcaa gggaaaccac cgatagcagt gtctagtagc aacctgtaaa 780 gccgcaatgc agcatcactg gaaaatacaa accaatggct aaaagtacat aagttaatgc 840 ctaaagaagt catataccag cggctaataa ttgtacaatc aagtggctaa acgtaccgta 900 atttgccaac ggcttgtggg gttgcagaag caacggcaaa gccccacttc cccacgtttg 960 tttcttcact cagtccaatc tcagctggtg atcccccaat tgggtcgctt gtttgttccg 1020 gtgaagtgaa agaagacaga ggtaagaatg tctgactcgg agcgttttgc atacaaccaa 1080 gggcagtgat ggaagacagt gaaatgttga cattcaagga gtatttagcc agggatgctt 1140 gagtgtatcg tgtaaggagg tttgtctgcc gatacgacga atactgtata gtcacttctg 1200 atgaagtggt ccatattgaa atgtaagtcg gcactgaaca ggcaaaagat tgagttgaaa 1260 ctgcctaaga tctcgggccc tcgggccttc ggcctttggg tgtacatgtt tgtgctccgg 1320 gcaaatgcaa agtgtggtag gatcgaacac actgctgcct ttaccaagca gctgagggta 1380 tgtgataggc aaatgttcag gggccactgc atggtttcga atagaaagag aagcttagcc 1440 aagaacaata gccgataaag atagcctcat taaacggaat gagctagtag gcaaagtcag 1500 cgaatgtgta tatataaagg ttcgaggtcc gtgcctccct catgctctcc ccatctactc 1560 atcaactcag atcctccagg agacttgtac accatctttt gaggcacaga aacccaatag 1620 tcaaccgcgg actgcgcatc 1640 <210> 2 <211> 1477 <212> DNA <213> Trichoderma reesei <220> <223> egl1 promoter <400> 2 tttcagcaat gcgtggcgtt ggcaggcgac ttcgcggcag tgcatgagcg atattcgggg 60 tcgttgaagc cgacgttcac gccggagatt ggcatgatcc cagtgcttta catcatcggg 120 gccaaatgcc ggcatcctgt tgtgcggcgg gaggccttgg gtcttttgag gcggcaaccg 180 atccgggagg cggtttggga tagcgttgtt gttgccaggg tagtggagag gataatggag 240 attgaggagg ttgggtttga gaagtgggaa atgatacaga gtatggaaca ggttccggtg 300 tggcagaggg ttgagacgct gtcttgggca catgtcgtcg tcgatggaca gtctgcgggc 360 agagtggaca ttaactatac gttctgcgcg cgagagggat cgcatattga gtctttcatg 420 atgtaataag cttgggcttg acagcgttct attgccagtg tatcaacgaa gtggtatgta 480 ccatcgtgct ctgtccagac gttttggtca ggtcgacaaa caggcttttc ttcctattct 540 ctttctttga tatatacacg cttagaaatg tgtcaaaaag aacagaaacc tcttttgatg 600 tagttatgcg catgctagac tgctcctgtt tcatgtggtt acaacaaaca gtctgatcga 660 cttcgaatac ttggactgat gaaggttgta cagattgctg acagatgtcg taatgcagag 720 caaggctgta gattccataa aaccagttgc ttcgcctgct gtggctctgg agaaccaaag 780 agacgtgtct cgggaggta agtggtatcg aatctatgag agaagcccag tctaagagag 840 gaccatctcg ccagggggaag atgaagctgg ttacaactga tttgttttcc cgtctgccac 900 catggtatag agcctggacc aatcaggcta aatcattgta tacaataagc ctagagaaaa 960 cctgaaatct gtcctcgtcc tttgtccgtt gtctaattat ccgttatttt cgaacgatga 1020 tacagtatga gttttgccga aattttgcta aaggtactat cgacggggga cacaagggtt 1080 gagtctgtat aacggctcga aacagcagct ggtagcagga atccaggccc gcgtttcatt 1140 tggattcatt ttcccatatt ccccttgcag aaggatacga cagtagcatt ggaaaccgta 1200 aatgacggca aaaagcatgg ttctgctcag atactccaag ccaacctatc gggtcctgga 1260 ggctatttcc aacatctcat agcctaacag aaataacgga agtcggcatc tgtatcgctc 1320 aaactgacca gacgagcccg ccatatcgag gcagagttac tctgtgttgc aaatccaact 1380 tataaagaca acaaccgcaa actttgtctt gtcgccatca gattgttcgc caagcaccct 1440 cccccccccc tatcttagtc cttcttgttg tcccaaa 1477 <210> 3 <211> 1084 <212> DNA <213> Trichoderma reesei <220> <223> xyn3 promoter <400> 3 gggaattccg tgattgacaa aatctattcg tatcaaacgt tcatgccgcc gtctccagtc 60 tcctcacgct atggtgcttg atatacataa gggggggacg ttgaatatat gttcccagtt 120 tacctccaca taagaaatat ctcctttgga cggtctctgc aattttgccc tcaagactct 180 gcagacaatc cctctgtgct ttaaaacccc cgagtatccc ggtcacctga tggcgcaagt 240 ctcaacttcc ccaggatgcc gtctcctcat ctccgtgatg gtaacaaccg catataaggg 300 acttttgtct tctttaggct tcggacgagg gggttctttc ccagatcaat gccttgaccg 360 aaactgtcaa gatggctata taggacactg tcaattttgc catttcagtc cgggtattta 420 gacttaaaag cacctagtat tttggttaa taaatctccg ggcaaaggtc tctttccgtg 480 cgtgtctgat gggttatgct aagctcatct ccgcagacag ggtagtaaca gaggtagccg 540 ttccttggaa agacggttaa ttgacttctt gactttgact gtccaattcg catggctaat 600 tgcggcaaaa atgatgccat atggccccgt gggcacaact ttctcacaag tctctggtgt 660 cttgactgag gtcgatgttg tgctctttct tcccaactat acaagtctaa actcctcagt 720 aaatcgatac aaggtaaatt taaactctct ggttactctt cctaccaaaa ggccctggtt 780 acatttcgtg tatacccgag gcggctgaat ctgggggact cacataggtg gatgcaatgt 840 gctattagcc agctacgcat atacaatcaa acattgaaaa tcaaaggata tacaacaact 900 ttgacgattt tccataaatt ggcatcatct ttctgagtcc tgatggatgt cagacagcaa 960 gcggacaagc tggctcatga ctcaatcctc cgaatacatc gcatcatcta ggagccattc 1020 tcacctcgaa acttctacca tctttccact gagtttcaat tgaggcggac accatggaag 1080 cacc 1084

Claims

1. A method for producing a protein, characterized in that: include: the step of culturing a microorganism in the presence of a glycosylamine, The glycosylamine is fed to the culture, The glycosylamine is β-D-glucopyranosamine, The microorganism is Trichoderma reesei, The protein is cellulase.

2. The manufacturing method according to claim 1, characterized in that: The microorganism is cultured in the presence of at least one selected from cellulose and cellobiose.

3. The manufacturing method according to claim 2, characterized in that: The microorganism contains a cellulose-inducible promoter or a cellobiose-inducible promoter.

4. The manufacturing method according to claim 3, characterized in that: A gene encoding the protein is linked downstream of the promoter.

5. The manufacturing method according to claim 4, characterized in that: The promoter is a filamentous fungus cellulase gene promoter.

6. The manufacturing method according to claim 1, characterized in that: The glycosylamines are added to the initial culture medium.

7. The manufacturing method according to claim 6, characterized in that: The amount of the glycosylamine added is 50 g or less per 1 L of the initial culture medium.

8. The manufacturing method according to any one of claims 1 to 7, characterized in that: The addition rate of the glycosylamine is less than 5 g / hr per 1 L of initial culture medium.

9. The manufacturing method according to any one of claims 1 to 7, characterized in that: During the culture, the pH of the culture is adjusted to 3-7.

10. The manufacturing method according to any one of claims 2 to 7, characterized in that: The total concentration of cellulose or cellobiose in the culture of the microorganism is 1 to 15 mass / volume %.

11. The production method according to any one of claims 1 to 7, characterized in that: Also includes: A step of recovering protein from the culture of said microorganism.

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