Method for growing microorganisms and method for producing material using microorganisms

Abstract

The present invention provides a method for growing microorganisms or a method for producing a material using microorganisms, said method being characterized by comprising culturing and growing microorganisms using, as a culture medium, a liquid containing an extract derived from nitrogen fixation algae, wherein the culture medium does not substantially contain a yeast extract.

Description

Method for cultivating microorganisms, and method for producing substances using microorganisms

[0001] The present invention relates to a method for cultivating microorganisms and a method for producing substances using microorganisms.

[0002] The production of amino acids and biopharmaceuticals using microorganisms is already in practical use. Furthermore, because the production cost of pharmaceuticals using microorganisms is significantly lower than that using mammalian cells, efforts are underway to produce antibody drugs inexpensively using microorganisms such as Escherichia bacteria (Escherichia coli), Corynebacterium bacteria, and Brevibacillus bacteria. These drugs are expected to have high therapeutic efficacy and reduced side effects, and antibody drugs produced using microorganisms are already in practical use (Non-Patent Literature 1-3). In addition, to reduce environmental impact and avoid food and protein crises associated with global population growth, dairy and egg products containing proteins such as casein, lactoglobulin, and ovalbumin produced by microorganisms are being put into practical use (precise fermentation technology) (Non-Patent Literature 4). Currently, microorganisms are being used in various fields such as food, pharmaceuticals, and genetic engineering, and further applications are expected in the future.

[0003] Heterotrophic microorganisms require nutrients such as glucose and amino acids derived from livestock and crops, and consequently, their cultivation requires chemical fertilizers and pesticides, resulting in an environmental burden (Figure 1). The livestock industry accounts for 15-18% of global greenhouse gas (GHG) emissions, which is more than the transportation industry. The production of ammonia, the main raw material for chemical fertilizers (Haber-Bosch process), requires a high-temperature (400-650°C) and high-pressure (20-40 MPa) treatment process using hydrogen derived from natural gas. More than 150 million tons are produced worldwide, and 1-2% of global energy consumption and 3-5% of global natural gas resources are used for ammonia production, contributing to global CO2 emissions. ２ This process accounts for 3% of emissions. Thus, the manufacturing process consumes vast amounts of resources and has a serious impact on the environment. Furthermore, most pesticide raw materials are petroleum-derived. With the increasing demand for microbial fermentation products containing amino acids and proteins, there is a risk of further increases in the environmental burden.

[0004] Recently, the inventors of this invention have succeeded in culturing animal cells using microalgae as a nutrient source for the sustainable production of cultured meat (Patent Documents 1-2, Non-Patent Documents 5-7). This research aims to establish a new animal cell culture system that reduces the risk of environmental impact and environmental changes, and microalgae have the potential to replace crop-dependent systems, which have a high environmental burden.

[0005] International Publication No. 2021 / 066113, International Publication No. 2022 / 270598

[0006] Matsuda, Y. et al. Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum. Microb. Cell Factories 13, 56 (2014).Mizukami, M. et al. Efficient production of trastuzumab Fab antibody fragments in Brevibacillus choshinensis expression system. Protein Expr. Purif. 150, 109-118 (2018).Rashid, M. H. Full-length recombinant antibodies from Escherichia coli: Production, characterization, effector function (Fc) engineering, and clinical evaluation. mAbs 14, 2111748 (2022).Nielsen, M. B., Meyer, A. S. and Arnau, J. The Next Food Revolution Is Here: Recombinant microbial production of milk and egg proteins by precision fermentation. Annu. Rev. Food Sci. Technol. 15, 173-187 (2024).Okamoto, Y., Haraguchi, Y., Sawamura, N., Asahi, T. and Shimizu, T. Mammalian cell cultivation using nutrients extracted from microalgae. Biotechnol. Prog. 36, e2941 (2020).Okamoto, Y. et al.Proliferation and differentiation of bovine myoblasts using Chlorella vulgaris extract for sustainable production of cultured meat. Biotechnol. Prog. 38, e3239 (2022).Yamanaka, K., Haraguchi, Y., Takahashi, H., Kawashima, I. and Shimizu, T. Development of serum-free and grain-derived-nutrient-free medium using microalga-derived nutrients-free medium using microalga-derived nutrients and mammalian Cell-secreted growth factors for sustainable cultured meat production. Sci. Rep. 13, 498 (2023).

[0007] The present invention aims to establish a novel method for cultivating microorganisms that reduces the risk of environmental burden and impact on environmental change, and a method for producing substances using microorganisms.

[0008] The inventors of this invention have conducted research and development from various angles to solve the above-mentioned problems. As a result, they have surprisingly found that when a liquid containing an extract derived from nitrogen-fixing algae, which has the ability to fix carbon dioxide and nitrogen, is used as a culture medium, microorganisms can be grown and substances can be produced by these microorganisms, even when the liquid does not contain yeast extract. In other words, the present invention includes the following embodiments.

[0009] [1] A method for culturing microorganisms, comprising culturing and culturing microorganisms using a liquid containing an extract derived from nitrogen-fixing algae as a culture medium, wherein the liquid substantially does not contain yeast extract. [2] The method according to item 1, wherein the liquid further substantially does not contain peptones other than the extract. [3] The method according to item 1 or 2, wherein the extract is an extract obtained by (i) hydrolysis, (ii) shaking, and / or (iii) heat treatment of the nitrogen-fixing algae. [4] The method according to item 3, wherein the hydrolysis treatment includes acid hydrolysis. [5] The method according to item 3 or 4, wherein the liquid is diluted with water and / or adjusted to a predetermined pH. [6] The method according to any one of items 1 to 5, wherein the nitrogen-fixing algae are cyanobacteria. [7] The method according to item 6, wherein the cyanobacteria belong to the order Nostocales. [8] The method according to any one of items 1 to 7, wherein the microorganism is selected from the group consisting of Bacillus bacteria, Corynebacterium bacteria, Brevibacillus bacteria, Escherichia bacteria, Lactobacillus, and Yeast.

[0010] [9] A method for producing a substance using microorganisms, comprising culturing microorganisms using a liquid containing an extract derived from nitrogen-fixing algae as a culture medium, wherein the liquid does not contain yeast extract.

[10] The method according to item 9, wherein the solution further substantially does not contain peptones other than the extract.

[11] The method according to item 9 or 10, wherein the extract is an extract obtained by (i) hydrolysis, (ii) shaking, and / or (iii) heat treatment of the nitrogen-fixing algae.

[12] The method according to item 11, wherein the hydrolysis includes acid hydrolysis.

[13] The method according to item 11 or 12, wherein the liquid is diluted with water and / or adjusted to a predetermined pH.

[14] The method according to any one of items 9 to 13, wherein the liquid contains a cell wall synthesis inhibitor.

[15] The method according to any one of items 9 to 14, wherein the nitrogen-fixing algae are cyanobacteria.

[16] The method according to item 15, wherein the cyanobacteria belong to the order Nostocales.

[17] The method according to any one of items 9 to 16, wherein the microorganism is selected from the group consisting of Bacillus bacteria, Corynebacterium bacteria, Brevibacillus bacteria, Escherichia bacteria, lactic acid bacteria, and yeast.

[18] The method according to any one of items 9 to 17, wherein the substance is selected from the group consisting of amino acids, peptides, proteins, nucleic acids, and vitamins.

[19] The method according to any one of items 9 to 18, wherein the microorganism is a genetically modified microorganism.

[0011] This invention makes it possible to provide a new method for culturing microorganisms that is inexpensive and reduces environmental impact, by using nutrients extracted from nitrogen-fixing algae as a substitute for nitrogen sources in conventional culture media.

[0012] A schematic diagram illustrating the problems of conventional microbial culture using nutrients from livestock and crops. Culture of Bacillus subtilis bacteria in anabaena extract obtained by acid hydrolysis. B. subtilis grew more efficiently in anabaena extract (AE) than in conventional culture medium (CM) (a: macroscopic observation, b: turbidity analysis). ISS (Inorganic Salt Solution): Inorganic salt solution (MgSO4) ４(Solution). B. subtilis actively consumed glucose (c) and protein-constituting amino acids (d, e) in the culture medium and anabaena extract. Downward arrows indicate undetectable levels. Data are shown as mean ± standard deviation (n=3). For statistical analysis of glucose and total amino acid concentrations, the results comparing (i) with and without microbial culture and (ii) without microbial culture are described. *: p < 0.05; ****: p < 0.0001. Note that in (e), each amino acid is shown from left to right in the following order: CM (no microorganisms), CM (with microorganisms), 10% AE (diluted with pure water, same applies below, no microorganisms), 10% AE (with microorganisms), 20% AE (no microorganisms), 20% AE (with microorganisms), 40% AE (no microorganisms), 40% AE (with microorganisms), 60% AE (no microorganisms), 60% AE (with microorganisms). The consumption or production of each nutrient by microorganisms during culture was estimated from the difference in each nutrient with and without the presence of microorganisms. (Ibid.) Changes in metabolism of Bacillus subtilis cultures during the growth phase and stationary phase using a 60% (v / v, diluted with pure water, same below) anabaena extract. B. subtilis hardly grew after penicillin G treatment (a). B. subtilis actively consumed glucose during the growth phase and stationary phase, and amino acids during the growth phase, but amino acids were significantly produced during the stationary phase (b, c). d-f: Metabolism of individual protein constituent amino acids (d: growth phase and stationary phase; e: growth phase; f: stationary phase). g-i: Metabolism of ornithine and arginine (g: growth phase and stationary phase; h: growth phase; i: stationary phase). j: Metabolism of γ-aminobutyric acid and hydroxyproline. Downward arrows indicate undetectable. Data are shown as mean ± standard deviation (n=3). *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. Note that in (d), for each amino acid, the data are shown from left to right in the order of 19h no microorganisms, 19h microorganisms present (growth phase), 72h no microorganisms, and 72h microorganisms present (stationary phase). (Same as above) (Same as above) (Same as above) Culture of Corynebacterium efficiens in anabaena extract induced by acid hydrolysis. C. efficiens grew efficiently in anabaena extract (AE) (a: macroscopic observation, b: turbidity analysis).C. efficiens actively consumed glucose in the anabaena extract (c) and tended to produce protein-constituting amino acids (d, e). Data are shown as mean ± standard deviation (n=3). ISS: Inorganic salt solution (MgSO4). ４). The statistical analysis results for glucose and total amino acid concentrations are described below, comparing the amounts with and without microbial culture (i) and without microbial culture (ii). *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. Note that in (e), for each amino acid, the results are shown from left to right in the order of 10% AE (no microorganisms), 10% AE (with microorganisms), 20% AE (no microorganisms), 20% AE (with microorganisms), 40% AE (no microorganisms), 40% AE (with microorganisms), 60% AE (no microorganisms), and 60% AE (with microorganisms). (Ibid.) Changes in metabolism of Corynebacterium efficens cultures during the growth phase and stationary phase using 40% (v / v) anabaena extract. C. C. efficiens hardly grew after penicillin G treatment (a). C. efficiens actively consumed glucose during the growth phase and stationary phase (b), tended to produce amino acids during the growth phase, and actively produced amino acids during the stationary phase (c). d-f: Metabolism of individual protein constituent amino acids (d: growth phase and stationary phase; e: growth phase; f: stationary phase). g: Metabolism of γ-aminobutyric acid and hydroxyproline. Downward arrows indicate undetectable. Data are shown as mean ± standard deviation (n=3). *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. Note that in (d), for each amino acid, the data are shown from left to right in the order of 19h no microorganisms, 19h microorganisms present (growth phase), 72h no microorganisms, and 72h microorganisms present (stationary phase). (Same as above) (Same as above) (Same as above) Culture of Escherichia bacteria (E. coli) in anabaena extract induced by acid hydrolysis. E. coli grew efficiently in anabaena extract (AE) (a: macroscopic observation, b: turbidity analysis). E. coli actively consumed glucose in anabaena extract (c), but consumed only protein-constituting amino acids in normal microbial culture media (d, e). Data are shown as mean ± standard deviation (n=3). ISS: inorganic salt solution (NaCl solution). For statistical analysis of glucose and total amino acid concentrations, the results comparing (i) with and without microbial culture and (ii) without microbial culture are described. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.In (e), each amino acid is shown from left to right in the following order: CM (microorganism-free), CM (microorganism-present), 10% AE (microorganism-free), 10% AE (microorganism-present), 20% AE (microorganism-free), 20% AE (microorganism-present), 40% AE (microorganism-free), 40% AE (microorganism-present), 60% AE (microorganism-free), and 60% AE (microorganism-present). (Ibid.) Culture of Brevibacillus bacteria (B. brevis) in anabaena extract induced by acid hydrolysis. B. brevis grew more efficiently in anabaena extract (AE) than in conventional medium (CM) (a: macroscopic observation, b: turbidity analysis). ISS: inorganic salt solution (MgSO4). ４). B. brevis consumed very little glucose (c), but actively consumed protein-constituting amino acids in the culture medium and anabaena extract (d, e). Data are shown as mean ± standard deviation (n=3). For the statistical analysis of glucose and total amino acid concentrations, the results comparing (i) with and without microbial culture and (ii) without microbial culture are described. **: p < 0.01, ***: p < 0.0001. Note that in (e), each amino acid is shown from left to right in the order of CM (no microorganisms), CM (with microorganisms), 10% AE (no microorganisms), 10% AE (with microorganisms), 20% AE (no microorganisms), 20% AE (with microorganisms), 40% AE (no microorganisms), 40% AE (with microorganisms), 60% AE (no microorganisms), 60% AE (with microorganisms). (Same as above) Culturing of Brevibacillus bacteria (B. brevis) in anabaena extract by shaking treatment. B. brevis grew more efficiently in anabaena extract by shaking method (AE(S)) than in conventional medium (CM) (a: macroscopic observation, b: turbidity analysis). B. brevis consumed glucose (c) and protein-constituting amino acids (d, e) in the anabaena extract. Data are shown as mean ± standard deviation (n=3). For the statistical analysis results of glucose and total amino acid concentrations, the results comparing (i) with and without microbial culture and (ii) without microbial culture are described. *: p < 0.05; ****: p < 0.0001. In (e), each amino acid is shown from left to right in the following order: CM (no microorganisms), CM (with microorganisms), 100% AE (undiluted with pure water, S) (no microorganisms), and 100% AE (S) (with microorganisms). (Same as above) Culture of genetically modified Brevibacillus bacteria (B. choshinensis) in anabaena extract by shaking treatment. Genetically modified B. choshinensis grew efficiently in anabaena extract by shaking treatment (AE(S)) as well as in conventional medium (CM) (a: macro observation, b: turbidity analysis). B. choshinensis consumed a small amount of glucose in anabaena extract (c) and actively consumed pyruvate and protein-constituting amino acids (d, e). Genetically modified B. choshinensis secreted bovine β-casein when cultured in anabaena extract as well as in conventional medium (f).A downward arrow indicates undetectable (less than 7.8 ng / mL). Data are shown as mean ± standard deviation (n=3). For the statistical analysis results of glucose, pyruvate, and total amino acid concentrations, the results comparing (i) with and without microbial culture and (ii) without microbial culture are described. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. In (f), for each amino acid, the order from left to right is CM (no microorganisms), CM (with microorganisms), 100% AE(S) (no microorganisms), and 100% AE(S) (with microorganisms). (Same as above) Recombinant B. choshinensis culture and human insulin-like growth factor I (IGF-I) production in anabaena extract by shaking treatment. Recombinant B. choshinensis grew efficiently in anabaena extract by shaking method (AE(S)) similar to conventional medium (CM) (a: turbidity analysis). Recombinant B. choshinensis secreted human IGF-I when cultured in anabaena extract similar to conventional medium (b). Downward arrows indicate undetectable levels (less than 0.5 ng / mL). Data are shown as mean ± standard deviation (n=3). ****: p < 0.0001. Microscopic observation of cyanobacteria and microorganisms. (a): Anabaena sp. PCC 7120 strain, (b): B. subtilis, (c): C. efficiens, (d): B. brevis, (e): E. coli, (f): Recombinant B. choshinensis. Arrow: heterocyst. Scale bar: 10 μm. Time course of free amino acid content in anabaena extract by shaking treatment. Comparison of growth rates of Brevibacillus bacteria (B. brevis) using anabaena extract by shaking treatment (1 day shaking or 3 days shaking). Comparison of nutritional factors (amino acids) of anabaena extract by acid treatment, shaking treatment, or conventional microbial media (802 medium or LB medium). Comparison of nutritional factors (carbohydrates, vitamins, inorganic salts) of anabaena extract by acid treatment, shaking treatment, or 802 medium or LB medium. Results of food and pharmaceutical protein production by microbial fermentation using anabaena extract. Culture results of casein-secreting Brebacillus bacteria using Scytonema sp. algal extract.

[0013] The embodiments of the present invention will be described below with reference to the drawings as necessary. The configurations of the embodiments are illustrative, and the present invention is not limited to the specific configurations of the embodiments.

[0014] In one embodiment, the present invention provides a method for cultivating microorganisms, comprising adding a solution to an extract derived from nitrogen-fixing algae, culturing and growing microorganisms using the liquid containing the extract as a culture medium, wherein the solution substantially does not contain yeast extract.

[0015] Furthermore, in one embodiment, the present invention provides a method for producing a substance using microorganisms, comprising adding a solution to an extract derived from nitrogen-fixing algae, culturing microorganisms using the liquid containing the extract as a culture medium, and causing the microorganisms to produce a substance, wherein the solution does not contain yeast extract.

[0016] In this specification, "microorganism" refers to microorganisms capable of producing any substance (e.g., amino acids, proteins, peptides, nucleic acids, vitamins, etc.) for use as food, pharmaceuticals, quasi-drugs, supplements, etc., or microorganisms that exert beneficial effects on living organisms (e.g., humans). Examples of such microorganisms include bacteria of the genera Bacillus, Corynebacterium, Brevibacillus, Escherichia, lactic acid bacteria, and yeast. In one embodiment, the microorganism applicable to the present invention may be one type or a mixture of more types. In another embodiment, the microorganism applicable to the present invention may be a microorganism that has been genetically transformed to produce a desired substance. The method of genetic transformation may follow known methods and is not particularly limited.

[0017] In one embodiment, the present invention provides a method for producing a substance using microorganisms. In this specification, "substance" refers to a substance produced by microorganisms that can be used as food, pharmaceuticals, quasi-drugs, supplements, etc., and may be selected from the group consisting of amino acids, peptides, proteins, nucleic acids, and vitamins. Examples of substances to be produced by microorganisms include amino acids such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, which are protein-constituting amino acids, as well as ornithine, citrulline, and γ-aminobutyric acid (GABA), which are not protein-constituting amino acids. Furthermore, the substance to be produced by microorganisms may be a peptide, for example, an oligopeptide in which 2 to 10 protein-constituting amino acids are linked together, or a polypeptide in which 10 to 100 protein-constituting amino acids are linked together, or any protein or a fragment thereof. Furthermore, the substances to be produced by microorganisms may include, for example, nucleic acids such as DNA and / or RNA having any chain length and / or sequence (e.g., plasmid vectors, phage vectors, cosmid vectors, BAC vectors, YAC vectors, etc.). Other examples of substances to be produced by microorganisms include vitamins such as vitamin D, vitamin K, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, pantothenic acid, biotin, or their precursors (synthetic intermediates). If the substances to be produced by microorganisms are peptides, proteins, and / or nucleic acids, they may be produced, for example, by genetically modified microorganisms. The genetic modification method can be carried out according to known methods and is not particularly limited.

[0018] This invention has found that, even without using conventional culture media containing yeast extract, as is used in in vitro culture of microorganisms, using a culture medium containing an extract derived from nitrogen-fixing algae can produce growth effects equivalent to or better than those of conventional culture media.

[0019] In this specification, "nitrogen-fixing algae" refers to algae that, in addition to their ability to take in carbon dioxide and synthesize sugars through photosynthesis, also possess the ability to take in and fix nitrogen. Examples include cyanobacteria. Cyanobacteria of the Nostocales order are examples of such cyanobacteria. Cyanobacteria of the Nostocales order have a filamentous morphology and form morphologically specialized nitrogen-fixing cells called heterocysts. The Nostocales order includes, for example, genera such as Coleodesmium sp., Fremyella sp., Microchaete sp., Rexia sp., Spiritrestis sp., Tolypothrix sp., Anabaena sp., Anabaenopsis sp., Aphanizomenon sp., Aulosira sp., Cyanospira sp., and Cylindrospermopsis. ), Cylindrospermum sp., Nodularia sp., Nostoc sp., Richelia sp., Calothrix sp., Gloeotricia sp., Scytonema sp., Stigonema sp., Hapalosiphon sp., Dolichospermum sp., Sphaerospermopsis This may include species of cyanobacteria such as Umezakia sp., Gloeotricia sp., Rivularia sp., or Cuspidothrix sp., and extracts of these cyanobacteria may be applied to the method of the present invention.Furthermore, cyanobacteria that do not possess heterocysts but are capable of nitrogen fixation under low-oxygen conditions or at night, such as the genus Synechococcus (e.g., Synechococcus sp. WH5701) of the order Chroococcales and the genus Trichodesmium (e.g., the order Oscillatoria), can also be applied to the present invention.

[0020] The nitrogen-fixing algae extract used in the method of the present invention allows for the growth of microorganisms to the same or even greater extent than conventional culture media, even without the use of environmentally harmful additives such as yeast extract or animal or grain-derived peptones, such as casein peptone, meat peptone, gelatin peptone, or soybean peptone, as is done in conventional culture media. Furthermore, the nitrogen-fixing algae extract used in the method of the present invention allows for the production of substances by microorganisms to the same or even greater extent than conventional culture media. This provides a new microbial culture system that can replace environmentally harmful additives and contribute to the reduction of greenhouse gases.

[0021] In this specification, "yeast extract" refers to commercially available products used for culturing microorganisms. In this specification, "peptone" is a general term for a mixture of amino acids and low molecular weight peptides obtained by hydrolyzing proteins, and includes, but is not limited to, casein peptone, meat peptone, gelatin peptone, and soy peptone. In one embodiment of the present invention, a culture medium used for culturing microorganisms may be one that does not contain peptone other than extracts derived from nitrogen-fixing algae.

[0022] In this specification, "substantially absent" means that, when carrying out the present invention, the substance in question is hardly present in the culture medium, but it does not mean that the substance in question is completely absent. For example, the substance in question may be present in a concentration of less than 10 mg / L. For example, the total amount of peptone-derived amino acids other than yeast extract and / or nitrogen-fixing algae extract may be present in a concentration of less than the lower limit of quantification, which is 0.1 mg / L.

[0023] In one aspect, the extract derived from nitrogen-fixing algae used in the present invention may be obtained by (i) hydrolysis treatment, (ii) shaking treatment, and / or (iii) heat treatment. The hydrolysis treatment may be an acid hydrolysis treatment or an alkali hydrolysis treatment, preferably an acid hydrolysis treatment.

[0024] Generally, biopolymers such as proteins and polysaccharides are hydrolyzed by mixing with an organic or inorganic strong acid (hydrochloric acid, sulfuric acid, trifluoroacetic acid, p-toluenesulfonic acid, methanesulfonic acid, etc.) or a strong alkali (sodium hydroxide, potassium hydroxide, etc.) with water added under heating (for example, 60°C to 200°C, preferably 70°C to 180°C, more preferably 80°C to 150°C), resulting in oligomers and monomers (peptides and amino acids in the case of proteins, oligosaccharides and monosaccharides in the case of polysaccharides). Therefore, in this specification, "acid hydrolysis treatment" refers to a hydrolysis treatment using a strong acid, and "alkali hydrolysis treatment" refers to a hydrolysis treatment using a strong alkali. In the present invention, known "acid hydrolysis treatment" and / or "alkali hydrolysis treatment" can be used. The reaction time of the "acid hydrolysis treatment" or "alkali hydrolysis treatment" can be adjusted according to conditions such as the type, amount, or concentration of the nitrogen-fixing algae used, or the pH and temperature of the reaction solution. For example, it may be carried out for 10 minutes to 72 hours, 1 hour to 48 hours, 12 hours to 36 hours, for example, about 24 hours.

[0025] In one embodiment, to obtain the extract derived from nitrogen-fixing algae, only the acid hydrolysis treatment may be carried out, only the alkali hydrolysis treatment may be carried out, or both the acid hydrolysis treatment and the alkali hydrolysis treatment may be carried out. When the acid hydrolysis treatment and the alkali hydrolysis treatment are carried out, alkali hydrolysis may be carried out following the acid hydrolysis treatment, or acid hydrolysis treatment may be carried out following the alkali hydrolysis treatment. A neutralization treatment may be carried out between the acid hydrolysis treatment and the alkali hydrolysis treatment.

[0026] In one embodiment, (i) the hydrolysis treatment may be carried out under atmospheric pressure or under pressure. As used herein, "under pressure" refers to a pressure condition higher than atmospheric pressure, i.e., 1 atmosphere, and may be carried out, for example, at 1.1 atmospheres or more, 1.5 atmospheres or more, 1.8 atmospheres or more, or 2 atmospheres or more. For example, it may be 1.1 to 300 atmospheres, 1.5 to 200 atmospheres, 1.8 to 100 atmospheres, 2 to 50 atmospheres, for example 2 to 20 atmospheres. The pressurization conditions may be carried out by any device or method, and for example, the pressurization conditions can be realized by using an autoclave.

[0027] The extract derived from nitrogen-fixing algae used in the present invention preferably neutralizes the hydrolysis product obtained by (i). Thereby, the hydrolysis product is neutralized and can be used for culturing microorganisms. For example, in (i), when acid hydrolysis is finally carried out, a basic substance or an aqueous solution thereof (for example, sodium hydroxide, potassium hydroxide or an aqueous solution thereof, etc.) may be added for neutralization treatment. For example, in (i), when alkali hydrolysis is finally carried out, an acidic substance or an aqueous solution thereof (hydrochloric acid, sulfuric acid, trifluoroacetic acid, p-toluenesulfonic acid, methanesulfonic acid, etc.) may be added for neutralization.

[0028] In other embodiments, (i) the hydrolysis treatment may be a method of mixing nitrogen-fixing algae suspended in a liquid (e.g., pure water) with a "solid acid catalyst" (also called "solid acid") and heating. For example, the treatment may be carried out using a solid acid catalyst described in International Publication No. 2022 / 270598. In this specification, "solid acid catalyst" refers to a catalyst consisting of an acid that is dispersed as a solid in the reaction solution. Examples include inorganic solid acids such as zeolite, alumina, silica, H-mordenite, and niobic acid, or organic materials such as resins such as strongly acidic ion exchangers (e.g., Amberlist™-15, Nafion™ NR-50, etc.) into which acidic groups such as sulfone groups, carboxyl groups, and phenolic hydroxyl groups have been introduced. Solid acid catalysts prepared in powder or granular form may be used to increase the contact surface area. By using a solid acid when hydrolyzing nitrogen-fixing algae, the neutralization treatment that was necessary when using a liquid acid is eliminated. This suppresses the increase in salt concentration due to salts that may be produced by the neutralization treatment. Furthermore, solid acid catalysts can be recovered and reused, which helps reduce the environmental impact.

[0029] In one embodiment, the solid acid catalyst applicable to the present invention may be a woody solid acid catalyst obtained by introducing sulfo groups into a carbide derived from a woody raw material and sulfonating it, or a resin solid acid catalyst obtained by introducing sulfo groups into a phenolic resin and sulfonating it.

[0030] In one embodiment, the woody solid acid catalyst applicable to the present invention may be obtained by carbonizing a woody raw material under temperature conditions that do not cause incineration to form a carbide, and then performing sulfonation to introduce a sulfo group (also referred to as a sulfonic acid group). As the woody solid acid catalyst, for example, solid acids disclosed in Japanese Patent No. 5528036 and J. Am. Chem. Soc 2008, vol. 130, No. 38, 12787-12793 can be used.

[0031] In one embodiment, the resin solid acid catalyst that can be used may be obtained by introducing sulfo groups into the raw material phenol resin and performing sulfonation.

[0032] In one embodiment, (ii) "shaking treatment" means suspending nitrogen-fixing algae in a liquid (e.g., pure water) and shaking while maintaining a predetermined temperature, for example, 4°C to 100°C, 10°C to 50°C, 20°C to 40°C, or 25°C to 35°C. It is believed that this process digests the nitrogen-fixing algae with enzymes such as proteases, thereby producing an extract containing peptides, amino acids, vitamins, inorganic salts, and / or sugars. The shaking rate is not particularly limited, but may be, for example, 10 rpm to 1000 rpm, 50 to 500 rpm, or 100 rpm to 300 rpm. The duration of the shaking treatment can be adjusted as appropriate, for example, 10 minutes to 72 hours, 1 hour to 48 hours, 12 hours to 36 hours, or for example, about 24 hours. Furthermore, the shaking treatment may be carried out for a period of time of preferably 12 to 168 hours, more preferably 24 to 120 hours, even more preferably 24 to 96 hours, or for example, about 72 hours.

[0033] In one embodiment, (iii) "heat treatment" means a process in which nitrogen-fixing algae are suspended in a liquid (e.g., pure water) and maintained at a predetermined temperature, for example, 60°C to 130°C, 80°C to 120°C, 90°C to 110°C, or for example, about 100°C. The time of the heat treatment can be adjusted as appropriate, for example, from 10 minutes to 72 hours, from 1 hour to 48 hours, from 12 hours to 36 hours, or for example, about 24 hours.

[0034] In one embodiment, the nitrogen-fixing algae extract used in step (1) may be subjected to a drying treatment before being subjected to the extraction treatments (i) to (iii) above, with freeze-drying being particularly preferred. Also in one embodiment, the nitrogen-fixing algae extract used in the present invention may be an extract obtained by any of the methods (i) to (iii) above, depending on the growth stage and nutritional requirements of the microorganism to be cultured, or two or more extracts obtained by the methods (i) to (iii) above may be mixed in any proportion.

[0035] In one embodiment, the nitrogen-fixing algae-derived extract used in the present invention may be provided in powder form after drying (e.g., freeze-drying, vacuum drying, vacuum drying, spray freeze-drying, or a combination thereof), or it may be provided as a liquid extract ("extract") obtained by performing any of the extraction treatments (i) to (iii) above. In one embodiment, the nitrogen-fixing algae-derived extract that can be used in the present invention may be prepared by adding a solution to the powdered nitrogen-fixing algae-derived extract and using the liquid containing the extract as a culture medium, or by adding a solution to the liquid extract (extract) and using the liquid containing the extract as a culture medium.

[0036] In one embodiment, the liquid containing the nitrogen-fixing algae extract used in the present invention may be used as a liquid culture medium, or as a gelled solid culture medium (for example, an agar medium).

[0037] In one embodiment, if the salt concentration and / or pH of the solution containing the nitrogen-fixing algae extract used in the present invention is not within the range of salt concentration and / or pH acceptable to the cultured microorganism, it may be adjusted to a predetermined salt concentration and / or pH by any method. For example, the solution containing the nitrogen-fixing algae extract may be further diluted with a liquid such as water or a buffer before use. For example, the volume ratio (v / v) of the nitrogen-fixing algae extract used in the present invention may be 10% to 100% (diluted with water; if 100%, no dilution is used; the same applies hereinafter). For example, in the case of a nitrogen-fixing algae extract obtained by hydrolysis (e.g., acid hydrolysis or alkaline hydrolysis), the volume ratio (v / v) of the nitrogen-fixing algae extract may be 10% to 60%, preferably 20% to 60%, and more preferably 40% to 60%. Furthermore, in the case of an extract derived from nitrogen-fixing algae obtained by shaking treatment, for example, the volume ratio (v / v) of the nitrogen-fixing algae extract may be 10% to 100%, preferably 50% to 100%, more preferably 70% to 100%, even more preferably 80% to 100%, or even 100%. The solution containing the nitrogen-fixing algae extract may also be adjusted to a predetermined pH by adding an acid or base. In the examples described below, pure water was used to dilute the solution containing the extract, but water with lower purity may be used as long as it does not hinder the cultivation of microorganisms or the production of substances.

[0038] In one embodiment, the liquid containing the nitrogen-fixing algae extract used in the present invention may also contain a cell wall synthesis inhibitor (for example, a β-lactam antibiotic (e.g., penicillin G, penicillin V, ampicillin, amoxicillin, cephalexin, cephalothin, cefazolin, etc.)). This allows for cultivation while substantially suppressing microbial growth and maintaining metabolic activity. As a result, substances can be preferentially produced by the microorganisms.

[0039] The present invention will be described in more detail below based on examples, but these examples are not intended to limit the present invention in any way.

[0040] 1. Materials and Methods 1-1. Cultivation of cyanobacteria and preparation of their extracts

[0041] 300 mL of the nitrogen-fixing filamentous cyanobacterium (Anabaena sp.) PCC 7120 strain (Figure 11a) (hereinafter referred to as "filamentous cyanobacterium") purchased from Pasteur Cultures of Cyanobacteria (Pasteur Institute, Paris, France) was placed in 500 mL Erlenmeyer flasks capped with silicone stoppers (AS ONE Corporation, Osaka, Japan) and maintained under continuous light in a plant growth chamber (Biotron; Nippon Medical Chemical Instruments Co., Ltd., Osaka, Japan) [temperature: 25 °C; CO ０ medium (25 mM NaHCO ３ ; 620 μM Mg ２ SO ４ ; 250 μM CaCl ２ ·2H ２ O; 190 μM Na ２ CO ３ ; 180 μM K ２ HPO ４ ·3H ２ O; 46 μM H ３ BO ３ ; 31 μM citric acid; 9.2 μM MnCl ２ ·4H ２ O; 6.1 μM FeCl ３ ; 2.3 μM Na ２ EDTA-Mg; 1.6 μM Na ２ MoO ４ ·2H ２ O; 0.77 μM ZnCl ２ ·7H ２ O; 0.32 μM CuSO ４ ·5H ２ O; 0.17 μM CoCl ３ ·5H ２ O) at a concentration of 1%, stirring speed of 60 rpm, photosynthetic photon flux density (PPFD): about 80 μmol / m ２ / s]. ２ / s].

[0042] Cyanobacteria were collected by centrifugation (10,000 x g, 5 minutes), and the supernatant was discarded. Subsequently, the culture medium components were removed by centrifugation (10,000 x g, 5 minutes) twice using pure water (Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan). The collected cyanobacteria were freeze-dried using a freeze-dryer (FDM-1000; Tokyo Rikakikai, Tokyo, Japan), and extracts were prepared from the cyanobacteria Anabaena strain PCC 7120 as follows (10 mL): (i) acid hydrolysis method (Haraguchi, Y., et al. Arch. Microbiol. 204, 615 (2022)), or (ii) the newly established shaking method.

[0043] (i) Acid hydrolysis treatment: Freeze-dried cyanobacteria were treated with 0.5 N hydrochloric acid (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) at a concentration of 50 g (dry weight) / L at 100°C for 24 hours using a heat block (Labnet, BM Equipment Co., Ltd., Tokyo, Japan). After neutralization with 19 N sodium hydroxide aqueous solution (pH 7.0), the mixture was centrifuged (10,000 × g, 5 minutes), and the supernatant was subjected to acid hydrolysis, biochemical analysis (analysis of microbial nutrient metabolism), and extracted for microbial culture (hereinafter referred to as "Anabaena extract by acid hydrolysis").

[0044] (ii) Shaking Treatment: Freeze-dried cyanobacteria were treated with pure water (Fujifilm Wako Pure Chemical Industries) at a concentration of 50 g (dry weight) / L at 30°C and 250 rpm for 0, 1, 3, 4, and 7 days using a shaker (BR-23FP, Taitec Co., Ltd., Saitama, Japan). After that, heat treatment (100°C, 10 minutes) was performed. The pH of the extract was approximately 6, so it was finely adjusted to pH 7.0 with 19 N sodium hydroxide aqueous solution. After centrifugation (10,000 g x 5 minutes), the supernatant was used as "Anabaena extract by shaking treatment" for biochemical analysis (analysis of microbial nutrient metabolism) and microbial culture. The pH value of the extract was measured using a pH meter (LAQUA twin, Horiba, Ltd., Kyoto, Japan).

[0045] Biochemical analysis of anabaena extract follows previous studies (Okamoto, Y., Haraguchi, Y., Sawamura, N., Asahi, T. and Shimizu, T. Mammarian cell cultivation using nutrients extracted from microalgae. Biotechnol. Prog. 36, e2941 (2020); Okamoto, Y. et al. Proqualification and difference of primary bovine myoblasts using The extraction of Chlorella vulgaris was carried out in accordance with the method described in *Biotechnol. Prog. 38, e3239 (2022)*. Glucose, pyruvate, and amino acid concentrations were measured using hexokinase, lactate oxidase, and liquid chromatography-mass spectrometry, respectively. These biochemical analyses were outsourced to SRL Corporation (Tokyo, Japan).

[0046] 1-2. Microbial Cultures: Bacillus bacteria (Bacillus subtilis) (B. subtilis subsp. subtilis) (Figure 10b, NBRC 13719, National Institute of Technology and Evaluation, Tokyo, Japan), Corynebacterium bacteria (C. efficiens) (Figure 10c, NBRC 100395, National Institute of Technology and Evaluation), or Brevibacillus bacteria (B. brevis) (Figure 10d, NBRC 100599, National Institute of Technology and Evaluation) are cultured in 802 medium (high polypeptone, Fujifilm Wako Pure Chemical Industries, Ltd., 10 g / L; yeast extract, Fujifilm Wako Pure Chemical Industries, Ltd., 2 g / L; MgSO4). ４ 7H ２(i) MgSO ４ Alternatively, the microorganisms were cultured in NaCl alone (inorganic salt solution, hereinafter referred to as "ISS"), (ii) 802 medium or LB medium (conventional medium), (iii) 10% (v / v), 20% (v / v), 40% (v / v), 60% (v / v) anabaena extract by acid hydrolysis, or 100% (v / v) anabaena extract by shaking. These microorganisms were cultured in a shaking incubator (BR-23FP) using Erlenmeyer flasks (AS ONE Corporation) with silicone stoppers (AS ONE Corporation, Osaka, Japan) [temperature: 30°C (Bacillus bacteria) or 37°C (Corynebacterium bacteria, Brevibacillus bacteria, Escherichia bacteria), shaking speed: 250 rpm]. After culturing, the cultures were centrifuged (4,000 × g, 10 minutes), and the culture supernatant was used for metabolite analysis. Microbial growth was measured using macro photographs taken with a digital camera (EOS M100, Canon Inc., Tokyo, Japan) and turbidity (optical density [OD] 660, OD) measured with a microplate reader. ６６０ The analysis was based on measurements of the following:

[0047] 1-3. Amino acid production by Bacillus bacteria (B. subtilis) and Corynebacterium bacteria (C. efficiens)

[0048] Bacillus subtilis or Corynebacterium efficiens were cultured in anabaena extract for 19 hours (growth phase). After culturing, bacterial growth was analyzed by measuring turbidity, and the culture supernatant was collected by centrifugation (4,000 × g, 10 minutes) for biochemical analysis. Anabaena extract containing 10 μM penicillin G (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the precipitate (microorganisms) and cultured for 72 hours (stationary phase). After culturing, turbidity was measured, and the culture supernatant was collected by centrifugation (4,000 × g, 10 minutes) for biochemical analysis.

[0049] 1-4. Culture of genetically modified Brevibacillus bacteria (B. choshinensis) and enzyme-linked immunosorbent assay (ELISA)

[0050] A pNCMO2-based vector containing a secretory signal peptide, six histidine residues (His-tag), a bovine β-casein gene (GenBank: AAA30431.1) or a human IGF-I gene (GenBank: CAA01954.1, sequence with methionine removed from the start codon), and a neomycin resistance gene was used as the expression vector. This vector was used to transform the bacterium B. choshinensis by electroporation. The resulting recombinant B. Choshinensis (Figure 10f) is PBN medium (Phyton Peptone, Thermo Fisher Scientific Inc., Waltham, MA, USA, 40 g / L; Glucose, Fujifilm Wako Pure Chemical Corporation, 20 g / L; Yeast Extract, Fujifilm Wako Pure Chemical Corporation, 5 g / L; FeSO4) ４ 7H ２ O, Fujifilm Wako Pure Chemical Corporation, 0.01 g / L; MnSO ４ ・5H ２ O, Fujifilm Wako Pure Chemical Corporation, 0.01 g / L; ZnSO ４ 7H ２ The cells were maintained with 0.001 g / L and 50 μg / mL neomycin (Fujifilm Wako Pure Chemical Corporation). The genetic recombination operation was outsourced to Protein Express Co., Ltd. (Chiba, Japan).

[0051] Cell proliferation and protein expression culture were performed using antibiotic-free PBN medium or anabaena extract, with shaking at 30°C and 250 rpm for 19 hours. After culturing, turbidity was measured, and the culture supernatant was collected by centrifugation (4,000 × g, 10 minutes). The concentration of secreted proteins in the supernatant was quantified using commercially available ELISA kits (bovine β-casein: Cloud-Clone Corp., Katy, TX, USA; human IGF-I: R&D Systems, Inc., Minneapolis, MN, USA). Biochemical analysis of the culture supernatant was performed as described above.

[0052] Anabaena strain PCC 7120, Bacillus (B. subtilis), Corynebacterium (C. efficiens), Brevibacillus (B. brevis), Escherichia (E. coli), and genetically modified Brevibacillus (B. choshinensis) were observed and photographed using a differential interference microscope (NIS-Elements L, Nikon Corporation, Tokyo, Japan).

[0053] 1-5. statistical analysis

[0054] Two-group comparisons were performed using unpaired Student's t-tests. Multiple-group comparisons were performed using GraphPad Prism software version 10.1.2 (GraphPad Software, Inc.). Multiple comparisons were performed using Tukey's test or Sidak's test as appropriate after analysis of variance. A p-value less than 0.05 (p < 0.05) was considered statistically significant.

[0055] 2. Experimental Results 2-1. (Example 1) Culture of Bacillus bacteria (Bacillus subtilis subsp. subtilis) using an anabaena extract obtained by acid hydrolysis.

[0056] First, we attempted to culture Bacillus bacteria (B. subtilis subsp. subtilis) using an anabaena extract obtained by acid hydrolysis. While Bacillus bacteria grew efficiently in conventional culture media, they did not grow in ISS (Figure 2a, b). Bacillus bacteria grew more efficiently in culture media containing 10% or more anabaena extract obtained by acid hydrolysis than in conventional media (Figure 2a, b). As the amount of extract increased, more efficient microbial growth was observed, and Bacillus bacteria increased 160-fold after 19 hours of culture with a 60% extract. The yield was more than three times that of conventional culture media.

[0057] Next, the metabolic activity of Bacillus bacteria was investigated. The anabaena extract obtained by acid hydrolysis contained more glucose than the conventional medium, and Bacillus bacteria completely consumed the glucose within 19 hours of incubation (Figure 2c). However, the amino acid content was higher in the conventional medium than in the diluted anabaena extract (10-60%). Bacillus bacteria also efficiently consumed amino acids (Figure 2d). Amino acid consumption was higher in the conventional medium than in the culture with the anabaena extract obtained by acid hydrolysis. Therefore, the consumption of each amino acid was analyzed. Bacillus bacteria actively consumed aspartic acid, serine, glutamic acid, proline, glycine, alanine, and arginine in both the conventional medium and the anabaena extract (Figure 2e). On the other hand, it is noteworthy that Bacillus bacteria hardly consumed valine, methionine, isoleucine, leucine, and histidine in the anabaena extract (in some cases they even excreted them), while these amino acids were actively consumed in the conventional medium.

[0058] 2-2. (Example 2) Amino acid production from Bacillus bacteria using Anabaena extract

[0059] Next, as shown in Figure 2b, we attempted to produce amino acids from Bacillus bacteria using a 60% (v / v) anabaena extract induced by acid hydrolysis, which showed optimal growth. Glutamic acid is produced and excreted into the culture supernatant when Corynebacterium glutamium is cultured and its growth is stopped by penicillin G treatment, etc. Therefore, we attempted to secrete and produce amino acids into the culture supernatant by culturing Bacillus bacteria with anabaena extract containing penicillin G. Bacillus bacteria actively consumed glucose in both the growth phase and the stationary phase, but hardly grew during the stationary phase (Figures 3a, b). This suggests that glucose was used as an energy source for purposes other than bacterial growth during the stationary phase. Bacillus bacteria consumed amino acids during the growth phase, but actively produced amino acids during the stationary phase (Figure 3c). Bacillus bacteria actively consumed aspartic acid and arginine during both the growth phase and the stationary phase (Figure 3d). During the growth phase, only four amino acids (valine, tyrosine, methionine, and phenylalanine) were produced, but during the stationary phase, 17 amino acids were produced by the microorganisms, with particularly significant production of 10 amino acids (threonine, glutamic acid, proline, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, and lysine) (Figure 3d-f). Bacillus bacteria also actively consumed aspartic acid (the amino acid most abundant in anabaena extract obtained by acid hydrolysis) during the stationary phase (Figure 3d-f). Aspartic acid family amino acids, such as threonine, methionine, isoleucine, and lysine, are synthesized from aspartic acid. Humans and many livestock animals cannot produce these amino acids except for aspartic acid. Therefore, threonine, methionine, isoleucine, and lysine are essential amino acids indispensable to the diets of humans and livestock animals. Lysine, methionine, and threonine are industrially produced worldwide, accounting for more than 50% of the amino acid market. Figure 3f shows that microorganisms actively consume aspartic acid during the stationary phase and also produce the important amino acids threonine, methionine, isoleucine, and lysine.Furthermore, glutamic acid family amino acids such as glutamic acid, arginine, citrulline, ornithine, proline, GABA, and hydroxyproline are widely used in the food, pharmaceutical, cosmetic, and animal feed industries. Therefore, these glutamic acid family amino acids were also analyzed. It was found that Bacillus bacteria consumed glutamic acid and proline during the growth phase, but active production of these amino acids was observed during the stationary phase after treatment with penicillin G (Figure 3d-f). Active production of ornithine, which was not observed during the growth phase, was also observed during the stationary phase (Figure 3g-i). At this time, consumption of arginine was also observed. Furthermore, Bacillus bacteria produced GABA and hydroxyproline during the stationary phase, but production of these amino acids was not observed during the growth phase (Figure 3j). In addition, production of citrulline, which was not present in the extract, was observed in both the growth and stationary phases. Using anabaena extract, it was shown that Bacillus bacteria produce amino acids from the aspartic acid family and glutamic acid family, making them a valuable source.

[0060] 2-3. (Example 3) Culturing of Corynebacterium efficens using Anabaena extract

[0061] Next, we attempted to culture Corynebacterium efficiens, a parent strain of bacteria used in the production of various amino acids including glutamic acid, using an anabaena extract obtained by acid hydrolysis. C. efficiens also grew efficiently in the anabaena extract, but did not grow in ISS (Figure 4a, b). Adding 40% (v / v) or 60% (v / v) extract increased C. efficiens 210-fold or 250-fold after 19 hours of culture, respectively (Figure 4b). Furthermore, C. efficiens grew more efficiently in the anabaena extract than in the conventional medium. C. efficiens actively consumed glucose (Figure 4c). In contrast, C. efficiens tended to produce amino acids, but no statistically significant increase was observed for any of the amino acids (Figure 4d, e).

[0062] 2-4. (Example 4) Amino acid production from Corynebacterium efficens using Anabaena extract

[0063] Next, we attempted to produce amino acids using C. efficiens. As shown in Figure 4b, the growth rate was higher with a 60% (v / v) anabaena extract obtained by acid hydrolysis than with a 40% (v / v) extract, but the variability was large. Therefore, we attempted to produce amino acids using a 40% (v / v) anabaena extract instead of a 60% (v / v) extract. C. efficiens produced a small amount of amino acids during the growth phase, but produced amino acids more actively during the stationary phase when penicillin G was added (Figure 5c). During the stationary phase, C. efficiens actively consumed aspartic acid while significantly producing 15 types of amino acids (threonine, serine, glutamic acid, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, tryptophan, lysine, and arginine) (Figures 5d-f). During the stationary phase, C. efficiens produced glutamic acid as well as threonine, isoleucine, methionine, and lysine, which are amino acids of the aspartic acid family. Furthermore, C. efficiens produced GABA during the stationary phase, which it does not produce during the growth phase (2.2 ± 0.3 μM, Figure 5g). These results were similar to those of Bacillus bacteria. On the other hand, unlike Bacillus bacteria, C. efficiens produced arginine but not ornithine. Furthermore, citrulline and hydroxyproline were not produced during either the growth or stationary phase (Figure 5g). It was shown that C. efficiens, when treated with penicillin G, can produce highly marketable aspartic acid and glutamic acid family amino acids using anabaena extract, making it a valuable source of these amino acids.

[0064] 2-5. (Example 5) Culture of Escherichia bacteria (E. coli) using Anabaena extract

[0065] Next, we attempted to culture Escherichia coli, a bacterium of the Escherichia genus widely used in the biosynthesis of proteins for food, medical, and industrial applications, using an anabaena extract obtained by acid hydrolysis. E. coli also grew efficiently with the anabaena extract, but did not grow in ISS (Figure 6a, b). When a 40% (v / v) extract was added, E. coli grew 61-fold in 15 hours of culture, which was faster than growth in conventional microbial media (56-fold in 15 hours of culture) (Figure 6b). E. coli also actively consumed glucose from the anabaena extract (Figure 6c). E. coli consumed amino acids in the conventional medium, but there was no change in the total amount of amino acids in the anabaena extract (Figure 6d). E. coli actively consumed aspartic acid, serine, and glutamic acid in the anabaena extract, but excreted alanine and leucine (Figure 6e). Alanine, an amino acid derived from pyruvate, was actively consumed in the conventional culture medium but not in the anabaena extract (Figure 6e). While it was suggested that E. coli utilizes alanine as an energy source in the conventional culture medium containing only trace amounts of glucose, it did not utilize it in the anabaena extract.

[0066] 2-6. (Example 6) Culture of Brevibacillus bacteria (B. brevis) using Anabaena extract

[0067] B. brevis proliferated efficiently in anabaena extract prepared by acid hydrolysis, but did not proliferate in ISS (Figure 7a, b). When 20% (v / v) or 40% (v / v) anabaena extract prepared by acid hydrolysis was added, cells proliferated 97-fold or 75-fold, respectively, after 19 hours of culture, which was lower than the growth rate in conventional microbial media (150-fold after 19 hours of culture) (Figure 7b). The proliferation rate of cells treated with 60% (v / v) anabaena extract prepared by acid hydrolysis was significantly reduced (Figure 7b). The cells actively consumed amino acids but not glucose (Figure 7c, d). Furthermore, they actively consumed aspartic acid, serine, glutamic acid, glycine, alanine, isoleucine, leucine, and arginine (Figure 7e). However, the 60% (v / v) anabaena extract prepared by acid hydrolysis did not consume glucose or amino acids. These results are consistent with the low growth rate of anabaena extract after acid hydrolysis.

[0068] When an anabaena extract prepared using 60% (v / v) acid hydrolysis was used, the growth rate decreased significantly. This was thought to be due to the effects of high salt concentration and osmotic pressure. Therefore, B. brevis was cultured using an extract without acid treatment (anabaena extract prepared by shaking). Almost no free glucose was detected in the extract before shaking, and only trace amounts of amino acids were detected. The amount of glucose in the anabaena extract increased with increasing shaking time, but it was significantly less than that of the acid hydrolysis extract. On the other hand, the amount of free amino acids reached a plateau in 3-4 days, and the amount was almost the same as or greater than that of the anabaena extract prepared by acid hydrolysis. This was thought to be due to the degradation of proteins by endogenous cyanobacteria proteases. Since the extract without acid treatment has a low salt concentration, it was thought that cultivation might be possible even with a 100% (v / v) extract, and B. brevis was cultured using a 100% extract. As a result, B. brevis grew in both the extract after shaking for 1 day and the extract after shaking for 3 days. Growth of brevis was observed. On the other hand, compared to anabaena extract shaken for one day, the anabaena extract shaken for three days (conditions: 30°C, 250 rpm) resulted in more efficient growth. Therefore, in the following experiments, anabaena extract subjected to three days of shaking was used. The difference in bacterial growth between extracts shaken for one day and extracts shaken for three days is thought to be due to the three-day shaking method resulting in a higher content of amino acids (as mentioned above). On the other hand, considering cost-effectiveness, anabaena extract subjected to the three-day shaking method was used, and anabaena extract subjected to longer shaking periods was not used. B. brevis grew faster using 100% (v / v) anabaena extract (3-day shaking treatment) than in conventional microbial media (280 times) (480 times after 19 hours of incubation) (Figure 8a, b). Furthermore, the microorganisms actively consumed almost all amino acids while consuming trace amounts of glucose (Figure 8c-e). These results indicate that, depending on the type of microorganism, using the optimal anabaena extract can enable more efficient growth than conventional media. B. brevis consumed more amino acids than glucose (Figure 7c, 7d, 8c, 8d). Thus, the appropriate extract can be selected during culture depending on the characteristics of the bacterial cell.As shown in Figures 2a and 2b, Bacillus bacteria that proliferated efficiently in acid hydrolysis extracts also proliferated efficiently in anabaena extracts obtained by 100% (v / v) shaking (322 ± 97 times growth after 19 hours of incubation, n = 3). Methods that do not use acid are considered to have a lower environmental impact.

[0069] 2-7. (Example 7) Culturing of genetically modified B. choshinensis using anabaena extract

[0070] Next, in order to produce recombinant protein using anabaena extract, we attempted to culture genetically modified B. choshinensis, which secretes β-casein, a bovine milk protein currently used as food. For recombinant B. choshinensis, we used anabaena extract (100% (v / v) concentration) obtained by shaking treatment, referring to previous results (Figures 7 and 8). B. choshinensis grew efficiently in the 100% (v / v) extract (240 times in 19 hours), and its growth rate was comparable to that of conventional microbial media (280 times in 19 hours of culture) (Figures 9a and 9b). B. choshinensis consumed trace amounts of glucose in the anabaena extract, but also actively consumed almost all protein-constituting amino acids, including pyruvate, aspartic acid, glycine, glutamic acid, alanine, valine, leucine, lysine, and arginine (Figure 9c-e). In the conventional medium, B. choshinensis actively consumed amino acids and a small amount of glucose, but conversely excreted pyruvate (Figure 9c-e). Furthermore, in the anabaena extract, amino acids were consumed more actively than in the conventional medium. This suggests that in the anabaena extract (shaking treatment), which has a low glucose content, not only pyruvate but also amino acids are utilized as an energy source. Importantly, B. choshinensis secreted bovine β-casein in the anabaena extract at a similar level to that in the conventional medium (40±8 μg / mL) (33±2 μg / mL) (Figure 9f). In this study, we successfully secreted and produced bovine casein protein without lysing the bacterial cells and without using conventional microbial culture media, by using Bovine casein gene-modified B. choshinensis and an anabaena extract.

[0071] Finally, we attempted to produce human IGF-I, which is used clinically as a biopharmaceutical and utilized in regenerative medicine and cell biology, using genetically modified B. choshinensis that secretes protein. B. choshinensis grew efficiently in 100% (v / v) extract, similar to conventional microbial culture media (Figure 10). B. choshinensis secreted human IGF-I in anabaena extract (176 ± 9 ng / mL), similar to conventional culture media (188 ± 7 ng / mL) (Figure 9f).

[0072] Anabaena extract contains more glucose and aspartic acid than conventional media, excluding PBN medium, and these were actively consumed by B. subtilis, C. efficiens, B. brevis, E. coli, and recombinant B. choshinensis. Glucose and aspartic acid served as energy sources for microorganisms and also provided other valuable amino acids, particularly those of the aspartic acid family. This represents a significant advantage of anabaena extract over conventional media.

[0073] 3. Additional Experiment 3-1. (Example 8) Increase in Free Amino Acid Amount Over Time with Shaking Treatment Time The effect of shaking treatment time on the amount of free amino acids in the anabaena extract was evaluated.

[0074] Following the "(ii) Shaking Treatment" described in 1-1. above, freeze-dried cyanobacteria were used as the raw material and subjected to shaking treatment at 30°C and 250 rpm. Samples were collected on day 1 and day 3 after the start of treatment, and the culture supernatant was obtained and the amount of free amino acids was measured (the method for measuring the amount of free amino acids was based on the previous research described in 1-1. (Okamoto et al., Biotechnol. Prog. 38, e3239 (2022))).

[0075] As a result, the 3-day processed extract showed a tendency for all analyzed free amino acids to increase compared to the 1-day processed extract (Figure 12).

[0076] 3-2. (Example 9) Growth of Brevibacillus bacteria using shaken extract The growth of Brevibacillus bacteria when the shaken extract was used as a culture medium was evaluated. Brevibacillus bacteria were shaken at 30°C using the shaken extract obtained in Example 8. Turbidity (OD) before incubation (0 hours) and after 19 hours ６６０ ) was measured.

[0077] The extract treated with shaking for three days showed a significant increase in the growth of Brevibacillus bacteria compared to the extract treated with shaking for one day. This result is thought to be due to the increased amount of each free amino acid confirmed in Example 8.

[0078] Figures 14 and 15 show the results of comparing the nutritional factors in acid-treated, shaking-treated (3 days), 802 medium, and LB medium. These results indicate that the anabaena extract contains each nutrient at a level comparable to or higher than that of conventional media. Furthermore, when comparing the different treatment methods for the anabaena extract, it was found that the acid-treated extract had high concentrations of glucose and aspartic acid, while the shaking-treated extract allowed for a balanced recovery of each protein-constituting amino acid and efficient recovery of vitamins.

[0079] 3-3. (Example 10) Production of various proteins by secretion using Anabaena extract As described in 1-2. Microbial culture, cells were cultured for 19 hours in Anabaena extract or conventional medium (PBN medium), the supernatant was collected, and the concentrations of each protein were analyzed using a commercially available ELISA kit. By using Anabaena extract, we succeeded in producing bovine β-casein, human α-lactalbumin, blazein, monellin, epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), albumin, and fibroblast growth factor (FGF-2) by secretion through microbial culture (Figure 16). The production efficiency was approximately the same as that of commercially available media.

[0080] 3-4. (Example 11) Cultivation using extract of a different species of nitrogen-fixing algae of the Nostocales order (Sytonema genus) An extract was prepared from bacteria of the genus Sytonema, and casein-secreting Brevibacillus bacteria were cultured in it. As a result, bacterial growth and casein secretion production were established in this extract as well. This demonstrates that the culture system of the present invention functions even when the raw material algae species are different (Figure 17).

Claims

1. A method for cultivating microorganisms, comprising culturing and growing microorganisms using a liquid containing an extract derived from nitrogen-fixing algae as a culture medium, characterized in that the liquid substantially does not contain yeast extract.

2. The method according to claim 1, characterized in that the liquid further substantially does not contain peptones other than the extract.

3. The method according to claim 1, wherein the extract is obtained by (i) acid hydrolysis, (ii) shaking, and / or (iii) heat treatment of the nitrogen-fixing algae.

4. The method according to claim 3, wherein the liquid is diluted with water and / or adjusted to a predetermined pH.

5. The method according to claim 1, wherein the nitrogen-fixing algae are cyanobacteria.

6. The method according to claim 6, wherein the cyanobacteria belong to the order Nostocales.

7. The method according to claim 1, wherein the microorganism is selected from the group consisting of bacteria of the genus Bacillus, bacteria of the genus Corynebacterium, bacteria of the genus Brevibacillus, bacteria of the genus Escherichia, lactic acid bacteria, and yeast.

8. A method for producing a substance using microorganisms, comprising culturing microorganisms using a liquid containing an extract derived from nitrogen-fixing algae as a culture medium, and causing the microorganisms to produce a substance, wherein the liquid does not contain yeast extract.

9. The method according to claim 8, characterized in that the liquid further substantially does not contain peptones other than the extract.

10. The method according to claim 8, wherein the extract is obtained by (i) acid hydrolysis, (ii) shaking, and / or (iii) heat treatment of the nitrogen-fixing algae.

11. The method according to claim 10, wherein the liquid is diluted with water and / or adjusted to a predetermined pH.

12. The method according to claim 8, characterized in that the solution contains a cell wall synthesis inhibitor.

13. The method according to claim 8, wherein the nitrogen-fixing alga is cyanobacteria.

14. The method according to claim 13, wherein the cyanobacteria belong to the order Nostocales.

15. The method according to claim 8, wherein the microorganism is selected from the group consisting of bacteria of the genus Bacillus, bacteria of the genus Corynebacterium, bacteria of the genus Brevibacillus, bacteria of the genus Escherichia, lactic acid bacteria, and yeast.

16. The method according to claim 8, wherein the substance is selected from the group consisting of amino acids, peptides, proteins, nucleic acids, and vitamins.

17. The method according to claim 8, wherein the microorganism is a genetically modified microorganism.

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