Method for producing useful substance, culture solution for culturing algae, apparatus for producing useful substance, and algae containing useful substance

Abstract

The present disclosure provides a method for producing a useful substance, the method comprising: a step for culturing algae in a culture solution containing a phosphorylation enzyme inhibitor so as to provide an algae culture; and a step for isolating the useful substance contained in the algae culture.

Description

Method for producing useful substances, culture medium for cultivating algae, apparatus for producing useful substances, and algae containing useful substances.

[0001] This disclosure relates to a method for producing a useful substance, a culture medium for cultivating algae, an apparatus for producing a useful substance, and algae containing a useful substance.

[0002] In recent years, the production of useful substances using algae has attracted attention. For example, astaxanthin, found in algae, is known as a functional substance with strong antioxidant properties. Astaxanthin is mainly produced by marine algae and accumulates in the bodies of predators through the food chain, giving a characteristic red color to seafood such as crabs and salmon.

[0003] Astaxanthin possesses antioxidant power approximately 1,000 times greater than vitamin E and 6,000 times greater than vitamin C, acting as a protective factor against ultraviolet radiation and lipid peroxidation. Furthermore, astaxanthin is known to protect the eyes from photodamage. Therefore, there is a need to establish efficient methods for producing astaxanthin, which possesses such high functionality.

[0004] Conventionally, a known method for producing astaxanthin in algae involves inducing astaxanthin accumulation by removing a nitrogen source from the culture medium (Non-Patent Literature 1). In addition, methods for improving the accumulation of useful substances by increasing the expression levels of genes such as the GPAT1 gene have also been reported (Non-Patent Literature 2).

[0005] However, methods involving nitrogen source removal make it difficult to perform large-scale cultivation at a practical level. Furthermore, since nitrogen is essential for cell proliferation, although the accumulation of astaxanthin per cell increases, an increase in cell density cannot be expected, and as a result, there are limitations to improving productivity.

[0006] On the other hand, methods involving gene function modification face challenges such as the difficulty of completely removing foreign genes, and the limitation of the available algal strains for gene function modification to model algae used in academia. Furthermore, identifying suitable target genes for modification requires considerable time and effort. In this context, there is a need for the development of simpler and more efficient methods for producing useful substances.

[0007] Li F, Cai M, Wu Y, Lian Q, Qian Z, Luo J, Zhang Y, Zhang N, Li C and Huang X (2022) Effects of Nitrogen and Light Intensity on the Astaxanthin Accumulation in Motile Cells of Haematococcus pluvialis. Front. Mar. Sci. 9:909237.Fukuda S, Hirasawa E, Takemura T, Takahashi S, Chokshi K, Pancha I, Tanaka K, Imamura S. Accelerated triacylglycerol production without growth inhibition by overexpression of a glycerol-3-phosphate acyltransferase in the unicellular red alga Cyanidioschyzon merolae. Sci Rep. 2018 Aug 17;8(1):12410.

[0008] This disclosure was made to solve the above-mentioned problems and aims to provide a method for producing useful substances, a culture medium for cultivating algae, an apparatus for producing useful substances, and algae containing useful substances.

[0009] One aspect of this disclosure is a method for producing a useful substance, comprising the steps of: culturing algae in a culture medium containing a phosphorylation enzyme inhibitor to provide an algal culture; and isolating a useful substance contained in the algal culture. Another aspect of this disclosure is a culture medium for culturing algae, which contains a phosphorylation enzyme inhibitor. Another aspect of this disclosure is a apparatus for producing a useful substance, which comprises a culture mechanism for culturing algae in a culture medium containing a phosphorylation enzyme inhibitor to provide an algal culture; and an isolation mechanism for isolating a useful substance from the algal culture. Another aspect of this disclosure is algae containing a useful substance, wherein the useful substance contains fatty acids, and the fatty acid content is 800 μg / 10 ８ It is an algae, larger than cells.

[0010] According to this disclosure, a method for producing a useful substance, a culture medium for cultivating algae, an apparatus for producing a useful substance, and algae containing the useful substance can be provided.

[0011] Figure 1 is an overall configuration diagram showing one embodiment of the apparatus for producing useful substances in this disclosure. Figure 2 shows the total amount of fatty acids in diatoms (Chaetoceros gracilis) one day after the addition of each inhibitor.

[0012] <Method for Producing Useful Substances> According to one embodiment of the present disclosure, a method for producing useful substances is provided, comprising the steps of: culturing algae in a culture medium containing a phosphorylation enzyme inhibitor to provide an algal culture; and isolating useful substances contained in the algal culture. According to this embodiment, the amount of useful substances accumulated in algae can be increased and the useful substances can be efficiently isolated by a simple method of culturing, for example, wild-type algae in a culture medium containing a phosphorylation enzyme inhibitor, without necessarily involving genetic modification.

[0013] The method for producing the useful substance of the embodiment includes the step of culturing algae in a culture medium containing a phosphorylation enzyme inhibitor to provide an algal culture.

[0014] The "useful substance" in the present disclosure is a substance having utility as a component such as pharmaceuticals, food additives, supplements, etc., utility in industry, utility in agriculture, and other industrial utilities. The useful substance can particularly be a substance having commercial value in an isolated state. The useful substance in the present disclosure can include low molecular weight compounds. The molecular weight of the "low molecular weight compound" in the present disclosure can be 1000 g / mol or less, 900 g / mol or less, or 800 g / mol or less. Also, the "low molecular weight compound" can be an organic compound. The "low molecular weight compound" in the present disclosure can be a compound having a molecular weight of 800 g / mol or less, or an organic compound having a molecular weight of 800 g / mol or less. The useful substance in the present disclosure can be lipophilic or water-soluble.

[0015] The useful substance in the embodiment can be one or more of 1,2-dipalmitoyl-glycero-3-phosphoglycerol (1,2-dipalmitoyl-glycero-3-phosphoglycerol), astaxanthin (astaxanthin), 7α-hydroxycholesterol (7α-hydroxy cholesterol), docosatrienoic acid (docosatrienoic acid; also referred to as FA(22:3)), γ-tocopherol (γ-tocopherol), α-tocopherol (α-tocopherol), prostaglandin J2 (prostaglandin J2), prostaglandin A2 (prostaglandin A2), trilaurin (trilaurin), oleamide (oleamide), flavanone (flavanone), 3-hydroxyhexadecanoic acid (3-hydroxyhexadecanoic acid), and cholesterol sulfate (cholesterol sulfate), or can include one or more of these. It is understood by those skilled in the art that these compounds are compounds that can be detected by LC-MS analysis using a reverse phase column such as an ODS column, and many can be classified as lipophilic useful substances.

[0016] The useful substances in the embodiments are ornithine, citrulline, nicotinamide mononucleotide (NMN), allantoin, maltobionic acid, methionine, histidine, glutathione (GSH), S-adenosylmethionine, valine, isoleucine-proline-proline, isoleucine, N-acetylglucosamine, β-alanine, decanoic acid, octanoic acid, leucine, phenylalanine, and citric acid. acid (citric acid), threonine, tryptophan, thiamine, γ-glutamyl-citrulline, inosine, N-acetyllysine, isoleucine-glycine, 3-methylcytidine, alanine, 3-amino-2-piperidone, N-acetylasparagine, N-acetylalanine, N-acetyl-β-alanine, arginine-glutamate, N-acetyltyrosine, guanosine, 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylic acid acid (5-amino-3,4-dihydro-2H-pyrrole-2-carboxylic acid), GDP-mannose, GDP-glucose, γ-glutamyl-alanine, arginine ethyl ester, acetyl-CoA, hypoxanthine, putrescine, asparagine, γ-glutamyl-lysine, N-(1-deoxy-1-fructosyl)glycine, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, glucose 1-phosphate, pyruvic acid, 2,4-diaminobutyric acid acid (2,4-diaminobutyric acid), pantothenic acid (pantothenic acid), arginine (arginine), N-acetylvaline (N-acetylvaline), isovalerylglycine (isovalerylglycine), N-valerylglycine (N-valerylglycine), N6,N6,N6-trimethyllysine (N6,N6,N6-trimethyllysine), ε-fructoselysine, γ-glutamyl-glutamine, glycine, N7-(1-carboxyethyl)arginine, MG-H2, methionine sulfoxide, asymmetric dimethylarginine (ADMA), 1-methyladenosine, nicotinamide riboside, 4-methylpentanoic It may be one or more of the following, or may contain one or more of these: acid (4-methylpentanoic acid), serine, β-alanyl-lysine, 1,3-diaminopropane, cystathionine, Nω-methylarginine, tyrosine, choline, 2-amino-2-methyl-1-propanol, 1-methylhistidine, 3-methylhistidine, myristoleic acid, γ-glutamyl-ornithine, N6,N6-dimethyllysine, and N6-formyllysine. These compounds can be detected by capillary electrophoresis-mass (CE-MS) analysis, and it is understood by those skilled in the art that many can be classified as water-soluble useful substances. Ornithine has been reported to exhibit effects such as growth hormone secretion and fatigue recovery (Pons, A., Bescos, R., Sureda, A., Tur,JA (2017). Metabolic Precursors of l-Arginine Supplementation in Sports: A Focus on l-Citrulline and l-Ornithine. In: Patel, V., Preedy, V., Rajendram, R. (eds) L-Arginine in Clinical Nutrition. Nutrition and Health. Humana Press, Cham. https: / / doi.org / 10.1007 / 978-3-319-26009-9_24; Moriyasu, K.; Nakajima, A.; Morita, M. Effects of Oral Ingestion of L-Ornithine on Mental Stress and Fatigue Based on the Trier Social Stress Test in Healthy Humans: A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Trial. J. Clin. Med. 2024, 13, 7583. https: / / doi.org / 10.3390 / jcm13247583. Citrulline improves blood flow and contributes to muscle and metabolic health (Allerton, TD; Proctor, DN; Stephens, JM; Dugas, TR; Spielmann, G.; Irving, BA l-Citrulline Supplementation: Impact on Cardiometabolic Health. Nutrients 2018, 10, 921. https: / / doi.org / 10.3390 / nu10070921). Nicotinamide mononucleotide (NMN) is known as an anti-aging substance (Nadeeshani H, Li J, Ying T, Zhang B,Lu J. Nicotinamide mononucleotide (NMN) as an anti-aging health product - Promises and safety concerns. J Adv Res. 2021 Aug 11;37:267-278. doi: 10.1016 / j.jare.2021.08.003. PMID: 35499054; PMCID: PMC9039735.)

[0017] The phosphorylating enzyme in the present disclosure is not limited as long as it is an enzyme that catalyzes a phosphorylation reaction. In an embodiment, the phosphorylating enzyme can be a phosphotransferase that transfers the γ-phosphate group of ATP to a substrate. The phosphorylation transfer reaction by the phosphorylating enzyme may depend on divalent cations such as Mg 2+ or Mn 2+ etc. The phosphorylating enzyme of the embodiment is preferably a protein kinase, more preferably a serine / threonine kinase. Examples of such serine / threonine kinases include the target of rapamycin (TOR).

[0018] The target of rapamycin (TOR) in the present disclosure can be TOR1 (the product of NCBI Gene ID: 853529), which is the catalytic subunit of a serine-threonine kinase complex identified as the target molecule of the immunosuppressant rapamycin in budding yeast, or TOR2 (the product of NCBI Gene ID: 853632), or the product of a corresponding gene (e.g., an ortholog) of a gene encoding these catalytic subunits. Alternatively, the target of rapamycin (TOR) can be a protein complex containing these gene products. The target of rapamycin in the present disclosure can be the product of an endogenous gene of algae, the product of a foreign gene, or both, or can include both.

[0019] The inhibitors in this disclosure are inhibitors that inhibit the activity of phosphorylation enzymes. The inhibitors in the embodiments are preferably small molecule compounds. The inhibitors in the embodiments may be protein kinase inhibitors, or serine / threonine kinase inhibitors. The inhibitors in the embodiments may also be ATP-competitive inhibitors. The inhibitors in the embodiments may be rapamycin target protein inhibitors, or inhibitors that bind to the catalytic domain contained in the rapamycin target protein. Examples of rapamycin target protein inhibitors that can be used in the embodiments include AZD8055, Torin 1, rapamycin, mTOR Inhibitor III (PP242), mTOR Inhibitor IV (Ku-63794), PI 3-K / mTOR Inhibitor III (PKI-179), mTOR Inhibitor XII (Torin2), mTOR Kinase Inhibitor II (WYE-354), PTK / PI 3-K / mTOR Inhibitor (PP121), or caffeine. Among these, caffeine is inexpensive and contained in food, making it cost-effective and suitable for use as an inhibitor for safely producing useful substances. Alternatively, the phosphorylation enzyme inhibitor in the embodiment may be a phosphatidylinositol-3 kinase (PI3K) inhibitor. Examples of PI3K inhibitors that can be used in the embodiment include PI 3-K / mTOR Inhibitor III (PKI-179), LY294002, wortmannin, AS604850, or SW30. It will be understood by those skilled in the art that at least some TOR inhibitors, including AZD8055, Torin 1, and PI 3-K / mTOR Inhibitor III (PKI-179), also have inhibitory activity against PI3K.

[0020] AZD8055 (AZD-8055) is also known as [5-[2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-2-methoxyphenyl]methanol ([5-[2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-2-methoxyphenyl]methanol) and is a compound having the structure shown below.

[0021] Torin 1 is also known as 1-[4-(4-propanoylpiperazin-1-yl)-3-(trifluoromethyl)phenyl]-9-quinolin-3-ylbenzo[h][1,6]naphthyridin-2-one and is a compound having the following structure.

[0022] The culture medium containing the phosphorylation enzyme inhibitor of the embodiment can be prepared by adding the inhibitor to a culture medium for algae. Alternatively, the culture medium containing the phosphorylation enzyme inhibitor of the embodiment may be prepared by adding the phosphorylation enzyme inhibitor to a pre-cultured culture in a medium that does not contain the phosphorylation enzyme inhibitor, so that the inhibitor is incorporated into the culture. The inhibitor may be dissolved in a suitable organic solvent such as DMSO beforehand and then added to the culture medium.

[0023] The concentration of the phosphorylation enzyme inhibitor in the embodiment may be a concentration that reduces the growth rate of algae by 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more compared to the growth rate in the control algal culture. The concentration of the phosphorylation enzyme inhibitor in the embodiment may be a concentration that reduces the growth rate of algae by 95% or less, 90% or less, 80% or less, 70% or less, 60% or less, or 50% or less compared to the growth rate in the control algal culture. Alternatively, the concentration of the phosphorylation enzyme inhibitor in the culture medium may be a concentration of the inhibitor that reduces the growth rate of algae by 10%, 20%, 30%, 40%, or 50%. By selecting such concentrations, it is possible to produce useful substances while maintaining the growth of algae.

[0024] The growth rate of algae is determined by the logarithmic growth phase (e.g., OD). 750 Algae with an O(OD) of 0.1–0.4, 0.25–0.3, or 0.27 can be cultured in the presence of an inhibitor and evaluated by measuring the increase in cell density after a certain period of time (12 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours). At this time, the cell density is OD 750 The optical density can be evaluated using such methods. Furthermore, the increase in cell density can be calculated as a relative value compared to a control culture. In this case, as the control algal culture, an algal culture to which only an organic solvent such as DMSO that dissolves the inhibitor has been added may be used. Specifically, it has been confirmed that when AZD8055 was used at a final concentration of 20 nM or Torin 1 at a final concentration of 0.6 μM for the diatom Chaetoceros gracilis, the increase in algal concentration (growth rate) after 24 hours decreased by approximately 50%. The optimal concentration of the inhibitor can be determined for other algal species and inhibitors by measuring the growth rate using a similar method. Alternatively, as the growth rate of the algae in the embodiment, the "specific growth rate," which is a coefficient for calculating the growth rate from the algal concentration, may be calculated and used by a method known to those skilled in the art.

[0025] In embodiments, the concentration of the phosphorylation enzyme inhibitor may be a concentration that inhibits TOR activity by 10%, 20%, 30%, 40%, or 50%. TOR activity can be measured by a kinase assay of an immunoprecipitate using a TOR-specific antibody. In this case, an immunoprecipitate prepared using an algal culture to which only an organic solvent such as DMSO that dissolves the inhibitor has been added can be used as a control. TOR activity can be calculated as a relative value to the kinase activity of the control sample.

[0026] The concentration of the phosphorylation enzyme inhibitor contained in the culture medium of the embodiment may be 0.001 nM or higher, 0.01 nM or higher, 0.05 nM or higher, 0.1 nM or higher, 1 nM or higher, 5 nM or higher or 10 nM or higher, 0.1 μM or higher, 0.25 μM or higher, 0.5 μM or higher or 0.6 μM or higher, and may be 100 μM or lower, 50 μM or lower or 20 μM or lower, 10 μM or lower, 5 μM or lower, 2 μM or lower or 0.6 μM or lower, 100 nM or lower or 10 nM or lower.

[0027] The concentration of AZD8055 contained in the culture medium of the embodiment may be 0.001 nM or higher, 0.01 nM or higher, 0.05 nM or higher, 0.1 nM or higher, 1 nM or higher, 5 nM or higher, or 10 nM or higher, and may be 100 μM or less, 50 μM or less, or 20 μM or less, 10 μM or less, 5 μM or less, 2 μM or less, 0.6 μM or less, 100 nM or less, or 10 nM or less. From the viewpoint of maintaining algal growth, preferably the concentration of AZD8055 is 0.001 nM or higher, 0.01 nM or higher, 0.05 nM or higher, 0.1 nM or higher, 1 nM or higher, 5 nM or higher, or 10 nM or higher, and 100 nM or less, or 30 nM or less.

[0028] The concentration of Torin 1 contained in the culture medium of the embodiment may be 0.001 nM or higher, 0.01 nM or higher, 0.05 nM or higher, 0.1 nM or higher, 1 nM or higher, 5 nM or higher, or 10 nM or higher, 0.1 μM or higher, 0.25 μM or higher, 0.5 μM or higher, or 0.6 μM or higher, and may be 100 μM or less, 50 μM or less, or 20 μM or less, 10 μM or less, 5 μM or less, 2 μM or less, or 0.6 μM or less. From the viewpoint of maintaining algal growth, preferably the concentration of Torin 1 is 0.001 nM or higher, 0.01 nM or higher, 0.05 nM or higher, 0.1 nM or higher, 1 nM or higher, 5 nM or higher, or 10 nM or higher, and 1 μM or less, or 0.6 μM or less.

[0029] The concentration of caffeine contained in the culture medium of the embodiment may be 1 mM or more, 5 mM or more, 10 mM or more, 20 mM or more, 40 mM or more, 60 mM or more, or 80 mM or more, and may be 120 mM or less, 100 mM or less, 80 mM or less, 60 mM or less, or 40 mM or less.

[0030] The types of algae in this disclosure are not limited. The algae may be non-sessile algae. The algae may be, for example, unicellular algae or microalgae. Examples of algae that may be used include any of the following classifications: Aurantiochytrium, Chlamydomonas, Chlorella, red algae Schizoan, Spirulina, Botryococcus, Euglena, haptophytes, Prasinophytes, green algae, brown algae, red algae, cyanobacteria, diatoms, yellow-green algae, golden algae, dinoflagellates, and seaweed. More specific examples of algae include algae of the genera Chaetoceros, Isochrysis, Pavlova, Pyramimonas, and Tisochrysis, with a preference for algae of the genus Chaetoceros, such as Chaetoceros gracilis. The algae in this disclosure may be a single species of algae or a population containing multiple species of algae. For example, a population of the same species of algae with genetic variation may be used. The algae of this disclosure may be algae derived from isolated algae. In the production methods of the embodiments, the algae may include algae of the genus Chaetoceros. In the production methods of the embodiments, it is preferable that the algae are algae having a gene encoding rapamycin target protein (TOR) or its catalytic subunit.

[0031] Algal cultivation can be carried out using culture media for algae known to those skilled in the art, seawater, other environmental water, diluted seawater, artificial seawater, mixtures thereof, or solutions with a partially common composition, depending on the properties of the control algae. The culture media for algae can be any aqueous solution in which natural algae can grow without particular limitations, or a specified aqueous medium for algae can be used. The medium may be an algal culture medium known to those skilled in the art, and may, for example, contain nutrients, carbon sources, rare metals, etc. Specific examples of the aqueous medium include IMK medium, SWM-3 medium, modified SWM-3 (mSWM-3) medium, modified media of these media, and media obtained by mixing these media or modified media. IMK medium contains 200mg / L NaNO3, 1.4mg / L Na2HPO4, 5mg / L K2HPO4, 2.68mg / L NH4Cl, 5.2mg / L Fe-EDTA, 0.332mg / L Mn-EDTA, 37.2mg / L Na2-EDTA, 0.023mg / L ZnSO4・7H2O, 0.014mg / L CoSO4・7H2O, 0.0073mg / L Na2MoO4・2H2O, 0.0025mg / L CuSO4・5H2O, 0.0017mg / L H2SeO3, 0.2mg / L Thiamin-HCl, 0.0015mg / L Biotin, 0.0015mg / L Vitamin B12 0.18mg / L A medium consisting of MnCl2・4H2O and the balance seawater. In particular, when culturing diatoms, 0.2–1 mM Na2SiO3 may be added to the IMK medium in addition to the above components.The mSWM-3 medium is a medium (pH 7.7 - 7.8) prepared by mixing 17 mg of NaNO3, 1.56 mg of NaH2PO4·2H2O, 5.68 mg of Na2SiO3·9H2O, 1.12 mg of Na2EDTA·2H2O, 0.084 mg of Fe-EDTA, 0.0346 μg of Na2SeO3, 1 ml of P-1 metal solution (618.3 mg of H3BO4, 69.25 mg of MnCl2·4H2O, 5.45 mg of ZnCl2, 238 μg of CoCl2·6H2O, 100 ml of distilled water), 0.2 μg of Vitamin B12, 1 mL of vitamin mixed solution S3 (5 mg of Thiamine HCl, 1 mg of Nicotinic acid, 1 mg of Calcium pantothenate, 0.1 mg of p-Aminobenzoic acid, 0.01 mg of Biotin, 50 mg of Inositol, 0.02 mg of Folic acid, 30 mg of Thymine, 100 mL of distilled water), 50 mg of Tris(hydroxymethyl)aminomethane and 98 mL of seawater. The cultivation of algae may be carried out under natural light or with light irradiation (for example, continuous white light irradiation of 50 μmol photon m -2 s -1 . Also, the cultivation of algae may be shake cultivation or may be carried out while bubbling air or sterilized air.

[0032] The method for producing a useful substance in the embodiment may further include a step of pre-culturing algae in a culture solution containing no inhibitor of phosphorylase. "Pre-culturing" may mean culturing algae in a culture solution containing no inhibitor of phosphorylase before culturing in a culture solution containing an inhibitor of phosphorylase. Specifically, for example, using a general algae culture medium such as IMK medium, at 25°C, under continuous irradiation of white light of 50 μmol photon m -2 s -1 , the cells can be cultured under the condition of stirring while bubbling sterilized air. In pre-culturing, when the algae reach a predetermined cell density (for example, about 1×10 in the logarithmic growth phase 6After reaching a cell / ml (or similar) concentration, the culture can be converted to a phosphorylation enzyme inhibitor (for example, a TOR inhibitor such as AZD8055 at a final concentration of 20 nM or Torin 1 at a final concentration of 0.6 μM) by adding the inhibitor to the culture medium. Alternatively, the conversion to culturing algae in a culture medium containing the phosphorylation enzyme inhibitor can be achieved by preparing a new culture medium from the culture obtained in the pre-culture stage through a medium exchange. In this case, the phosphorylation enzyme inhibitor may be added to the fresh medium used for the medium exchange.

[0033] The method for producing useful substances in the embodiment includes a step of isolating useful substances contained in an algal culture. In this disclosure, "isolation" of a useful substance may mean separating and purifying the useful substance contained in the algal culture from the culture. The culture may be subjected to solid-liquid separation such as centrifugation, and depending on whether the useful substance is contained in the solid components such as cells in the culture or in the liquid components such as the culture medium, the supernatant or pellet may be subjected to subsequent isolation steps. The pellet containing cells may be washed with a suitable washing solution such as sterile artificial seawater, and then dissolved with a dissolving solution containing an organic solvent such as ethanol to extract the useful substance. At this time, cell disruption may be performed by physical means such as sonication. Furthermore, the culture supernatant or the sample after cell disruption may be subjected to pretreatment including one or more of stirring, centrifugation, filtration, ultrafiltration, deproteinization, and solid-phase extraction (SPE).

[0034] Pre-treated or unpre-treated samples can be separated by high-performance liquid chromatography (HPLC). Columns suitable for HPLC include reversed-phase columns such as C18 columns (e.g., octadecylsilyl (ODS) columns (2 × 50 mm, 2 μm)), normal-phase columns such as silica columns, and gel filtration columns. Those skilled in the art will understand that these columns can be appropriately selected depending on the target small molecule, such as 1,2-dipalmitoyl-glycero-3-phosphoglycerol, astaxanthin, 7α-hydroxycholesterol, docosatrienoic acid, γ-tocopherol, α-tocopherol, prostaglandin J2, prostaglandin A2, trilaurin, oleamide, flavanone, 3-hydroxyhexadecanoic acid, and cholesterol sulfate. The mobile phase used in HPLC can be appropriately selected by those skilled in the art depending on the column and target molecule. For example, the above substance can be eluted using a gradient elution method with an ODS column and mobile phase A (water / 0.1% formic acid) and mobile phase B (isopropanol:acetonitrile:water = 65:30:5, 0.1% formic acid, 2 mM ammonium formate). The eluted useful substance can be fractionated, and after removing the solvent, it can be recovered as a dry powder. The purity of the recovered useful substance can be confirmed, for example, by analysis using a high-resolution mass spectrometer. If the purity does not reach the target value (e.g., 95% or higher), separation and purification may be performed again by HPLC.

[0035] Detection in HPLC can utilize detection methods known to those skilled in the art, such as mass spectrometry (MS), tandem mass spectrometry (MS / MS), ultraviolet-visible spectroscopy (UV-Vis), fluorescence spectroscopy (FL), differential refractive index (RI), and evaporative light scattering detection (ELS). Mass spectrometers used in MS and MS / MS include Fourier transform mass spectrometers (FT-MS) and time-of-flight mass spectrometers (TOF-MS). In particular, using high-resolution mass spectrometers such as FT-MS enables highly accurate metabolite identification with a mass error of ±10 ppm or less.

[0036] <Culture medium for culturing algae> According to another embodiment of the present disclosure, a culture medium for culturing algae can be provided, which includes a phosphorylation enzyme inhibitor. The elements of this embodiment (e.g., algae, culture, culture medium, phosphorylation enzyme, or inhibitor) can be described in the section on <Method for producing useful substances>. The culture medium for culturing algae can be prepared using a general algae culture medium such as IMK medium as a base medium. Examples of phosphorylation enzyme inhibitors used in this embodiment include TOR inhibitors such as AZD8055, Torin 1, rapamycin, mTOR Inhibitor III (PP242), mTOR Inhibitor IV (Ku-63794), PI 3-K / mTOR Inhibitor III (PKI-179), mTOR Inhibitor XII (Torin2), mTOR Kinase Inhibitor II (WYE-354), PTK / PI 3-K / mTOR Inhibitor (PP121), or caffeine. Alternatively, the phosphorylation enzyme inhibitor in the embodiment may be a phosphatidylinositol-3 kinase (PI3K) inhibitor. Examples of PI3K inhibitors that can be used in the embodiment include LY294002, wortmannin, AS604850, or SW30. The concentration of the phosphorylation enzyme inhibitor in the culture medium of the embodiment may be a concentration that reduces the growth rate of algae by 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more compared to the growth rate of the control algal culture. The concentration of the phosphorylation enzyme inhibitor in the embodiment may be a concentration that reduces the growth rate of algae by 95% or less, 90% or less, 80% or less, 70% or less, 60% or less, or 50% or less compared to the growth rate of the control algal culture. Alternatively, the concentration of the phosphorylation enzyme inhibitor in the culture medium may be a concentration of the inhibitor that reduces the growth rate of algae by 10%, 20%, 30%, 40%, or 50%.

[0037] Alternatively, the concentration of the phosphorylation enzyme inhibitor in the culture medium in the embodiment may be a concentration of the inhibitor that inhibits the activity of the phosphorylation enzyme by 10%, 20%, 30%, 40%, or 50%. In the embodiment, the concentration of the phosphorylation enzyme inhibitor in the culture medium may be a concentration of the inhibitor that inhibits the activity of TOR by 10%, 20%, 30%, 40%, or 50%. These inhibitors can be added to the culture medium dissolved in a suitable organic solvent such as DMSO.

[0038] The culture medium of this embodiment can promote the production of lipid-soluble useful substances such as 1,2-dipalmitoyl-glycero-3-phosphoglycerol, astaxanthin, 7α-hydroxycholesterol, docosatrienoic acid, γ-tocopherol, α-tocopherol, prostaglandin J2, prostaglandin A2, trilaurin, oleamide, flavanone, 3-hydroxyhexadecanoic acid, cholesterol sulfate, and fatty acids while maintaining the growth of algae. These lipid-soluble useful substances are known to have preventive and therapeutic effects against neonatal respiratory syndrome, antioxidant effects, or functions to prevent the infiltration of inflammatory cells as components of lipid membranes.

[0039] Fatty acids include myristic acid (C14:0), pentadecanoic acid (C15:0), palmitic acid (C16:0), palmitoleic acid (C16:1), heptadecanoic acid (C17:0), heptadecenoic acid (C17:1), stearic acid (C18:0), elaidic acid (C18:1n9t), oleic acid (C18:1n9c), linoleic acid (C18:2n6c), γ-linolenic acid (C18:3n6), α-linolenic acid (C18:3n3), and arachidic acid. Examples include acid (arachidic acid; C20:0), eicosenoic acid (eicosenoic acid; C20:1n9), dihomo-γ-linolenic acid (dihomo-γ-linolenic acid; C20:3n6), arachidonic acid (arachidonic acid; C20:4n6), behenic acid (behenic acid; C22:0), erucic acid (erucic acid; C22:1n9), lignoceric acid (lignoceric acid; C24:0), and one or more of the following: docosahexaenoic acid (docosahexaenoic acid; DHA; C22:6n3), decanoic acid, octanoic acid, and myristoleic acid.

[0040] The culture medium of this embodiment maintains the growth of algae while containing ornithine, citrulline, nicotinamide mononucleotide (NMN), allantoin, maltobionic acid, methionine, histidine, glutathione (GSH), S-adenosylmethionine, valine, isoleucine-proline-proline, isoleucine, N-acetylglucosamine, β-alanine, decanoic acid, octanoic acid, leucine, phenylalanine, and citric acid. acid (citric acid), threonine, tryptophan, thiamine, γ-glutamyl-citrulline, inosine, N-acetyllysine, isoleucine-glycine, 3-methylcytidine, alanine, 3-amino-2-piperidone, N-acetylasparagine, N-acetylalanine, N-acetyl-β-alanine, arginine-glutamate, N-acetyltyrosine, guanosine, 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylic acid acid (5-amino-3,4-dihydro-2H-pyrrole-2-carboxylic acid), GDP-mannose, GDP-glucose, γ-glutamyl-alanine, arginine ethyl ester, acetyl-CoA, hypoxanthine, putrescine, asparagine, γ-glutamyl-lysine, N-(1-deoxy-1-fructosyl)glycine, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, glucose 1-phosphate, pyruvic acid, 2,4-diaminobutyric acid acid (2,4-diaminobutyric acid), pantothenic acid (pantothenic acid), arginine (arginine), N-acetylvaline (N-acetylvaline), isovalerylglycine (isovalerylglycine), N-valerylglycine (N-valerylglycine), N6,N6,N6-trimethyllysine (N6,N6,N6-trimethyllysine), ε-fructoselysine, γ-glutamyl-glutamine, glycine, N7-(1-carboxyethyl)arginine, MG-H2, methionine sulfoxide, asymmetric dimethylarginine (ADMA), 1-methyladenosine, nicotinamide riboside, 4-methylpentanoic This can promote the production of useful substances such as acid (4-methylpentanoic acid), serine, β-alanyl-lysine, 1,3-diaminopropane, cystathionine, Nω-methylarginine, tyrosine, choline, 2-amino-2-methyl-1-propanol, 1-methylhistidine, 3-methylhistidine, myristoleic acid, γ-glutamyl-ornithine, N6,N6-dimethyllysine, and N6-formyllysine. Many of these water-soluble beneficial substances are known to have functions such as promoting growth hormone secretion, improving fatigue, improving blood flow, maintaining muscle and metabolic health, anti-aging effects, antioxidant effects, promoting wound healing, promoting bone mineralization, anti-diabetic effects, improving hypertension, anti-inflammatory effects, immunomodulatory effects, improving athletic performance, and providing essential amino acids.

[0041] The culture medium of the embodiment can be prepared by adding a phosphorylation enzyme inhibitor to fresh culture medium. Alternatively, the culture medium of the embodiment can be prepared by adding a pre-cultured culture in a medium that does not contain a phosphorylation enzyme inhibitor, for example, algae, to the logarithmic growth stage (e.g., about 1 × 10⁻⁶). 6 The preparation may also be carried out in the culture by adding a phosphorylation enzyme inhibitor when the cell / ml level reaches a certain point.

[0042] <Apparatus for Manufacturing Useful Substances> According to yet another embodiment of the present disclosure, an apparatus for manufacturing useful substances can be provided, comprising: a culture mechanism for culturing algae in a culture medium containing a phosphorylation enzyme inhibitor to provide an algal culture; and an isolation mechanism for isolating a useful substance from the algal culture. The elements of this embodiment (e.g., algae, culture, culture medium, phosphorylation enzyme, or inhibitor, etc.) can be described in the sections <Method for Manufacturing Useful Substances> and <Culture Medium for Cultivating Algae>.

[0043] The culture mechanism of the embodiment (Figure 1) comprises a culture tank for culturing algae, in which algae are cultivated in a culture medium containing a phosphorylation enzyme inhibitor. The culture mechanism of the embodiment may include a temperature control unit (e.g., including a heater and temperature sensor capable of maintaining a temperature suitable for algae cultivation, such as 20°C to 35°C), a light irradiation unit (e.g., including an LED light capable of continuously irradiating with white light), and an aeration and stirring unit (e.g., including an air pump and bubbling device capable of supplying sterile air via a sterile filter). The culture mechanism may also include a turbidimeter for measuring the cell density in the culture tank and various sensors for measuring the culture environment, such as pH and dissolved oxygen.

[0044] The isolation mechanism of the embodiment (Figure 1) is a mechanism for isolating useful substances from algal cultures. The isolation mechanism of the embodiment may include a solid-liquid separation unit (e.g., including a centrifuge), a cell disruption unit (e.g., including an ultrasonic disruptor), and a useful substance separation unit (e.g., including an HPLC system equipped with a separation column such as an ODS column, capable of gradient elution, and a high-resolution mass spectrometer).

[0045] The apparatus for producing useful substances in the embodiment may have a pre-culture mechanism for pre-culturing algae. The pre-culture mechanism in the embodiment (Figure 1) includes a pre-culture tank and may include a temperature control unit, a light irradiation unit, and an aeration and stirring unit similar to those of the culture mechanism described above. Furthermore, the pre-culture mechanism may have a cell density of a predetermined value (for example, about 1 × 10⁻⁶). 6 The pre-culture mechanism may include a turbidimeter capable of detecting when the cell / ml level reaches a certain point, and a control unit capable of automatically transferring one of the following pre-cultures to the culture mechanism based on the detection signal. In another embodiment, the pre-culture mechanism may include a control unit capable of automatically adding a phosphorylation enzyme inhibitor to the pre-culture tank, or automatically exchanging the pre-culture with fresh medium containing the phosphorylation enzyme inhibitor when transferring the pre-culture to the culture mechanism.

[0046] In this embodiment, the pre-culture mechanism, the culture mechanism, and the isolation mechanism are connected to each other by piping or the like, allowing for the automatic transfer of cultures or samples. Furthermore, the operation of each mechanism may be centrally controlled by a central control unit.

[0047] According to the manufacturing apparatus of this embodiment, a series of processes, from the addition of phosphorylation enzyme inhibitors to the isolation of useful substances, can be carried out in an automated manner, minimizing human intervention and enabling the efficient and highly reproducible production of useful substances.

[0048] <Algae containing useful substances> According to yet another embodiment of the present disclosure, algae containing a useful substance, wherein the useful substance contains a fatty acid, and the fatty acid content is 800 μg / 10 ８ Algae with a cell count of 1 or more are provided. The descriptions in the sections on <Method for producing useful substances> and <Culture medium for culturing algae> may be applied to the elements of this embodiment (e.g., algae, useful substances, etc.).

[0049] The algae of one embodiment may be algae cultured in a caffeine-containing culture medium. The algae of this embodiment have a characteristic fatty acid composition profile compared to the same type of algae cultured in a caffeine-free culture medium. The total fatty acid content in the algae of this embodiment is 1000 μg / 108 cells or more, 1200μg / 10 8 cells or more, 1500μg / 10 8 More than 1 cell, or 1800 μg / 10 8 It may be more than 10 cells. The total fatty acid content is 10,000 μg / 10 8 cells or less, 5000μg / 10 8 Cells or less or 3000 μg / 10 8 It may be smaller than cells.

[0050] The algae in one embodiment may be a diatom, and may belong to the genus Chaetoceros. The algae in the embodiment may be Chaetoceros gracilis.

[0051] The algae of one embodiment exhibit a change in fatty acid composition specific to caffeine treatment. Specifically, the algae of this embodiment have a palmitic acid (C16:0) content of 150 μg / 10 compared to the same type of algae cultured in a caffeine-free culture medium. 8 The number of cells is greater than 180 μg / 10 8 The algae may be more than 10 μg / 10 8 For cells larger than 10, the arachidic acid (C2O:0) content is 6 μg / 10 8 For cells larger than 10, the behenic acid (C22:0) content is 6 μg / 10 8 More than 10 cells, or containing 5 μg / 10 of lignoceric acid (C24:0). 8 The algae may satisfy at least one of the above conditions. The algae of one embodiment exhibit the above content for all of these long-chain saturated fatty acids. Caffeine treatment differs from other TOR inhibitor treatments such as AZD8055 or Torin 1 in that it can achieve a high content of these long-chain saturated fatty acids.

[0052] The algae in this embodiment have a myristic acid (C14:0) content of 50 μg / 10 8The levels are below 1 / 10, or erucic acid (C22:1n9) is substantially undetectable (e.g., 1 μg / 10). 8 It may have the following characteristics (below cells). Such a decrease or disappearance of specific fatty acids may result from specific metabolic regulation by caffeine.

[0053] The following examples illustrate the concept, but this disclosure is not limited to the examples described below.

[0054] Example 1: Analysis of metabolite changes in the diatom Chaetoceros gracilis after treatment with a TOR inhibitor.

[0055] Diatom Chaetoceros gracilis undergoes logarithmic growth phase in IMK medium (approximately 1 × 10⁻⁶). 6 The cells were cultured up to the limit (cells / ml). Culture conditions were 25°C, white light with 50 μmol photons, and the cells were agitated by bubbling air. The cultured cells were supplemented with AZD8055 at a final concentration of 10 nM or Torin 1 at a final concentration of 0.6 μM. In preliminary experiments, the growth rate of C. gracilis decreased by approximately 50% at these concentrations. The growth rate of C. gracilis is determined by the logarithmic growth phase (e.g., OD). 750 C. gracilis (=0.27) was cultured in the presence or absence of the inhibitor, and the OD (Oxygen Demand) was measured after 24 hours. 750 The evaluation was performed by measuring [the relevant parameter]. An equal amount of DMSO, the solvent for the inhibitor, was added to the control.

[0056] After 24 hours of inhibitor or DMSO addition, cells were collected by centrifugation (25°C, 16,000 g, 10 minutes). The collected cells were washed twice with artificial seawater, and then an ethanol solution containing 1 μM of internal standard was added, followed by sonication for 30 seconds. Milli-Q water was then added and the mixture was stirred, followed by centrifugation (2,300 × g, 4°C, 5 minutes), and the supernatant was collected. The collected supernatant was allowed to dry and redissolved in 50% isopropanol aqueous solution (v / v) to prepare the sample for measurement.

[0057] Metabolites were measured by CE-MS or LC-MS. CE-MS was performed according to the measurement method described in Japanese Patent No. 6106864. LC-MS was performed using a Thermo Fisher Scientific Vanquish Flex UHPLC System and Orbitrap Exploris 240. An ODS column (2 × 50 mm, 2 μm) was used as the analytical column, and gradient analysis was performed using mobile phase A (water / 0.1% formic acid) and mobile phase B (isopropanol:acetonitrile:water = 65:30:5, 0.1% formic acid, 2 mM ammonium formate). Measurements were performed in both positive and negative modes. Unless otherwise specified, in the following examples, metabolite measurements were performed by CE-MS or LC-MS using the same apparatus configuration, analytical column, mobile phase, and gradient conditions as described above, and ionization was performed in both positive and negative modes.

[0058] The measurement data was analyzed using MasterHands ver.2.20.0.1 (developed by Keio University), and peaks with an S / N ratio of 3 or higher were automatically extracted. The obtained peak area values ​​were corrected using the area values ​​of internal standards and converted to relative area values. Metabolite identification was performed by matching with an HMT metabolite library under conditions of mass error ±10 ppm and retention time error ±0.15 minutes.

[0059] As a result, 1,2-dipalmitoyl-glycero-3-phosphoglycerol, astaxanthin, 7α-hydroxycholesterol, docosatrienoic acid (FA(22:3)), γ-tocopherol, α-tocopherol, prostaglandin J2 / prostaglandin A2, trilaurin, oleamide, flavanone, 3-hydroxyhexadecanoic acid, and cholesterol sulfate were identified as metabolites that significantly increased upon treatment with Torin 1 or AZD8055 (Table 1). In particular, 1,2-dipalmitoyl-glycero-3-phosphoglycerol (peak 1; one of two corresponding peaks observed) showed a 21.0-fold increase with Torin 1 treatment and a 3.5-fold increase with AZD8055 treatment. These metabolites are known to have preventive and therapeutic effects against neonatal respiratory syndrome, antioxidant effects, or functions that prevent the infiltration of inflammatory cells as components of lipid membranes.

[0060]

[0061] The results described above illustrate that treatment of algae with TOR inhibitors increases the production of various useful substances. In particular, treatment with Torin 1 revealed a 2.0 to 21.0-fold increase in the production of useful substances. These results suggest that controlling algal metabolism using TOR inhibitors may be applicable to the efficient production of useful substances.

[0062] Example 2: Analysis of changes in water-soluble metabolites of the diatom Chaetoceros gracilis after treatment with a TOR inhibitor.

[0063] Diatom Chaetoceros gracilis undergoes logarithmic growth phase in IMK medium (approximately 1 × 10⁻⁶). 6 The cells were cultured up to (cells / ml). Culture was carried out at 25°C under white light with 50 μmol photons m -2 s -1The cells were agitated by bubbling air while irradiating them with a light source. The cultured cells were then treated with either AZD8055 at a final concentration of 20 nM or Torin 1 at a final concentration of 0.6 μM. An equal volume of DMSO, the solvent for the inhibitor, was added to the control.

[0064] Cells were collected by centrifugation (25°C, 16,000 g, 10 minutes) one and three days after the addition of the inhibitor or DMSO. Metabolites were extracted from the collected cells, and the amount of metabolites was measured by CE-FTMS.

[0065] Table 2 shows the ratio (induction rate) of the content of each useful substance in inhibitor-treated algae to the content in control algae. Allantoin, maltobionic acid, ornithine, methionine, citrulline, histidine, glutathione, S-adenosylmethionine, valine, isoleucine-proline-proline, isoleucine, N-acetylglucosamine, β-alanine, decanoic acid, octanoic acid, leucine, phenylalanine, nicotinamide mononucleotide, citric acid, threonine, and tryptophan were identified as water-soluble metabolites that significantly increased with AZD8055 or Torin 1 treatment. In particular, allantoin showed a remarkable increase of 343.04 times with AZD8055 treatment (3 days) and 133.72 times with Torin 1 treatment (3 days). ornithine showed an 85.98-fold increase after AZD8055 treatment (3 days) and a 71.23-fold increase after Torin 1 treatment (3 days). These metabolites are known to have functions such as promoting growth hormone secretion, improving fatigue, anti-aging effects, antioxidant effects, promoting wound healing, promoting bone mineralization, and providing essential amino acids.

[0066]

[0067] Example 3: Analysis of changes in lipid-soluble metabolites of the diatom Chaetoceros gracilis due to caffeine treatment.

[0068] Diatom Chaetoceros gracilis undergoes logarithmic growth phase in IMK medium (approximately 1 × 10⁻⁶). 6 The cells were cultured up to (cells / ml). Culture was carried out at 25°C under white light with 50 μmol photons m -2 s -1 The process was carried out while irradiating with light and agitating the cells by bubbling air. Final concentrations of 10 mM, 20 mM, 40 mM, or 80 mM caffeine were added to the culture medium during the logarithmic growth phase. Nothing was added to the control culture medium.

[0069] Cells were collected 24 hours after caffeine addition by centrifugation (25°C, 16,000 g, 10 minutes). Metabolites were extracted from the collected cells, and their amounts were measured by LC-FTMS.

[0070] Table 3 shows the induction rates of each useful substance in caffeine-treated algae compared to the control. Caffeine treatment induced the accumulation of 1,2-dipalmitoyl-glycero-3-phosphoglycerol, astaxanthin, 7α-hydroxycholesterol, γ-tocopherol, α-tocopherol, prostaglandin J2 / prostaglandin A2, trilaurin, oleamide, 3-hydroxyhexadecanoic acid, cholesterol sulfate, cholesterolone, and β-cryptoxanthin. In particular, γ-tocopherol increased 48.75-fold with 20 mM caffeine treatment, and cholesterolone increased 40.05-fold with 80 mM caffeine treatment. These results suggest that caffeine functions as a TOR inhibitor and exhibits a useful substance accumulation induction effect similar to that of AZD8055 and Torin 1.

[0071]

[0072] Example 4: Analysis of lipid-soluble metabolite changes in the red alga Cyanidioschyzon merolae after rapamycin treatment

[0073] Red algae Cyanidioschyzon merolae SF12 strain in MA2 medium during logarithmic growth phase (OD 750 The culture was carried out at 40°C under white light with 50 μmol photons. -2 s -1 The procedure was performed while irradiating the cells and bubbling air through them. Rapamycin was added to the culture medium to a final concentration of 2 μM. An equal volume of DMSO, the solvent for rapamycin, was added to the control.

[0074] Cells were collected by centrifugation (25°C, 16,000 g, 10 minutes) one and three days after the addition of rapamycin or DMSO. Metabolites were extracted from the collected cells, and the amount of metabolites was measured by LC-FTMS.

[0075] Table 4 shows the induction rates of useful substances in Cyanidisoschyzon merolae treated with rapamycin. Rapamycin treatment induced the accumulation of glucosylceramide (d18:1 / 24:1), 1,2-dipalmitoyl-glycero-3-phosphoglycerol, sitosterol, 3-hydroxytetradecanoic acid, α-tocopherol, and zeaxanthin. In particular, glucosylceramide (d18:1 / 24:1) showed a remarkable 16.1-fold increase after 3 days of treatment. Zeaxanthin has a function of protecting the eyes, and α-tocopherol is known to have antioxidant effects as vitamin E.

[0076]

[0077] Example 5: Analysis of changes in water-soluble metabolites of the red alga Cyanidioschyzon merolae after rapamycin treatment

[0078] Cyanidioschyzon merolae SF12 strain was cultured under the same conditions as in Example 4 and treated with rapamycin. Metabolites were extracted from the recovered cells, and the amount of metabolites was measured by CE-FTMS.

[0079] Rapamycin treatment induced the accumulation of numerous metabolites (Table 5). Particularly significant increases were observed in thiamine (7.4 times after 1 day treatment, 16.9 times after 3 days treatment), γ-glutamyl-citrulline (6.2 times after 1 day treatment, 9.5 times after 3 days treatment), inosine (5.1 times after 1 day treatment), citrulline (4.8 times after 1 day treatment, 5.5 times after 3 days treatment), 3-methylcytidine (11.8 times after 3 days treatment), and ornithine (2.0 times after 1 day treatment). These metabolites are known to have functions such as providing nutrition as vitamin B1, improving blood flow, maintaining muscle and metabolic health, and promoting the secretion of growth hormone.

[0080]

[0081] Example 6: Changes in the fatty acid composition of diatoms due to TOR inhibitor treatment

[0082] The diatom Chaetoceros gracilis was cultured under the same conditions as in Examples 1-3 and treated with AZD8055 (10 nM), Torin 1 (0.6 μM), or caffeine (40 mM) for 24 hours. Fatty acids were extracted from the harvested cells, and the fatty acid composition was analyzed by gas chromatography-mass spectrometry (GC-MS).

[0083] Treatment with TOR inhibitors AZD8055, Torin 1, and caffeine increased the total fatty acid content compared to the control (Figure 2). In all TOR inhibitor treatments, the total fatty acid content was 800 μg / 10 8 The total fatty acid content exceeds 1000 μg / 10 cells, and especially with Torin 1 and caffeine treatment, the total fatty acid content is 1000 μg / 10 8 It went beyond cells.

[0084] Treatment-dependent changes were also observed in the fatty acid profiles of algae (Table 6). For example, palmitic acid (C16:0) increased to 242.5 μg / 10 after Torin 1 treatment. 8 Cells increased to approximately 2.5 times that of the control, and palmitoleic acid (C16:1) was 207.2 μg / 10 after caffeine treatment. 8The cells increased to approximately 1.5 times the control group.

[0085] Caffeine showed a specific change in fatty acid composition among TOR inhibitors. Specifically, in the caffeine-treated group, C16:0 was 197.6 μg / 10 8 cells and C16:1 207.2 μg / 10 8 Cells showed high levels, and C18:0 was 11.8 μg / 10 8 cells, C20:0 6.9 μg / 10 8 cells, C22:0 6.6 μg / 10 8 cells, C24:0 5.4 μg / 10 8 The levels rose to the cells, and C14:0 was 48 μg / 10 8 C22:1n9 was undetectable due to suppression by cells. In contrast, AZD8055 and Torin 1 detected 5.9 μg / 10 of C18:0, respectively. 8 cells and 9.0 μg / 10 8 cells, C20:0: 3.2 μg / 10 8 cells and 4.3 μg / 10 8 cells, C22:0: 3.1 μg / 10 8 cells and 4.2 μg / 10 8 cells, C24:0: 2.7 μg / 10 8 cells and 3.6 μg / 10 8 It remained in cells and did not exhibit a similar profile.

[0086] The above results illustrate that culturing algae in a culture medium containing AZD8055, Torin 1, and a rapamycin-targeted protein inhibitor containing caffeine can promote the production and / or accumulation of useful substances by algae.

[0087] This disclosure includes the following embodiments: [Claim 1] A method for producing a useful substance, comprising the steps of: culturing algae in a culture medium containing a phosphorylation enzyme inhibitor to provide an algal culture; and isolating a useful substance contained in the algal culture. [Claim 2] The method according to Claim 1, further comprising the step of pre-culturing the algae in a culture medium not containing a phosphorylation enzyme inhibitor. [Claim 3] The method according to Claim 1 or 2, wherein the phosphorylation enzyme is a rapamycin target protein. [Claim 4] The method according to any one of Claims 1 to 3, wherein the inhibitor is AZD8055 or Torin 1. [Claim 5] The method according to any one of Claims 1 to 3, wherein the inhibitor is caffeine. [Claim 6] The method according to any one of Claims 1 to 5, wherein the concentration of the inhibitor in the culture medium is such that the growth rate of the algae is reduced by 5% or more and 80% or less compared to the growth rate of a control algal culture. [Item 7] The useful substances include astaxanthin, 1,2-dipalmitoyl-glycero-3-phosphoglycerol, 7α-hydroxycholesterol, docosatrienoic acid, γ-tocopherol, α-tocopherol, prostaglandin J2, prostaglandin A2, trilaurin, oleamide, flavanone, 3-hydroxyhexadecanoic acid, cholesterol sulfate, cholesterolone, β-cryptoxanthin, myristic acid (C14:0), pentadecanoic acid (C15:0), palmitic acid (C16:0), palmitoleic acid (C16:1), heptadecanoic acid (C17:0), heptadecenoic acid (C17:1), stearic acid (C18:0), elaidic acid (elaidic acid; C18:1n9t), oleic acid (oleic acid; C18:1n9c), linoleicacid (linoleic acid; C18:2n6c), γ-linolenic acid (γ-linolenic acid; C18:3n6), α-linolenic acid (α-linolenic acid; C18:3n3), arachidic acid (arachidic acid; C20:0), eicosenoic acid (eicosenoic acid; C20:1n9), dihomo-γ-linolenic acid (dihomo-γ-linolenic acid; C20:3n6), arachidonic acid (arachidonic acid; C20:4n6), behenic acid (behenic acid; C22:0), erucic acid (erucic acid; C22:1n9), lignoceric acid (lignoceric acid; C24:0), and docosahexaenoic acid (docosahexaenoic acid; DHA; C22:6n3), decanoic acid (decanoic acid), octanoic A method for producing a product according to any one of items 1 to 6, comprising one or more of the following: acid (octanoic acid) and myristoleic acid. [Item 8] The useful substances include ornithine, citrulline, nicotinamide mononucleotide (NMN), allantoin, maltobionic acid, methionine, histidine, glutathione (GSH), S-adenosylmethionine, valine, isoleucine-proline-proline, isoleucine, N-acetylglucosamine, β-alanine, decanoic acid, octanoic acid, leucine, phenylalanine, and citric acid.acid (citric acid), threonine, tryptophan, thiamine, γ-glutamyl-citrulline, inosine, N-acetyllysine, isoleucine-glycine, 3-methylcytidine, alanine, 3-amino-2-piperidone, N-acetylasparagine, N-acetylalanine, N-acetyl-β-alanine, arginine-glutamate, N-acetyltyrosine, guanosine, 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylic acid acid (5-amino-3,4-dihydro-2H-pyrrole-2-carboxylic acid), GDP-mannose, GDP-glucose, γ-glutamyl-alanine, arginine ethyl ester, acetyl-CoA, hypoxanthine, putrescine, asparagine, γ-glutamyl-lysine, N-(1-deoxy-1-fructosyl)glycine, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, glucose 1-phosphate, pyruvic acid, 2,4-diaminobutyric acid acid (2,4-diaminobutyric acid), pantothenicpantothenic acid, arginine, N-acetylvaline, isovalerylglycine, N-valerylglycine, N6,N6,N6-trimethyllysine, ε-fructoselysine, γ-glutamyl-glutamine, glycine, N7-(1-carboxyethyl)arginine, MG-H2, methionine sulfoxide, asymmetric dimethylarginine (ADMA), 1-methyladenosine, nicotinamide riboside, 4-methylpentanoic A method for producing a product according to any one of claims 1 to 6, comprising one or more of the following: acid (4-methylpentanoic acid), serine, β-alanyl-lysine, 1,3-diaminopropane, cystathionine, Nω-methylarginine, tyrosine, choline, 2-amino-2-methyl-1-propanol, 1-methylhistidine, 3-methylhistidine, myristoleic acid, γ-glutamyl-ornithine, N6,N6-dimethyllysine, and N6-formyllysine. [Item 9] The method for producing algae according to any one of items 1 to 8, wherein the algae are diatoms or red algae. [Item 10] A culture medium for culturing algae, comprising a phosphorylation enzyme inhibitor. [Item 11]Apparatus for producing useful substances, comprising: a culture mechanism for culturing algae in a culture medium containing a phosphorylation enzyme inhibitor to provide an algal culture; and an isolation mechanism for isolating a useful substance from the algal culture. [Item 12] Algae containing a useful substance, wherein the useful substance contains a fatty acid, and the fatty acid content is 800 μg / 10 ８ Algae having cells or more. [Clause 13] The algae according to Claim 12, wherein the algae are diatoms or red algae. [Clause 14] The algae according to Claim 12 or 13, wherein the algae are algae cultured in a caffeine-containing culture medium.

[0088] While this disclosure has been described with reference to several embodiments described above, this disclosure is not limited to the examples given in these embodiments. Various modifications can be made to the configuration and details of the present invention within the scope of this disclosure.

Claims

1. A method for producing a useful substance, comprising the steps of: culturing algae in a culture medium containing a phosphorylation enzyme inhibitor to provide an algal culture; and isolating a useful substance contained in the algal culture.

2. The manufacturing method according to claim 1, further comprising the step of pre-culturing the algae in a culture medium that does not contain a phosphorylation enzyme inhibitor.

3. The manufacturing method according to claim 1 or 2, wherein the inhibitor is caffeine.

4. The manufacturing method according to claim 3, wherein the concentration of the inhibitor in the culture medium is such that the growth rate of the algae is reduced by 5% or more and 80% or less compared to the growth rate of the control algae culture.

5. The above useful substances include astaxanthin, 1,2-dipalmitoyl-glycero-3-phosphoglycerol, 7α-hydroxycholesterol, docosatrienoic acid, γ-tocopherol, α-tocopherol, prostaglandin J2, prostaglandin A2, trilaurin, oleamide, flavanone, 3-hydroxyhexadecanoic acid, cholesterolone, β-cryptoxanthin, cholesterol sulfate, myristic acid (C14:0), pentadecanoic acid (C15:0), palmitic acid (C16:0), palmitoleic acid (C16:1), heptadecanoic acid (C17:0), heptadecenoic acid (C17:1), stearic acid (C18:0), elaidic acid (elaidic acid; C18:1n9t), oleic acid (oleic acid; C18:1n9c), linoleic acid (linoleic acid; C18:2n6c), γ-linolenic acid (γ-linolenic acid; C18:3n6), α-linolenic acid (α-linolenic acid; C18:3n3), arachidic acid (arachidic acid; C20:0), eicosenoic acid (eicosenoic acid; C20:1n9), dihomo-γ-linolenic acid (dihomo-γ-linolenic acid; C20:3n6), arachidonic acid (arachidonic acid; C20:4n6), behenic acid (behenic acid; C22:0), erucic acid (erucic acid; C22:1n9), lignoceric acid (lignoceric acid; C24:0), and docosahexaenoic acid Acids (docosahexaenoic acid; DHA;A method for producing a substance according to claim 1 or 2, comprising one or more of the following: C22:6n3), decanoic acid, octanoic acid, and myristoleic acid.

6. The above-mentioned useful substances include ornithine, citrulline, nicotinamide mononucleotide (NMN), allantoin, maltobionic acid, methionine, histidine, glutathione (GSH), S-adenosylmethionine, valine, isoleucine-proline-proline, isoleucine, N-acetylglucosamine, β-alanine, decanoic acid, octanoic acid, leucine, phenylalanine, and citric acid. acid (citric acid), threonine, tryptophan, thiamine, γ-glutamyl-citrulline, inosine, N-acetyllysine, isoleucine-glycine, 3-methylcytidine, alanine, 3-amino-2-piperidone, N-acetylasparagine, N-acetylalanine, N-acetyl-β-alanine, arginine-glutamate, N-acetyltyrosine, guanosine, 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylic acid acid (5-amino-3,4-dihydro-2H-pyrrole-2-carboxylic acid), GDP-mannose, GDP-glucose, γ-glutamyl-alanine, arginine ethyl ester, acetyl-CoA, hypoxanthine, putrescine, asparagine, γ-glutamyl-lysine, N-(1-deoxy-1-fructosyl)glycine, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, glucose 1-phosphate, pyruvic acid, 2,4-diaminobutyric acid acid (2,4-diaminobutyric acid), pantothenic acid (pantothenic acid), arginine (arginine), N-acetylvaline (N-acetylvaline), isovalerylglycine (isovalerylglycine), N-valerylglycine (N-valerylglycine), N6,N6,N6-trimethyllysine (N6,N6,N6-trimethyllysine), ε-fructoselysine, γ-glutamyl-glutamine, glycine, N7-(1-carboxyethyl)arginine, MG-H2, methionine sulfoxide, asymmetric dimethylarginine (ADMA), 1-methyladenosine, nicotinamide riboside, 4-methylpentanoic A method for producing a product according to claim 1 or 2, comprising one or more of the following: acid (4-methylpentanoic acid), serine, β-alanyl-lysine, 1,3-diaminopropane, cystathionine, Nω-methylarginine, tyrosine, choline, 2-amino-2-methyl-1-propanol, 1-methylhistidine, 3-methylhistidine, myristoleic acid, γ-glutamyl-ornithine, N6,N6-dimethyllysine, and N6-formyllysine.

7. Algae containing a useful substance, wherein the useful substance contains a fatty acid, and the fatty acid content is 800 μg / 10 ８ Algae that are larger than or equal to cells.

8. The algae according to claim 7, wherein the algae is a diatom or a red alga.

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