Microorganism having hyaluronic acid-producing ability, variant thereof, method for producing hyaluronic acid using same, medium composition for microorganism, and method for producing hyaluronic acid using same

Abstract

The present invention relates to a Streptococcus zooepidemicus SMT013 strain having an increased ability to produce hyaluronic acid, a variant thereof, and a method for producing hyaluronic acid using same. The Streptococcus zooepidemicus SMT013 strain according to an aspect has an increased ability to produce hyaluronic acid, and thus can be used to produce hyaluronic acid. Also, provided are a medium composition for a microorganism producing hyaluronic acid, the medium composition comprising arginine and uridine, and a method for producing hyaluronic acid using same. The medium composition for a microorganism producing hyaluronic acid, according to an aspect, can be used to produce hyaluronic acid with increased efficiency.

Description

Microorganisms capable of producing hyaluronic acid, variants thereof, methods for producing hyaluronic acid using the same, culture medium compositions for the said microorganisms, and methods for producing hyaluronic acid using the same

[0001] The present invention relates to a Streptococcus zooepidemicus SMT013 strain having the ability to produce hyaluronic acid, a variant thereof, and a method for producing hyaluronic acid using the same.

[0002] The present invention relates to a culture medium composition for microorganisms that produce hyaluronic acid, and a method for producing hyaluronic acid using the same.

[0003] Hyaluronic acid is a colorless, transparent, high-viscosity polysaccharide with a molecular weight of 50,000 to 13,000,000 Da, in which glucuronic acid and N-acetylglucosamine are alternately linked in (1-3) and (1-4). Hyaluronic acid is a substance secreted by living organisms for self-protection purposes and is widely used, especially in the medical and cosmetic fields, due to its excellent moisturizing, lubricating, and protective effects against the invasion of bacteria.

[0004] Hyaluronic acid can be extracted from biological tissues such as chicken combs, skin, umbilical cords, joint fluid, and bovine eyes, but these tissues contain impurities such as chondroitin sulfate and glucose aminoglycans, which require high costs to separate and purify the product by excluding them, and the yield is also low, making it uneconomical. In contrast, producing hyaluronic acid using microorganisms allows for the production of high-yield hyaluronic acid at a relatively low cost.

[0005] Microorganisms known as hyaluronic acid-producing strains include, most notably, Streptococcus disgalactiae, *Zeuepidemicus*, *Equi*, *Piogenes*, *Equisimilis*, and *Faecalis* of the genus Streptococcus, but these microorganisms are known to have relatively low hyaluronic acid productivity. There is still a demand for Streptococcus *Zeuepidemicus* strains with increased hyaluronic acid production capacity.

[0006] Korean Patent No. 10-0258237 discloses a method for producing hyaluronic acid by culturing Streptococcus epidemicus ATCC 35246 in a medium containing 0.1 to 5 g / L of uridine. Additionally, Japanese Patent Publication No. 1987-289198 discloses a method for producing hyaluronic acid by culturing Streptococcus equi in a medium containing arginine and glutamic acid, wherein hyaluronic acid production is significantly increased in a medium containing arginine and glutamic acid compared to a medium containing only arginine or glutamic acid (Table 3). Japanese Patent Publication No. 1988-141594 discloses a method for producing hyaluronic acid by culturing Streptococcus equi ATCC9527 in a medium containing 1.0 to 4.0 g / L of arginine (Tables 1 and 2). However, the above patent documents do not disclose producing hyaluronic acid by culturing Streptococcus epidemicus in a medium containing arginine and uridine.

[0007] Therefore, there is still a need for an alternative culture medium composition for culturing microorganisms capable of producing hyaluronic acid, even with conventional technology, and a method for producing hyaluronic acid using the same.

[0008] One aspect provides one or more microorganisms capable of producing hyaluronic acid selected from the Streptococcus zooepidemicus SMT013 strain (accession number KCCM13522P) and variants thereof.

[0009] Another aspect provides a composition containing the above-mentioned microorganism.

[0010] Another aspect provides a kit containing the above-mentioned microorganism.

[0011] Another aspect provides a method for producing hyaluronic acid using the above-mentioned microorganism.

[0012] Another aspect provides hyaluronic acid produced by a method for producing hyaluronic acid using the above-mentioned microorganism.

[0013] One aspect provides a culture medium composition for microorganisms that produce hyaluronic acid, comprising arginine and uridine.

[0014] Another aspect provides a method for producing hyaluronic acid, comprising the step of culturing the microorganism in the microorganism medium composition.

[0015] One aspect provides a microorganism capable of producing hyaluronic acid selected from one or more of the Streptococcus zooepidemicus SMT013 strain (accession number KCCM13522P) and variants thereof.

[0016] The above SMT013 strain is a strain selected from Streptococcus zooepidemicus SMT001 strain isolated from horse respiratory tract by inducing random mutations using EMS.

[0017] The above SMT013 strain has significantly increased hyaluronic acid production capacity compared to the parent strain S. zooepidemicus SMT001.

[0018] The above variant may contain further genetic modifications in the SMT013 strain.

[0019] As used herein, the term "genetic modification" includes changing the composition or structure of the genetic material of a cell.

[0020] The above genetic modification may be caused by random mutation induction or by artificial genetic manipulation, for example, by site-specific mutation induction.

[0021] The above random mutation may be induced by exposing or contacting the cell with physical, chemical, and molecular mutagens. The physical mutagens may be, for example, ultraviolet (UV), gamma rays, atmospheric and room temperature plasma (ARTP), or a combination thereof. The mutagens may be ethanemethylsulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (NTG), 5-bromouracil (5-BU), methylmethane sulfonic acid (MMS), or a combination thereof. The molecular mutagens may be transposons.

[0022] The above genetic modification may be performed by genetic manipulation. Genetic manipulation may be induced by homologous recombination, allelic exchange, Cre-loxP-based system, FLP-FRT recombination system, CRISPR-Cas9 system, RNA interference (RNAi), episomal plasmid-mediated gene overexpression, or a combination thereof.

[0023] The above genetic modification may be one or more of the following: a genetic modification that reduces the toxicity of the SMT013 strain, a genetic modification that increases the hyaluronic acid production capacity, a genetic modification that increases the molecular weight of the hyaluronic acid, and a genetic modification that increases the viscosity of the hyaluronic acid.

[0024] The toxicity of the SMT013 strain may be due to hemolysin. Genetic modifications that reduce the toxicity of the SMT013 strain may be genetic modifications that reduce the expression of hemolysin or its biosynthetic pathway proteins.

[0025] In this specification, the term "hemolysin" may be a substance that causes the lysis of blood cells, such as red blood cells, by destroying cell membranes, such as lipids and proteins. The hemolysin may form pores in the lipid bilayer of blood cells or hydrolyze the phospholipids of the bilayer. The hemolysin may cause β-hemolysis.

[0026] The above hemolytic toxin may be streptolysin S (SLS), streptolysin O (SLO), NADase (NAD+glycohydrolase), or a combination thereof.

[0027] In this specification, the term “genetic modification that reduces expression” includes the complete elimination or reduction of expression. The reduction of expression may be due to a deletion or disruption of a gene. The “deletion” or “disruption” of a gene may be a mutation in part or all of the gene, or a mutation in part or all of the regulatory sequence of the gene, such as a promoter or terminator, which causes the gene to be not expressed or to be expressed at a reduced level compared to the natural gene product, or to express a gene product (e.g., an enzyme) having no activity or reduced activity. The mutation may include the addition, substitution, insertion, deletion, or conversion of one or more nucleotides of the gene.

[0028] The above-mentioned decrease in expression may be a decrease of about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 55% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 100% compared to parent cells or "wild-type" cells.

[0029] The above hemolytic toxin may have an amino acid sequence of any one of SEQ ID NOs 1 to 9 or sequence identity of 80% or more, for example, 85% or more, 90% or more, 92.5% or more, 95.0% or more, or 97.5% or more. Genetic modifications that reduce the expression of the above hemolytic toxin may reduce the expression of streptolysin S (SLS), streptolysin S-like peptides, and genes for their biosynthetic pathway proteins. The above hemolytic toxin gene may be sagA, sagB, sagC, sagD, sagE, sagF, sagG, sagH, or sagI.

[0030] The genetic modification that increases the hyaluronic acid production capacity, hyaluronic acid molecular weight, or hyaluronic acid viscosity may be one or more of the genetic modifications that increase the expression of genes related to hyaluronic acid biosynthesis and the genetic modifications that decrease the expression of genes related to one or more of hyaluronic acid degradation and byproduct generation.

[0031] In this specification, increased expression may be due to the introduction of a polynucleotide encoding an enzyme or polypeptide into a cell, an increase in its copy number, or a mutation in the regulatory region of said polynucleotide. The cell into which said gene is introduced may or may not contain said gene. said gene may be operably linked to a regulatory sequence that enables its expression, for example, a promoter, an enhancer, a polyadenylation site, or a combination thereof. The polynucleotide introduced from the outside or whose copy number is increased may be endogenous or exogenous. The endogenous gene refers to a gene that existed on the genetic material contained within a microorganism. The exogenous gene refers to a gene introduced into a cell from the outside, and the introduced gene may be homologous or heterologous with respect to the host cell into which it is introduced. "Heterologous" may mean foreign rather than native.

[0032] In this specification, the term "copy number increase" may be due to the introduction or amplification of the gene, and includes cases where a gene not present in the unmanipulated cell is acquired through genetic manipulation. The introduction of the gene may be mediated by a vehicle such as a vector. The introduction may be a transient introduction in which the gene is not integrated into the genome, or an insertion into the genome. The introduction may be achieved, for example, by introducing a vector into which a polynucleotide encoding the target polypeptide has been inserted into the cell, and then the vector is replicated within the cell or the polynucleotide is integrated into the genome.

[0033] In the present specification, the increase in expression may be increased by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 50% or more, about 60% or more, about 70% or more, or about 100% or more compared to the parent cell or wild-type cell.

[0034] The aforementioned genes related to hyaluronic acid biosynthesis are hyaluronan synthase, UDP-glucose dehydrogenase, UDP-glucose pyrophosphorylase, phosphoglucomutase, phosphoglucoisomerase, glutamine amidotransferase, phosphoglucosamine mutase, acCoA acetyl transferase, UDP-N-acetylglucosamine pyrophosphorylase, or vitreoscilla hemoglobin (VHb). It could be a gene, or a combination of them.

[0035] The gene involved in the degradation of hyaluronic acid may be a hyaluronidase gene. The gene involved in the formation of the byproduct may be a lactate dehydrogenase gene.

[0036] The above hyaluronidase may have the amino acid sequence of SEQ ID NO. 10 or sequence identity of 80% or more, for example, 85% or more, 90% or more, 92.5% or more, 95.0% or more, or 97.5% or more. The above hyaluronidase gene may be a hylB or hyl gene.

[0037] In one embodiment, the SMT013 strain may further include genetic modifications that reduce the expression of one or more of the streptolysin S gene and the hyaluronidase gene.

[0038] The above SMT013 strain may further include genetic modifications that increase or decrease the expression of one or more genes related to sugar metabolism and cell wall synthesis.

[0039] The genes related to glucose metabolism mentioned above may be associated with one or more of glycolysis, the TCA cycle, and glucose uptake. These genes may be, but are not limited to, genes encoding citrate lyase, fructose-bisphosphate aldolase, fumarase glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, glucokinase, pyruvate dehydrogenase kinase, pyruvate kinase, or enzymes involved in the PTS system.

[0040] The above-mentioned cell wall synthesis-related genes may be, but are not limited to, genes encoding UDP-GlcNAc enol pyruvate transferase, DP-N-acetyl allylacetone glucosamine reductase, and decadecenyl diphosphate-MurNAc-pentapeptide-UDP-GlcNAcGlcNAc transferase.

[0041] Another aspect provides a composition containing the above-mentioned microorganism.

[0042] The above composition may include a carrier. The carrier may be a culture medium, a buffer, an excipient, a stabilizer, or a combination thereof.

[0043] Another aspect provides a kit containing the microorganism. The kit may include a carrier. The carrier may be a culture medium, a buffer, an excipient, a stabilizer, or a combination thereof.

[0044] Another aspect provides a method for producing hyaluronic acid comprising the step of producing hyaluronic acid by culturing the above-mentioned SMT013 strain (accession number KCCM13522P) or a variant thereof in a medium.

[0045] In the above-mentioned culture step, the medium may contain a carbon source, a nitrogen source, and amino acids.

[0046] The carbon source may be glucose, fructose, maltose, galactose, glycerol, or a combination thereof. The nitrogen source may be yeast extract, casein peptone, casein acid hydrolysate, casein enzyme hydrolysate, bactopeptone, neopeptone, or a combination thereof. The amino acid may be glutamine, lysine, cysteine, arginine, methionine, aspartic acid, glycine, or a combination thereof.

[0047] The carbon source is glucose, and the nitrogen source may be yeast extract.

[0048] The above medium may not contain animal-derived components. The above medium may further contain glutamic acid or its salt, arginine or its salt, and uridine or its salt. The concentrations of glutamic acid or its salt, arginine or its salt, and uridine or its salt may be 0.1 to 15.0 g / L, 0.5 to 10.0 g / L, 0.5 to 10.0 g / L, and 0.1 to 5.0 g / L, respectively.

[0049] The pH of the above medium may be 6.0 to 8.0, for example, 6.5 to 7.5.

[0050] The above culture may be performed at 34 to 38 ℃.

[0051] The above culture may be carried out under aerobic conditions, for example, under aeration of 0.1 to 2.0 vvm. The above culture may be carried out under stirring.

[0052] The above method may include the step of separating hyaluronic acid from a culture. The step of separating hyaluronic acid from a culture may include the step of removing cells from the culture to obtain a culture medium. Cell removal may be by centrifugation, filtration, precipitation, or a combination thereof.

[0053] The step of separating hyaluronic acid from the culture may include the step of separating hyaluronic acid from the culture medium. The step may involve, but is not limited to, performing one or more of the following processes on the hyaluronic acid present in the culture medium: ethanol precipitation, activated carbon treatment, alumina treatment, and ultrafiltration.

[0054] Another aspect provides hyaluronic acid produced by the above method.

[0055] The hyaluronic acid may have a molecular weight of 0.1 to 5.0 MDa, for example, 0.5 to 4.0 MDa, or 1.0 to 2.5 MDa.

[0056]

[0057] Another aspect provides a culture medium composition for microorganisms that produce hyaluronic acid, comprising arginine and uridine.

[0058] In this specification, the term “arginine, glutamic acid, or uridine” includes salts thereof. Arginine may be L-arginine or a hydrochloride salt thereof. Glutamic acid may be L-glutamic acid or a sodium salt thereof, for example, monosodium glutamic acid (MSG). Uridine includes uridine monophosphate, uridine diphosphate, or uridine triphosphate.

[0059] In the above culture medium composition, the concentration of the arginine or its salt may be 0.1 to 15.0 g / L, 0.5 to 10.0 g / L, 0.5 to 8.0 g / L, 0.5 to 6.0 g / L, 0.5 to 4.0 g / L, 0.5 to 3.0 g / L, 0.5 to 2.5 g / L, 1.0 to 2.5 g / L, 1.5 to 2.5 g / L, or about 2.0 g / L.

[0060] In the above culture medium composition, the concentration of uridine or its salt may be 0.01 to 10.0 g / L, 0.1 to 5.0 g / L, 0.1 to 4.0 g / L, 0.1 to 3.0 g / L, 0.1 to 2.0 g / L, 0.1 to 1.0 g / L, 0.2 to 1.0 g / L, 0.3 to 1.0 g / L, 0.3 to 0.9 g / L, 0.4 to 0.9 g / L, 0.5 to 0.8 g / L, or about 0.75 g / L.

[0061] The above medium composition may further comprise glutamic acid or a salt thereof. In the above medium composition, the concentration of the glutamic acid or a salt thereof may be 0.1 to 15.0 g / L, 0.5 to 10.0 g / L, 0.5 to 8.0 g / L, 0.5 to 6.0 g / L, 0.5 to 4.0 g / L, 0.5 to 3.0 g / L, 0.5 to 2.5 g / L, 1.0 to 2.5 g / L, 1.5 to 2.5 g / L, or about 2.0 g / L.

[0062] The above-described culture medium composition may be used to culture the microorganism in a medium having a pH of 6.0 to 8.0. For example, the pH may be 6.2 to 7.8, 6.4 to 7.6, 6.6 to 7.4, 6.8 to 7.2, or 7.0 to 8.0.

[0063] The above culture medium composition may be used to culture the microorganism at a temperature of 30 to 38 ℃, for example, 32 to 38 ℃, 33 to 37 ℃, 34 to 38 ℃, 32 to 36 ℃, or 34 to 36 ℃. The above culture medium composition may be used to produce hyaluronic acid from the microorganism.

[0064] In one embodiment, the medium composition may contain arginine or its salt and uridine or its salt at concentrations of 0.1 to 15.0 g / L and 0.05 to 10.0 g / L, or 0.5 to 10.0 g / L and 0.1 to 5.0 g / L, respectively.

[0065] In one embodiment, the medium composition may contain arginine, uridine, and glutamic acid at concentrations of 0.1 to 15.0 g / L, 0.05 to 10.0 g / L, and 0.1 to 15.0 g / L, respectively, or 0.5 to 10.0 g / L, 0.1 to 5.0 g / L, and 0.5 to 10.0 g / L.

[0066] The above-mentioned culture medium composition may additionally include a carbon source. The carbon source may be a known carbon source. The carbon source may be a monosaccharide or a polysaccharide. The polysaccharide may be a disaccharide, a trisaccharide, or starch. The carbon source may be a single component or a complex component. The carbon source may be, for example, glucose, starch, sucrose, galactose, fructose, or a combination thereof. The carbon source may not be of animal origin.

[0067] The carbon source may be 40 to 120 g / L, 60 to 120 g / L, 70 to 120 g / L, 80 to 120 g / L, 90 to 120 g / L, 90 to 110 g / L, 95 to 105 g / L, 80 to 110 g / L, 80 to 105 g / L, or about 100 g / L.

[0068] The above culture medium composition may additionally include a nitrogen source. The nitrogen source may be a known nitrogen source. The nitrogen source may be an organic or inorganic nitrogen source. The nitrogen source may be a nitrogen source containing a single component or a complex component. The nitrogen source may be, for example, ammonium sulfate, ammonium nitrate, sodium nitrate, casamino acid, yeast extract, soybean extract such as peptone, tryptone, and soytone, or a combination thereof. The carbon source may not be of animal origin.

[0069] The above nitrogen source may be an amount necessary to maintain the growth of the microorganism or to produce hyaluronic acid. The above nitrogen source may be, for example, 5 to 100 g / L, 10 to 50 g / L, 20 to 40 g / L, 20 to 30 g / L, 5 to 50 g / L, 5 to 40 g / L, 5 to 30 g / L, 5 to 20 g / L, 5 to 10 g / L, 5 to 30 g / L, 10 to 30 g / L, or 15 to 30 g / L.

[0070] In one embodiment, the culture medium composition may include yeast extract and soybean hydrolysate as nitrogen sources. In the culture medium composition, the yeast extract and soybean hydrolysate may be included at concentrations of 5 to 20 g / L and 5 to 20 g / L, respectively. The culture medium composition may not include any other complex components other than yeast extract and soybean hydrolysate as nitrogen sources.

[0071] The above culture medium composition may additionally include inorganic salts. The inorganic salts may be macro or trace elements necessary for the growth of microorganisms. The inorganic salts may include, for example, sodium chloride, sodium monophosphate, sodium diphosphate, ferrous sulfate, potassium chloride, magnesium sulfate, potassium diphosphate, or a combination thereof.

[0072] The above culture medium composition may include glucose as a carbon source. The above culture medium composition may include glucose as a carbon source and may not include other complex components other than yeast extract and soybean hydrolysate.

[0073] The above medium composition may not contain glutamic acid. The above medium composition may not contain glutamic acid at a concentration of 0.5 to 10.0 g / L.

[0074] The above-mentioned culture medium composition may be a culture medium for producing hyaluronic acid.

[0075] The above-described culture medium composition may be used in a method for producing hyaluronic acid by culturing microorganisms, for increasing the molecular weight, yield, or a combination thereof. The method may involve making the content of arginine and uridine, or glutamic acid, arginine, and uridine equal to that of the above-described culture medium composition. The method may involve adjusting the ratio of each component among arginine and uridine, or arginine and uridine, within the range of the content of the above-described culture medium composition.

[0076] In the above method, arginine and uridine, or glutamic acid, arginine and uridine, may be added before culture or at any point during culture.

[0077] The above microorganism may be a Streptococcus genus microorganism or a variant thereof. The Streptococcus genus microorganism may be Streptococcus zooepidemicus, for example, strain SMT013 (KCCM13522P), or a variant thereof. Additionally, the Streptococcus genus microorganism may be a known strain, for example, strain KCCM 40304, strain KCCM 40305, strain ATCC 39920, strain ATCC 35246, or a variant thereof.

[0078] In this specification, the term “variant” includes “genetic modification” compared to the parent strain. The term “genetic modification” includes changing the composition or structure of the genetic material of a cell.

[0079] The above genetic modification may be caused by random mutation induction or by artificial genetic manipulation, for example, by site-specific mutation induction.

[0080] The above random mutation may be induced by exposing or contacting the cell with physical, chemical, and molecular mutagens. The physical mutagens may be, for example, ultraviolet (UV), gamma rays, atmospheric and room temperature plasma (ARTP), or a combination thereof. The mutagens may be ethanemethylsulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (NTG), 5-bromouracil (5-BU), methylmethane sulfonic acid (MMS), or a combination thereof. The molecular mutagens may be transposons.

[0081] The above genetic modification may be performed by genetic manipulation. Genetic manipulation may be induced by homologous recombination, allelic exchange, Cre-loxP-based system, FLP-FRT recombination system, CRISPR-Cas9 system, RNA interference (RNAi), episomal plasmid-mediated gene overexpression, or a combination thereof.

[0082] In one embodiment, the carbon source in the medium composition is glucose, and the concentration of glucose is 40 to 120 g / L, and arginine or its salt, glutamic acid or its salt, or uridine or its salt may be included at concentrations of 0.5 to 10.0 g / L, 0.5 to 10.0 g / L, and 0.1 to 5.0 g / L, respectively.

[0083] The culture medium composition may be used to produce hyaluronic acid with a molecular weight of 0.5 to 3.0 MDa, for example, 0.5 to 3.0 MDa.

[0084] Another aspect provides a method for producing hyaluronic acid, comprising the step of culturing the microorganism in a culture medium composition.

[0085] The composition of the culture medium is as described above. The culture may be batch, fed-batch, or continuous culture. The culture may have a pH of 6.0 to 8.0, for example, the pH may be 6.2 to 7.8, 6.4 to 7.6, 6.6 to 7.4, 6.8 to 7.2, or 7.0 to 8.0. Additionally, the culture may be performed at a temperature of 30 to 38°C, for example, 32 to 38°C, 33 to 37°C, 34 to 38°C, 32 to 36°C, or 34 to 36°C. In one embodiment, the culture may be performed at a pH of 6.0 to 8.0 and a temperature of 34 to 38°C.

[0086] The above method may increase the molecular weight, yield, or combination thereof of the hyaluronic acid produced. The above method may make the content of arginine and uridine, or glutamic acid, arginine, and uridine equal to that of the medium composition. The above method may include adjusting the ratio of each component among arginine and uridine, or arginine and uridine, within the range of the content of the medium composition.

[0087] In the above method, arginine and uridine, or glutamic acid, arginine and uridine may be added at any time during culture, for example, at the start, early, or during culture.

[0088] In the above method, the culture may be performed for 6 to 48 hours, for example, 6 to 36 hours, 6 to 24 hours, 6 to 18 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24 hours, or 12 to 18 hours.

[0089] In the above method, the culture may be performed by transferring the strain to the main culture medium and culturing it after seed culture. The seed culture may be 3 to 12, 5 to 9, 7 to 12, or about 7 hours. The seed culture medium may be the same as the main culture medium.

[0090] The above culture may be an aerobic culture. The above culture may be performed under stirring or aeration. The aeration may be performed in an amount of 0.5 to 3 vvm, for example, 0.5 to 5 vvm, 0.5 to 2.5 vvm, 0.5 to 2.0 vvm, 0.5 to 1.5 vvm, 0.8 to 1.2 vvm, or about 1.0 vvm.

[0091] The above culture may be carried out in a bioreactor, for example, a bioreactor with a volume of 1.0 to 5.0 L, 1.0 to 4.0 L, 1.0 to 3.0 L, 1.5 to 3.0 L, or about 2.5 L. The bioreactor may be equipped with a stirrer to provide aeration.

[0092] The above method may further include a step of separating hyaluronic acid from the culture. The separation step may be by a known method. The step may include separating the culture medium containing hyaluronic acid from the cells. The separation step may include filtration, precipitation, centrifugation, or a combination thereof. The separation step may include, for example, a step of mixing the culture with water, diatomaceous earth, and activated carbon to obtain a mixture, and then filtering the mixture using a filter, for example, a filter press, to obtain a supernatant. Additionally, the supernatant may be concentrated and buffered by performing filtration, for example, transverse flow filtration (TFF), using a molecular weight cutoff filter, for example, a 30 kDa filter. The buffered hyaluronic acid solution may be sterilized. The sterilization may be performed by filtration using a sterilization filter, for example, a 0.2 μm sterilization filter.

[0093] The above steps may include one or more of the steps of mixing a sterilized hyaluronic acid solution with ethanol to precipitate hyaluronic acid, washing the precipitated hyaluronic acid with ethanol, curing the washed hyaluronic acid using ethanol, and drying the cured hyaluronic acid particles, for example, drying under reduced pressure.

[0094] A Streptococcus zooepidemicus SMT013 strain or a variant thereof according to one aspect has increased hyaluronic acid production ability.

[0095] A composition or kit according to a different aspect can be used in a method for producing hyaluronic acid.

[0096] According to a method for producing hyaluronic acid according to different aspects, hyaluronic acid can be produced efficiently.

[0097] Hyaluronic acid produced by the above method according to a different aspect has improved physical properties.

[0098] A culture medium composition for microorganisms that produce hyaluronic acid according to one aspect can be used to produce hyaluronic acid with increased efficiency.

[0099] According to the method for producing hyaluronic acid according to one aspect, hyaluronic acid can be produced efficiently.

[0100] Figure 1 is a photograph showing the appearance of SMT001 and SMT013 strains after cultivation inoculated onto an agar medium.

[0101] Figure 2 is a photograph showing the results of confirming a mutant strain of SMT013 with a deleted sagA gene on blood agar medium.

[0102] Figure 3 is an electrophoresis image showing the PCR results confirming sagA gene deletion when using SMT013 ΔsagA strain genome DNA as a template.

[0103] Figure 4 is an electrophoresis image showing the PCR results confirming hylB deletion when the SMT013ΔhylBΔsagA strain genome DNA was used as a template.

[0104] Figure 5 shows the results of evaluating the hyaluronic acid production capacity of SMT013ΔsagA and SMT013ΔhylBΔsagA strains compared to SMT013.

[0105] The present invention will be explained in more detail below through examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0106] Example 1. Isolation of S. zooepidemicus SMT001 strain from horse respiratory samples

[0107] 1. Method for isolating strains capable of producing hyaluronic acid from horse respiratory samples

[0108] A strain of S. zooepidemicus with hyaluronic acid-producing ability was isolated from horse respiratory samples. S. zooepidemicus is a facultative anaerobic strain and possesses hemolytic activity induced by hemolysin.

[0109] First, respiratory samples were collected from 24 horses in the Jeju Island region. The samples were evenly streaked onto blood agar medium using swabs. The medium was incubated for 24 hours in a static incubator at 37°C under anaerobic conditions. Among the resulting colonies, those exhibiting hemolytic activity and a slightly shiny, viscous texture were selected. Genomic DNA was extracted from the selected colonies, and the 16S rRNA gene was amplified using PCR with primer sets of SEQ ID NOs. 11 and 12, using the DNA as a template, and its nucleotide sequence was confirmed. Analysis of one of the colonies revealed that the nucleotide sequence of the 16S rRNA gene was as shown in SEQ ID NO. 13, and genetic identity was analyzed using the NCBI BLAST program. As a result, it was confirmed that the 16S rRNA gene sequence had 98% homology with the 16S rRNA gene sequence of the standard strain ATCC 43079 of Streptococcus zooepidemicus.

[0110] The identified strain was named S. zooepidemiucsSMT001.

[0111] 2. Confirmation of Carbon Metabolism Characteristics of SMT001 Strain

[0112] The carbon source metabolic activity of the SMT001 strain was analyzed using the API 50 CH kit (Biomerieux, cat. 50300). As a result, ribose, galactose, glucose, fructose, mannose, sorbitol, N-acetylglucosamine, esculin, salicin, cellobiose, maltose, lactose, sucrose, starch, and glycogen could be used as carbon sources.

[0113] No. Carbon Source SMT0010 Control Group - 1 Glycerol - 2 Erythritol - 3 D-Arabinose - 4 L-Arabinose - 5 Ribose + 6 D-Xylose - 7 L-Xylose - 8 Adonitol - 9 Methyl-β-D-Xylopyranoside - 10 Galactose + 11 Glucose + 12 Fructose + 13 Mannose + 14 Sorbose - 15 Rhamnose - 16 Dulcitol - 17 Inositol - 18 Mannitol - 19 Sorbitol + 20 Methyl-α-D-Mannopyranoside - 21 Methyl-α-D-Glucoside - 22 N-Acetyl-Glucosamine + 23 Aminoside Gdalin-24 Arbutin-25 Esculin+26 Salicin+27 Cellobios+28 Maltose+29 Lactose+30 Melivios-31 Sucrose+32 Trehalose-33 Inulin-34 Melesitose-35 Raffinose-36 Starch+37 Glycogen+38 Xylitol-39 Genthiobios-40 D-Turanos-41 D-Lysose-42 D-Tagatose-43 D-Fucose-44 L-Fucose-45 D-Arbitol-46 L-Arbitol-47 Glucuronate-482 Keto-Gluconic Acid-495 Keto-Gluconic Acid-

[0114] Example 2. Selection of SMT013 strain with high-concentration hyaluronic acid production ability

[0115] 1. Selection of mutant strains capable of producing high concentrations of hyaluronic acid

[0116] A mutant strain of the SMT001 strain with improved hyaluronic acid production capacity using ethyl methanesulfonate (EMS) was selected. The SMT001 strain was cultured at 37°C in TSB medium (tryptone 17 g / L, soytone 3 g / L, sodium chloride 5 g / L, disodium phosphate 2.5 g / L, glucose 2.5 g / L) and then centrifuged to precipitate the cells. After removing the supernatant, the cell pellet was washed with phosphate-buffered saline (PBS) and centrifuged, and this process was repeated twice. After adding PBS, EMS was added at various concentrations and cultured at 37°C for 30 minutes, after which the cells were washed twice with PBS. 100 μL of each treatment group was taken and plated on TSA medium (tryptone 17 g / L, soytone 3 g / L, sodium chloride 5 g / L, disodium phosphate 2.5 g / L, glucose 2.5 g / L, agar 20 g / L).

[0117] Strains with improved hyaluronic acid production ability were selected based on the viscosity of the generated colonies. After primarily selecting colonies that were significantly larger and more viscous compared to the SMT001 strain, hyaluronic acid production ability was reconfirmed in flask culture using MH1 medium (glucose 70 g / L, magnesium sulfate 2.5 g / L, yeast extract 10 g / L, soybean hydrolysate 10 g / L, potassium phosphate 1.4 g / L, and anhydrous sodium phosphate 6.5 g / L). By repeating this selection process, the mutant strain SMT013, capable of producing high concentrations of hyaluronic acid, was finally obtained. When seed cultures of SMT001 and SMT013 were shaken and cultured on TSB and dropped onto TSA and cultured at 35°C for 24 hours, it was confirmed that SMT013 exhibited a phenotype that was shiny in light and viscous (Fig. 1).

[0118] 2. Confirmation of Hyaluronic Acid Production Capacity of SMT013 Strain

[0119] After incubating SMT013 and SMT001 strains in TSB liquid medium for 24 hours, 20 ml of MH1 medium was placed in a 125 ml Erlenmeyer flask, and each strain was inoculated with the OD at 600 nm adjusted to 0.02. Subsequently, the culture was stirred for 28 hours at 35 ℃ and 230 rpm. The cell pellet was removed from the culture medium, and the filtrate was obtained by filtering through a 0.22 μm syringe filter. The hyaluronic acid production capacity of the two strains was then measured using the CTAB turbidimetric method (Oueslati N et al., Carbohydr Polym. 2014 Nov 4;112: 102-8). This method quantitatively analyzes the hyaluronic acid-CTAB (HA-CTAB) complex, formed by the binding of a hyaluronic acid polymer and CTAB (cetyltrimethylammonium bromide), using turbidity, and quantifies hyaluronic acid based on this analysis. As a result, strains SMT013 and SMT001 produced 652.5 mg / L and 274.4 mg / L of hyaluronic acid, respectively (Table 2). Strain SMT013 exhibits a significantly increased hyaluronic acid production capacity of approximately 2.4 times compared to SMT001.

[0120] Streptococcus zooepidemicus SMT013 was deposited with the Korean Culture Collection of Microorganisms (KCCM) on October 10, 2024, and was assigned deposit number KCCM13522P.

[0121] Strain Hyaluronic Acid Concentration (mg / L) SMT001274.4 SMT013652.5

[0122] Example 3. Preparation of a mutant strain of SMT013 with weakened hemolytic activity

[0123] 1. Vector construction and transformation verification for the production of strains with weakened hemolytic activity

[0124] Hemolytic activity is a trait required for S. zooepidemicus to infect animals and cause disease. Although animals are the primary hosts of S. zooepidemicus, humans also pose a risk of opportunistic infection; therefore, for the industrial use of this bacterium, its hemolytic activity must be weakened to ensure safety. The hemolytic activity of S. zooepidemicus is mediated by the sag operon. Hemolytic activity was eliminated in SMT013 by deleting the sagA gene via homologous recombination. The sagA gene codes for streptolysin S (SLS), which induces hemolysis. To achieve this, a gene deletion system based on the pSET4s::sacB vector was utilized (Sun et al., Appl Microbiol Biotechnol. 2013 97(19):8629-36). A 1 kb flanking region of the sagA gene was cloned and added to both the 5' and 3' ends of the vector, and an erythromycin resistance gene was introduced as a selection marker instead of the chloramphenicol resistance gene to construct a gene disruption vector. This vector was then transformed into the SMT013 strain to obtain a transformant with the disruption vector inserted into the chromosome. Subsequently, the selection of the sagA gene disruption strain was carried out following the method described in the aforementioned reference. The finally selected transformant was cultured at 37°C, diluted, and plated on blood agar solid medium.

[0125] Figure 2 is a photograph showing the results of confirming the SMT013 mutant strain with the sagA gene deletion on blood agar medium. As shown in Figure 2, hemolysis occurred in the SMT013 strain, whereas hemolysis did not occur in the SMT013ΔsagA strain.

[0126] 2. Confirmation of sagA gene deletion in SMT013ΔsagA strain

[0127] To confirm whether the gene at the chromosomal sagA locus of the SMT013 strain was properly destroyed, PCR was performed using the genomic DNA of the SMT013ΔsagA strain as a template and primer sets of SEQ ID NOs. 14 and 15 that attach to the flanking regions of both the 5' and 3' ends of the sagA gene, and the products were analyzed by electrophoresis.

[0128] Figure 3 is an electrophoresis image confirming the sagA gene deletion in SMT013 through the amplification of the sagA gene. As shown in Figure 3, in the SMT013ΔsagA strain, a product was detected with a size reduced by the size of the sagA gene compared to the parent strain, which indicates that the sagA gene has been removed.

[0129] As shown in Figures 2 and 3, a SMT013ΔsagA strain that did not undergo hemolysis was finally obtained.

[0130] Example 4. Preparation of the SMT013ΔsagAΔhylB strain derived from SMT013, which simultaneously attenuates hemolysis and hyaluronidase.

[0131] 1. Vector construction and transformation verification for the production of hyaluronidase-inhibited strains

[0132] A strain with weakened hyaluronidase activity was constructed by deleting the hylB gene in the SMT013ΔsagA strain via homologous recombination. The hylB gene is a gene that codes for hyaluronate lyase (also called "hyaluronidase").

[0133] To this end, a gene deletion system based on the pSET4s::sacB vector, as described in Example 4, "1. Vector construction and transformation identification for the production of strains with weakened hemolytic activity," was utilized. A vector was constructed by removing the 5' and 3' flanking regions of sagA from the pSET4s::sacB-sagA gene-destruction vector and introducing 1000 bp each of the 5' and 3' portions of the hylB gene to delete the central portion (amino acid 374 to 839) containing the catalytic domain of the hylB gene. This vector was then transformed into the SMT013ΔsagA strain to obtain a transformation in which this vector was inserted into the chromosome. Subsequently, the selection of hylB gene-destruction strains was carried out following the method described in the above reference, and colonies without erythromycin resistance were finally selected.

[0134] 2. Confirmation of hylB gene deletion

[0135] The gene was amplified by PCR using primer sets of SEQ ID NOs. 16 and 17 that bind to the hylB gene regions corresponding to the N and C terminals, using chromosomal DNA extracted from selected colonies as a template, and the amplified product was analyzed by electrophoresis. From the electrophoresis results, it was confirmed whether a portion of the hylB gene was properly destroyed.

[0136] Figure 4 is an electrophoresis image showing the PCR results using the genomic DNA of the SMT013ΔhylBΔsagA strain as a template. As shown in Figure 4, a product of 2.185 kb with a reduced size compared to the parent strain was detected in the SMT013ΔhylBΔsagA strain, indicating that the hylB gene was deleted. In other words, the production of the SMT013ΔhylBΔsagA strain was confirmed by the additional deletion of hylB in the host SMT013ΔsagA.

[0137] Example 5. Evaluation of Hyaluronic Acid Production Capacity of SMT013ΔsagA and SMT013ΔhylBΔsagA Strains

[0138] 1. Evaluation of flask culture productivity

[0139] The hyaluronic acid productivity of the SMT013ΔsagA strain and the SMT013ΔhylBΔsagA strain was evaluated by comparing them with SMT013. Strain culture and hyaluronic acid quantification were carried out in the same manner as described in "2. Confirmation of Hyaluronic Acid Production Ability of SMT013 Strain" of Example 2.

[0140] Figure 5 shows the results of evaluating the hyaluronic acid production capacity of SMT013, SMT013ΔsagA strains, and SMT013ΔhylBΔsagA. It was confirmed that hyaluronic acid production increased by approximately 2.77% and 5.04% in SMT013ΔsagA strain (670.6 mg / L) and SMT013ΔhylBΔsagA strain (685.4 mg / L) compared to the parent strain SMT013 (652.5 mg / L).

[0141] 2. Evaluation of Fermenter Culture Productivity

[0142] Cultures of the SMT013, SMT013ΔsagA, and SMT013ΔhylBΔsagA strains, respectively, were prepared by seed culture in TSB liquid medium at 230 rpm at 37 ℃ for 15 hours. 250 mL of the obtained seed culture was inoculated into a 2.5 L fermenter (BioFlo320, Eppendorf) containing 1 L of MH1 medium and cultured for 8 hours at pH 7.0, 34 ℃, 1 vvm, and 600 rpm. After culture, the strain pellet was removed by centrifugation, and the viscosity of the filtered culture medium was measured using a viscometer (DV2T, Brookfield). The molecular weight of the produced hyaluronic acid was confirmed by size exclusion chromatography, and the results are shown in Table 3. When only sagA was deleted from SMT013, there was no effect on the viscosity and molecular weight of the culture medium, but when hylB was also deleted, it was confirmed that the viscosity and molecular weight increased significantly.

[0143] Strain Culture Viscosity (cp) Molecular Weight (kDa) SMT0132 1.61,355 SMT013ΔsagA2 7.41,396 SMT013ΔhylBΔsagA16 7.42,034

[0144]

[0145] Example 6. Effects of Arginine, Glutamic Acid, and Uridine on Hyaluronic Acid Production

[0146] Streptococcus zooepidemicus SMT013 (accession number: KCCM13522P) was inoculated into 250 mL of TSB (tryptone 17 g / L, soytone 3 g / L, sodium chloride 5 g / L, disodium phosphate 2.5 g / L, glucose 2.5 g / L) and cultured at 37 ℃ at 200 rpm for 18 hours. Next, 250 mL of the culture medium after cultivation was completed was inoculated into a 2.5 L fermenter (Eppendorf BIOFLO320) containing 1 L of sterile seed culture medium (glucose 50 g / L, magnesium sulfate 2.5 g / L, yeast extract 10.0 g / L, soybean hydrolysate 10.0 g / L, potassium phosphate 1.4 g / L, sodium phosphate 6.5 g / L, and antifoaming agent J673A (Strucktol) 0.1 g / L), and cultured for 7 hours at pH 7.5, temperature 35℃, 600 rpm, and aeration 1.0 vvm. The above antifoaming agent

[0147] 50 mL of the culture medium after cultivation was completed was inoculated into a 2.5 L fermenter (Eppendorf BIOFLO320) containing 1 L of sterile main culture medium (glucose 70 g / L, magnesium sulfate 2.5 g / L, yeast extract 10.0 g / L, soybean hydrolysate 10.0 g / L, potassium phosphate 1.4 g / L, sodium phosphate 6.5 g / L, and antifoaming agent 0.1 g / L) (hereinafter referred to as 'MH1'), and cultured for 18 hours at pH 7.0, temperature 34℃, 700 rpm, and aeration 1.0 vvm. The antifoaming agent concentration was 0.1 g / L.

[0148] In addition, the same method was used to culture in a medium supplemented with 4.5 g / L arginine, 3.5 g / L glutamic acid, 1.2 g / L uridine, or a combination thereof in MH1 medium. The specific medium composition is shown in Table 1. The arginine and glutamic acid are based on arginine hydrochloride and MSG, respectively, and in this case, the standard concentrations of arginine and glutamic acid are approximately 3.6 g / L and approximately 3.1 g / L, respectively.

[0149] Name Addition Wave Result MSGL-Arginine Uridine Glucose Consumption (g / L) Viscosity (cp) OD (600 nm) HA (g / L) Average Molecular Weight (MDa) Comparison Group 1---67.22 3.80 7.15 4.05 0.36 Comparison Group 2+-+68.02 1.68.80 4.07- Experimental Group 1-++67.01 71.47.85 6.67- Experimental Group 2+++69.33 16.89.82 6.48 0.84

[0150] * + : Addition, - : No addition, MSG: Monosodium glutamate. The above addition may involve combining MH1 medium with arginine, glutamic acid, uridine, or a combination of one or more of these during the medium preparation stage prior to culture. Alternatively, it may involve adding arginine, glutamic acid, uridine, or a combination of one or more of these to MH1 medium during culture. In this example, the combination was performed prior to culture. During culture, the pH was maintained at pH 7.0 using 5N NaOH, and after 18.5 hours of culture, the culture medium viscosity and OD 600 Viscosity and hyaluronic acid concentrations were each checked. Viscosity and hyaluronic acid concentrations were measured according to the manufacturer's protocol using a rotational viscometer (Brookfield, DV2T) and the CTAB HA concentration analysis method.

[0151] Table 4 shows the viscosity of the culture medium, hyaluronic acid concentration, and the degree of cell growth according to the composition of the medium.

[0152] As shown in Table 4, hyaluronic acid concentration, OD in Experimental Group 1 containing arginine and uridine and Experimental Group 2 containing a combination of arginine, glutamic acid, and uridine, compared to Comparison Group 1 not containing arginine, glutamic acid, uridine, or a combination thereof, or Comparison Group 2 containing glutamic acid and uridine. 600 and viscosity increased significantly.

[0153] In addition, compared to Comparison Group 1, viscosity and HA production in Comparison Group 2 did not significantly increase. This indicates that the medium containing glutamic acid and uridine does not significantly affect viscosity and HA production. Since the viscosity is correlated with the molecular weight of HA, this also indicates that the medium containing glutamic acid and uridine does not significantly affect viscosity and HA production. This is an unexpected effect compared to Japanese Patent Publication No. 1987-289198, which discloses a method for producing hyaluronic acid by culturing Streptococcus equi in a medium containing arginine and glutamic acid, wherein hyaluronic acid production is significantly increased in a medium containing arginine and glutamic acid compared to a medium containing only arginine or glutamic acid.

[0154] In addition, compared to control group 2, viscosity and HA production significantly increased in experimental groups 1 and 2. This indicates that the combination of arginine and uridine has a significant effect on viscosity and HA production. Since the increase in viscosity indicates an increase in the molecular weight of HA, this also indicates an unexpected effect that the combination of arginine and uridine increases not only the production of HA but also the molecular weight.

[0155] In addition, the viscosity in experimental group 2 increased significantly compared to experimental group 1. This indicates that glutamic acid combined with arginine and uridine affects the increase in HA molecular weight. Considering that there was no difference in viscosity and HA production in comparative group 2, which used a medium containing glutamic acid and uridine, compared to comparative group 1, the results of experimental group 2 indicate that the three-component combination of glutamic acid, arginine, and uridine acts synergistically to increase the production of HA and the molecular weight of HA.

[0156] Example 7: Effect of Glucose Concentration

[0157] In this example, the effect of the culture medium composition with different glucose concentrations on hyaluronic acid production in the experimental group 2 medium of Example 6 was confirmed. Table 5 shows the results of measuring hyaluronic acid viscosity, OD, and hyaluronic acid concentration according to glucose concentration and culture time in the experimental group 2 medium. The culture was performed using the same process as in Example 1, except for glucose concentration and culture time, and hyaluronic acid viscosity, OD, and hyaluronic acid concentration were measured.

[0158] Glucose Concentration (g / L) Result Incubation Time (h) Viscosity (cp) OD (600 nm) HA (g / L) Average Molecular Weight (MDa) 70 18.5 316.8 9.8 26.4 80.8 48 522.5 568.8 6.9 87.05 - 100 24.06 78.06 40 6.5 60.96

[0159] As shown in Table 5, it was confirmed that the viscosity and concentration of hyaluronic acid in the culture end solution increased with increasing glucose content, and the medium composition with a glucose concentration of 100 g / L was named MH1-3.

[0160] Example 8: Production and Evaluation of Hyaluronic Acid

[0161] The culture solution produced in Example 7 was mixed with water, diatomite, and activated carbon. Water was mixed at a ratio of 50 to 300 (w / v)% relative to the culture solution, diatomite at a ratio of 0.5 to 10.0 (w / v)% relative to the culture solution, and activated carbon at a ratio of 0.5 to 10.0 (w / v)% relative to the culture solution, and the supernatant was separated using a filter press.

[0162] The separated supernatant was concentrated using a tangential flow filtration (TFF) system equipped with a 30kD cassette filter, and buffer exchange was performed. Concentration was carried out until the volume of the supernatant was reduced to 20 to 50%, and buffer exchange was performed after the concentration was finished. A third-order buffer was used, and the remaining concentrate was recovered after processing with 3 to 5 times the volume of the concentrate.

[0163] Sodium chloride, which is necessary for hyaluronic acid precipitation, and pH 8.0 EDTA, which removes trace metal ions, were mixed into the obtained concentrate to a final concentration of 0.2 to 2.0 M and 0.01 to 5.0 mM, respectively, and reacted for 1 to 2 hours. After the reaction was completed, sterilization was performed by passing the mixture through a 0.2 μm sterilization filter.

[0164] Hyaluronic acid particles were precipitated by adding 99.9% ethanol at a volume of 1 to 3 times that of the sterilization filtrate for 2 to 5 hours. After precipitation was complete, the particles were washed 1 to 3 times using 65 to 75% ethanol, and then a curing process was performed 1 to 3 times using 99.9% ethanol to improve the stability of the hyaluronic acid particles by removing moisture surrounding them. The hyaluronic acid particles after the curing process were dried under reduced pressure for 24 to 48 hours.

[0165] The intrinsic viscosity was measured using the produced hyaluronic acid powder, and the molecular weight of the hyaluronic acid was determined using the measured intrinsic viscosity value. The measurement of intrinsic viscosity is performed by measuring the time it takes for hyaluronic acid solutions prepared at a constant concentration to pass through a capillary viscometer, and utilizing the correlation between the passage time and the concentration of the hyaluronic acid solution. Using the calculated intrinsic viscosity value, the average molecular weight can be calculated through the correlation between the average molecular weight and the intrinsic viscosity. The above intrinsic viscosity measurement was conducted according to the procedure specified in European Pharmacopoeia 11.0, SODIUM HYALURONATE monography.

[0166] As a result, it was confirmed that the molecular weight of the produced hyaluronic acid was 0.80 to 1.20 MDa.

[0167]

Claims

A microorganism capable of producing hyaluronic acid selected from one or more of the Streptococcus zooepidemicus SMT013 strain (accession number KCCM13522P) and variants thereof. A microorganism according to claim 1, wherein the variant has a genetic variation caused by random mutation, or has a genetic variation caused by genetic manipulation, or has both a genetic variation caused by random mutation and a genetic variation caused by genetic manipulation. A microorganism according to claim 1, wherein the variant comprises one or more of a genetic modification that reduces the expression of toxins including hemolytic toxins, a genetic modification that increases the ability to produce hyaluronic acid, a genetic modification that increases the molecular weight of hyaluronic acid, and a genetic modification that increases the viscosity of hyaluronic acid. In claim 3, the genetic modification that reduces hemolytic toxin is a genetic modification that reduces the expression of streptolysin or its biosynthetic pathway protein, and A microorganism in which a genetic modification that increases hyaluronic acid production capacity or molecular weight is a genetic modification that increases the expression of a hyaluronic acid biosynthetic pathway protein, or a genetic modification that decreases the expression of a protein involved in hyaluronic acid degradation. In claim 3, the toxin is streptolysin S, a streptolysin S biosynthetic pathway protein, a protein that is one of sagABCDEFGHI, or a combination thereof, and The above hyaluronic acid biosynthetic pathway protein is hyaluronan synthase, UDP-glucose dehydrogenase, UDP-glucose pyrophosphorylase, phosphoglucomutase, phosphoglucoisomerase, glutamine amidotransferase, phosphoglucosamine mutase, acetyl-CoA acetyltransferase, UDP-N-acetylglucosamine pyrophosphorylase, vitreoscilla hemoglobin (VHb), or a combination thereof. Microorganisms in which the protein involved in the degradation of hyaluronic acid is hyaluronidase. A microorganism according to claim 5, wherein the toxin gene is the sagA gene and the hyaluronidase gene is the hylB gene. The microorganism of claim 1, wherein the hyaluronic acid has a molecular weight of 0.1 to 5.0 MDa. A composition comprising the microorganism of claim 1. A kit comprising the microorganism of claim 1. A method for producing hyaluronic acid comprising the step of culturing the microorganism of claim 1 in a culture medium to produce hyaluronic acid. A culture medium composition for microorganisms producing hyaluronic acid comprising arginine or its salt and uridine or its salt. A culture medium composition according to claim 11, further comprising glutamic acid or a salt thereof. A culture medium composition according to claim 11, wherein the concentration of the arginine or its salt is 0.5 to 10.0 g / L. A culture medium composition according to claim 11, wherein the concentration of uridine or a salt thereof is 0.1 to 5.0 g / L. A culture medium composition according to claim 12, wherein the concentration of glutamic acid or a salt thereof is 0.5 to 10.0 g / L. A culture medium composition according to claim 11, wherein the microorganism is a microorganism of the genus Streptococcus. A culture medium composition according to claim 11, wherein the microorganism is Streptococcus epidemicus. A culture medium composition according to claim 11, intended for use in culturing microorganisms to produce hyaluronic acid. A method for producing hyaluronic acid, comprising the step of culturing a microorganism capable of producing hyaluronic acid in a medium composition comprising arginine or a salt thereof and uridine or a salt thereof. A method according to claim 19, wherein the culture is performed at a pH of 6.0 to 8.

0. The method of claim 19, wherein the culture is performed at a temperature of 34 to 38 ℃. A method according to claim 19, wherein the arginine or its salt and uridine or its salt, or arginine or its salt, uridine or its salt and glutamic acid or its salt are added before or during cultivation.

Images