Method for producing gelatin, additives and culture medium

Abstract

To provide a method for producing gelatin, including culturing a methanol-utilizing yeast having a gelatin gene, in which the amount of gelatin production is improved; and to provide an additive and a medium for use in the above-mentioned method for producing gelatin, which can improve the amount of gelatin production.SOLUTION: Provided is a method for producing gelatin, comprising adding methanol and at least two groups of components selected from group 1: at least one of glycine and serine, group 2: pyruvic acid or a salt thereof, group 3: glutathione, and group 4: at least one of arginine and lysine to a medium; and culturing a methanol-utilizing yeast having a gelatin gene.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing gelatin, which comprises culturing a methanol-utilizing yeast having a gelatin gene. The present invention further relates to an additive for use in the above-mentioned method for producing gelatin, and a medium for use in the production of gelatin by the methanol-utilizing yeast. [Background technology]

[0002] Methanol-utilizing yeasts such as Pichia pastoris are known as useful expression systems for heterologous proteins. A feature of this expression system is the use of a strong promoter such as the alcohol oxidase gene (AOX1) that is induced by methanol. By introducing a gene encoding a heterologous protein downstream of this promoter, the desired heterologous protein such as gelatin can be expressed and secreted.

[0003] There are known efforts to study culture conditions to improve the yield of heterologous proteins. For example, Patent Documents 1 to 3 describe culture under conditions containing multiple amino acids, but the effect on gelatin production is unclear.

[0004] In Patent Documents 4 and 5, with regard to gelatin production, it is described that a collagen-producing methanol-utilizing yeast is cultured in the presence of sodium pyruvate and glutathione, but combinations with other components are not fully examined. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent Application Publication US2020 / 0199523 [Patent Document 2] U.S. Patent Application Publication US2015 / 0299648 [Patent Document 3] China Patent Application Publication CN112501041A [Patent Document 4] China Patent Application Publication CN106256911A [Patent Document 5] China Patent Application Publication CN114045321A Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a method for producing gelatin, which includes culturing a methanol-utilizing yeast having a gelatin gene, and which improves the production amount of gelatin.A further object of the present invention is to provide an additive for use in the above-mentioned method for producing gelatin, which can improve the production amount of gelatin.A further object of the present invention is to provide a medium for use in the production of gelatin by a methanol-utilizing yeast, which can improve the production amount of gelatin. [Means for solving the problem]

[0007] As a result of intensive research conducted by the present inventors to solve the above problems, they found that the production amount of gelatin can be improved by culturing a methylotrophic yeast having a gelatin gene using components from at least two groups selected from Group 1: at least one of glycine and serine, Group 2: pyruvic acid or a salt thereof, Group 3: glutathione, and Group 4: at least one of arginine and lysine. The present invention was completed based on the above findings.

[0008] According to the present invention, the following inventions are provided. <1> Group 1: At least one of glycine and serine Group 2: Pyruvic acid or its salts Group 3: Glutathione, and Group 4: At least one of arginine and lysine and methanol to a medium, and culturing a methylotrophic yeast having a gelatin gene. <2> The at least two groups of components are Group 1, Group 2, and Group 3 components; <1> A method for producing gelatin according to the above item. <3> At least two of the above groups of ingredients (1) glycine or serine, pyruvic acid or a salt thereof, and glutathione, (2) glycine, and pyruvic acid or a salt thereof, (3) pyruvic acid or a salt thereof, and glutathione, (4) pyruvic acid or a salt thereof, and arginine, (5) glycine and glutathione, or (6) serine and lysine, Either <1> or <2> A method for producing gelatin according to the above item. <4> The total amount of glycine added is 1.0 g or less per liter of medium, or the total amount of serine added is 1.0 g or less per liter of medium. <1> ~ <3> 10. The method for producing gelatin according to any one of the above. <5> The total amount of pyruvic acid or its salts added per liter of medium is 4.6 g or less. <1> ~ <4> 10. The method for producing gelatin according to any one of the above. <6> The total amount of glutathione added per liter of medium is 6.2 g or less. <1> ~ <5> 10. The method for producing gelatin according to any one of the above. <7> A total amount of arginine added is 0.7 g or less, or a total amount of lysine added is 2.3 g or less per liter of medium; <1> ~ <6> 10. The method for producing gelatin according to any one of the above. <8> A methanol-inducible promoter is operably linked to a gelatin gene. <1> ~ <7> 10. The method for producing gelatin according to any one of the above. <9> The gelatin has an amino acid sequence of the following formula (1): <1> ~ <8> 10. The method for producing gelatin according to any one of the above. A-[(Gly-XY) n ]m -B formula (1) In the formula, A represents one or more any amino acid residues, B represents one or more any amino acid residues, Gly represents a glycine residue, n Xs each independently represent any amino acid residue, and n Ys each independently represent any amino acid residue. m represents an integer of 2 to 10, n represents an integer of 3 to 100, and n Gly-XYs may be all or partly the same or different from each other. <10> The gelatin is (A) a polypeptide having the amino acid sequence shown in SEQ ID NO:1; (B) a polypeptide having an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO:1 and having bioaffinity; or (C) a polypeptide having an amino acid sequence in which one or several amino acid residues are deleted, substituted or added relative to the amino acid sequence shown in SEQ ID NO:1 and having bioaffinity Either <1> ~ <9> 10. The method for producing gelatin according to any one of the above. <11> Group 1: At least one of glycine and serine Group 2: Pyruvic acid or its salts Group 3: Glutathione, and Group 4: At least one of arginine and lysine comprising at least two groups of components selected from <1> ~ <10> 1. An additive for use in the method for producing gelatin according to any one of the above. <12> The at least two groups of components are Group 1, Group 2, and Group 3 components; <11> The additive described in <13> At least two of the above groups of ingredients (1) glycine or serine, pyruvic acid or a salt thereof, and glutathione, (2) glycine, and pyruvic acid or a salt thereof, (3) pyruvic acid or a salt thereof, and glutathione, (4) pyruvic acid or a salt thereof, and arginine, (5) glycine and glutathione, or (6) serine and lysine, Either <11> or <12> The additive described in <14> Group 1: At least one of glycine and serine Group 2: Pyruvic acid or its salts Group 3: Glutathione, and Group 4: At least one of arginine and lysine at least two of the following groups of ingredients: methanol; Nitrogen source; Minerals; and vitamin A medium for use in producing gelatin by a methylotrophic yeast, comprising: <15> The at least two groups of components are Group 1, Group 2, and Group 3 components; <14> The medium described in <16> At least two of the above groups of ingredients (1) glycine or serine, pyruvic acid or a salt thereof, and glutathione, (2) glycine, and pyruvic acid or a salt thereof, (3) pyruvic acid or a salt thereof, and glutathione, (4) pyruvic acid or a salt thereof, and arginine, (5) glycine and glutathione, or (6) serine and lysine, Either <14> or <15> The medium described in <17> The total amount of glycine added is 1.0 g or less per liter of medium, or the total amount of serine added is 1.0 g or less per liter of medium. <14> from <16> 10. A medium according to any one of the preceding claims. <18> The total amount of pyruvic acid or its salts added per liter of medium is 4.6 g or less. <14> from <17> 10. A medium according to any one of the preceding claims. <19> The total amount of glutathione added per liter of medium is 6.2 g or less. <14> from <18> 10. A medium according to any one of the preceding claims. <20> A total amount of arginine added is 0.7 g or less, or a total amount of lysine added is 2.3 g or less per liter of medium; <14> from <19> 10. A medium according to any one of the preceding claims. Effect of the Invention

[0009] According to the present invention, when gelatin is produced by culturing a methylotrophic yeast having a gelatin gene, the yield of gelatin can be improved. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows the protein yield for each example. [Diagram 2] FIG. 2 shows the protein yield for each example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] An example of an embodiment of the present disclosure will be described below. However, the present disclosure is not limited to the following embodiment, and can be implemented with appropriate modifications within the scope of the object of the present disclosure. In this specification, a numerical range indicated using "~" means a range that includes the numerical values ​​before and after "~" as the minimum and maximum values, respectively.

[0012] The method for producing gelatin of the present invention comprises the steps of: Group 1: At least one of glycine and serine Group 2: Pyruvic acid or its salts Group 3: Glutathione, and Group 4: At least one of arginine and lysine and methanol to a medium, and culturing a methylotrophic yeast having a gelatin gene.

[0013] Methanol-utilizing yeasts are microorganisms that can grow using methanol as the sole carbon source, including yeasts belonging to the genera Pichia, Ogataea, Candida, Torulopsis, and Komagataella.

[0014] Examples of the Pichia genus include Pichia methanolica, the Ogataea genus include Ogataea angusta, Ogataea polymorpha, Ogataea parapolymorpha, and Ogataea minuta, the Candida genus includes Candida boidinii, and the Komagataella genus includes Komagataella pastoris and Komagataella phaffii. Both Komagataella pastoris and Komagataella phaffii are also known as Pichia pastoris. In the present invention, the genus Pichia or Komagataella is preferred.

[0015] Specific examples of methanol-utilizing yeast strains include Komagataella pastoris X-33, which can be obtained from the American Type Culture Collection, Thermo Fisher Scientific, and the like.

[0016] The methanol-utilizing yeast used in the present invention has a gelatin gene. Gelatin may be natural gelatin, or may be a mutant or recombinant gelatin having at least one amino acid residue different from the natural gelatin. Natural gelatin means gelatin made from naturally occurring collagen, or a polypeptide having the same amino acid sequence as gelatin made from naturally occurring collagen. Unless otherwise specified, in this specification, mutant or recombinant gelatin is collectively referred to as recombinant gelatin. Natural gelatin or recombinant gelatin may be derived from animals such as fish and mammals, and natural gelatin or recombinant gelatin of mammals is preferable. Mammalian animals include, for example, humans, horses, pigs, mice, rats, etc., and humans or pigs are more preferable. Natural gelatin is preferably derived from pigs or humans, and recombinant gelatin is preferably derived from humans.

[0017] Gelatin refers to a polypeptide containing six or more consecutive amino acid sequences represented by Gly-XY, and the polypeptide may contain one or more other amino acid residues in addition to the amino acid sequence represented by Gly-XY. In Gly-XY, Gly represents a glycine residue, and X and Y represent any amino acid residues other than glycine residues. The amino acid sequence represented by Gly-XY corresponds to an amino acid sequence derived from a partial amino acid sequence of collagen, and the repetition of this sequence means a sequence characteristic of collagen.

[0018] A plurality of Gly-XY in one gelatin molecule may be the same or different. In addition, X and Y in the Gly-XY sequence are independent for each repeating unit and may be the same or different. It is preferable that X and Y contain a large amount of imino acid residues, i.e., proline residues or oxyproline residues. The content of such imino acid residues is preferably 10% to 45% in one gelatin molecule. The content of Gly-XY in one gelatin molecule is preferably 80% or more of the total, more preferably 95% or more, and most preferably 99% or more.

[0019] The gelatin is preferably a recombinant gelatin obtained by introducing into an appropriate host a base sequence or amino acid sequence in which one or more bases or amino acid residues have been changed from the base sequence or amino acid sequence of a gene encoding a collagen having six or more consecutive amino acid sequences represented by Gly-XY, and expressing the changed base sequence or amino acid sequence in a conventional manner. The use of such recombinant gelatin has the advantages of improving bone regeneration ability and expressing various properties compared to the use of natural gelatin, and of avoiding unfavorable effects such as rejection by the living body.

[0020] As recombinant gelatins, those disclosed in, for example, EP1014176A2, US6992172B1, WO2004 / 85473A2, WO2008 / 103041A1, JP-T2010-519293, JP-T2010-519252, JP-T2010-518833, JP-T2010-519251, WO2010 / 128672A1, and WO2010 / 147109A1 can be particularly preferably used.

[0021] The molecular weight of the recombinant gelatin is preferably 2 kDa or more and 100 kDa or less, more preferably 5 kDa or more and 90 kDa or less, and even more preferably 10 kDa or more and 90 kDa or less.

[0022] From the viewpoint of biocompatibility, the recombinant gelatin preferably further comprises a cell adhesion signal, and more preferably has two or more cell adhesion signals in one molecule. Such cell adhesion signals can include RGD sequence, LDV sequence, REDV sequence, YIGSR sequence, PDSGR sequence, RYVVLPR sequence, LGTIPG sequence, RNIAEIIKDI sequence, IKVAV sequence, LRE sequence, DGEA sequence, and HAV sequence, and preferably include RGD sequence, YIGSR sequence, PDSGR sequence, LGTIPG sequence, IKVAV sequence, and HAV sequence, and RGD sequence is particularly preferred. Among RGD sequences, ERGD sequence is more preferred.

[0023] Regarding the arrangement of RGD sequences in recombinant gelatin, the number of amino acid residues between RGD sequences is preferably 0 to 100, more preferably 25 to 60. Moreover, the RGD sequences are preferably arranged non-uniformly within the above range of the number of amino acid residues.

[0024] It is preferable that the ratio of RGD sequences to the total number of amino acid residues in the recombinant gelatin (total number of amino acid residues in RGD sequences / total number of amino acid residues × 100) is at least 1.2%, and if the recombinant gelatin contains 250 or more amino acid residues, it is preferable that each stretch of 250 amino acid residues contains at least one RGD sequence.

[0025] More preferably, the recombinant gelatin contains at least two RGD sequences per 250 amino acid residues, more preferably at least three RGD sequences, and even more preferably at least four RGD sequences.

[0026] The sequence of the recombinant gelatin preferably has the following features: (1) does not contain serine and threonine residues, (2) does not contain serine, threonine, asparagine, tyrosine, or cysteine ​​residues, and (3) does not contain the amino acid sequence Asp-Arg-Gly-Asp. The recombinant gelatin may have any one of the preferred sequence features (1) to (3) alone, or may have two or more of them in combination.

[0027] The gelatin preferably has an amino acid sequence of the following formula (1): A-[(Gly-XY) n ] m -B formula (1) In the formula, A represents one or more any amino acid residues, B represents one or more any amino acid residues, Gly represents a glycine residue, n Xs each independently represent any amino acid residue, and n Ys each independently represent any amino acid residue. m represents an integer of 2 to 10, preferably an integer of 3 to 5. n represents an integer of 3 to 100, preferably an integer of 15 to 70, and more preferably an integer of 50 to 60. n Gly-XYs may be all or partly the same, or may be different from each other.

[0028] More preferably, the recombinant gelatin is Gly-Ala-Pro-[(Gly-XY) 63 ]3-Gly, in which 63 X's each independently represent any amino acid residue, and 63 Y's each independently represent any amino acid residue. The 63 Gly-XY's may all be identical, may be partially identical, or may be different from one another.

[0029] The repeating unit of recombinant gelatin is preferably formed by combining a portion of the amino acid sequence of naturally occurring collagen as one unit. The naturally occurring collagen referred to here is preferably type I, type II, type III, type IV, or type V. More preferably, it can be type I, type II, or type III. The collagen is preferably derived from human, horse, pig, mouse, or rat, and more preferably from human.

[0030] The isoelectric point of the recombinant gelatin is preferably 5 to 10, more preferably 6 to 10, and further preferably 7 to 9.5.

[0031] The gelatin is preferably (A) a polypeptide having the amino acid sequence shown in SEQ ID NO:1; (B) a polypeptide having an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO:1 and having bioaffinity; or (C) a polypeptide having an amino acid sequence in which one or several amino acid residues are deleted, substituted or added relative to the amino acid sequence shown in SEQ ID NO:1 and having bioaffinity Either:

[0032] The polypeptide having the amino acid sequence shown in SEQ ID NO:1 is the recombinant gelatin described in WO2008 / 103041A1, and is also referred to as CBE3. CBE3: Molecular weight: 51.6kD Structure: GAP[(GXY)63]3G Number of amino acids: 571 RGD sequence: 12 Imino acid content: 33% Nearly 100% of the amino acids have the repeating structure GXY. CBE3 has the ERGD sequence. The amino acid sequence of CBE3 does not contain any serine, threonine, asparagine, tyrosine or cysteine ​​residues. Isoelectric point: 9.34 Hydrophilic repeat unit ratio in polymer: 26.1%

[0033] (SEQ ID NO:1) 3G

[0034] From the viewpoint of the bioaffinity of gelatin, the sequence identity in (B) can be more preferably 90% or more, and even more preferably 95% or more.

[0035] The partial amino acid sequence in the sequence of (B) is a partial amino acid sequence that corresponds to the repeating unit of the sequence shown in SEQ ID NO: 1. When the polypeptide of (B) has a plurality of partial amino acid sequences that correspond to the repeating units, the polypeptide can contain one, preferably two or more, repeating units with a sequence identity of 80% or more.

[0036] It is preferable that the polypeptide defined in (B) contains a partial sequence having 80% or more sequence identity with the partial amino acid sequence corresponding to the repeating unit, which accounts for 80% or more of the total number of amino acid residues.

[0037] The length of the polypeptide defined in (B) can be 151 to 2260 amino acid residues, and from the viewpoint of degradability after crosslinking, it is preferable that the length is 193 or more amino acid residues, and from the viewpoint of stability, it is preferable that the length is 944 or less amino acid residues, and it is more preferable that the length is 380 to 756 amino acid residues.

[0038] The number of amino acid residues deleted, substituted or added in the polypeptide defined in (C) may be one or several, and varies depending on the total number of amino acid residues in the recombinant gelatin, but can be, for example, 2 to 15, preferably 2 to 5.

[0039] In the present invention, a methanol-utilizing yeast having the above-mentioned gene encoding gelatin (i.e., gelatin gene) is used.

[0040] Methanol-utilizing yeast having a gelatin gene can be prepared, for example, by introducing a recombinant vector containing a gelatin gene into the methanol-utilizing yeast. A vector is an artificially constructed nucleic acid molecule that usually contains a base sequence derived from a heterologous organism.

[0041] The vector may contain a nucleotide sequence encoding a signal peptide that enables gelatin to be secreted from yeast. Specific examples of nucleotide sequences encoding signal sequences include α-Mating Factor (αMF) from Saccharomyces cerevisiae, acid phosphatase (PHO1) from Ogataea angusta, acid phosphatase (PHO1) from Komagataella pastoris, invertase (SUC2) from Saccharomyces cerevisiae, PLB1 from Saccharomyces cerevisiae, bovine serum albumin (BSA), human serum albumin (HSA), and immunoglobulin signal sequences.

[0042] In the vector, a base sequence encoding a signal peptide may be placed at the 5' end of the gelatin gene.

[0043] The gelatin gene can be preferably contained in the vector in the form of being inserted into an expression cassette. The expression cassette refers to an expression system that contains the gelatin gene and can make it expressible as a polypeptide. The expressible state refers to a state in which the base sequence contained in the expression cassette is placed under the control of elements necessary for gene expression so that it can be expressed in the host yeast.

[0044] Elements necessary for gene expression include, for example, promoters and terminators.

[0045] Preferably, the promoter can be operably linked to the gelatin gene. The promoter refers to a base sequence region located upstream of the base sequence to be expressed, and various transcriptional regulatory factors involved in promoting or suppressing transcription, as well as RNA polymerase, bind to or act on the promoter to read the base sequence to be expressed and transcribe complementary RNA. The promoter is not particularly limited as long as it is an appropriate promoter that can cause expression in the selected carbon source.

[0046] More preferably, a methanol-inducible promoter can be operably linked to the gelatin gene. Examples of methanol-inducible promoters include, but are not limited to, AOX1 promoter, AOX2 promoter, CAT promoter, DHAS promoter, FDH promoter, FMD promoter, GAP promoter, MOX promoter, etc. Among the above, the AOX1 promoter can be particularly preferably used.

[0047] When a terminator is used, the terminator is located downstream of the gelatin gene. The terminator can be appropriately selected depending on the promoter and host yeast used.

[0048] The vector may further include a base sequence of a selection marker gene (an auxotrophy complementing gene, a drug resistance gene, etc.). An example of the drug resistance gene is a Zeocin resistance gene, but is not particularly limited thereto. The vector may further include an autonomous replication sequence (ARS), a centromere DNA sequence, or a telomere DNA sequence depending on the host.

[0049] Examples of vectors include circular vectors, linear vectors, and artificial chromosomes, and are preferably plasmid vectors.

[0050] As a plasmid vector, pPICZαA, B, C vectors (Invitrogen) can be used, but are not limited to these. pPICZαA, B, C vectors can be used for the expression and secretion of recombinant proteins in Pichia pastoris. Recombinant proteins are expressed as fusions to an N-terminal peptide encoding the Saccharomyces cerevisiaea-factor secretion signal. The ppPICZα vector contains the AOX1 promoter for methanol-inducible expression, the α-factor secretion signal to induce secretory expression of recombinant proteins, a Zeocin resistance gene for selection in both E. coli and Pichia, and a C-terminal peptide containing a c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of recombinant fusion proteins.

[0051] The gelatin gene can be introduced into yeast by any known transformation method, including, but not limited to, electroporation, the lithium acetate method, the spheroplast method, etc. Among these, electroporation is preferred.

[0052] The thus obtained methanol-utilizing yeast having the gelatin gene can be used for the production of gelatin after being appropriately cultured and grown by a conventional method. In the present invention, it is preferable to culture the methanol-utilizing yeast to a high density state using a carbon source other than methanol, such as glycerol, before producing gelatin by culturing with the addition of components of at least two groups selected from groups 1 to 4.

[0053] In the method for producing gelatin of the present invention, Group 1: At least one of glycine and serine Group 2: Pyruvic acid or its salts Group 3: Glutathione, and Group 4: At least one of arginine and lysine and methanol are added to the medium, and a methylotrophic yeast having a gelatin gene is cultured.

[0054] According to another aspect of the present invention, Group 1: At least one of glycine and serine Group 2: Pyruvic acid or its salts Group 3: Glutathione, and Group 4: At least one of arginine and lysine An additive for use in the method for producing gelatin according to the present invention is provided, comprising at least two groups of components selected from:

[0055] According to yet another aspect of the present invention, Group 1: At least one of glycine and serine Group 2: Pyruvic acid or its salts Group 3: Glutathione, and Group 4: At least one of arginine and lysine at least two of the following groups of ingredients: methanol; Nitrogen source; Minerals; and vitamin The present invention provides a medium for use in the production of gelatin by a methylotrophic yeast, comprising:

[0056] In the method for producing gelatin, additives and culture medium according to the present invention, specifically, any of the combinations of Group 1 and Group 2, Group 1 and Group 3, Group 1 and Group 4, Group 2 and Group 3, Group 2 and Group 4, Group 3 and Group 4, Group 1 and Group 2 and Group 3, Group 1 and Group 2 and Group 4, Group 1 and Group 3 and Group 4, Group 2 and Group 3 and Group 4, and Group 1 and Group 2 and Group 3 and Group 4 can be used.

[0057] In the method, additive and medium for producing gelatin according to the present invention, the components of at least two groups are preferably components of Group 1, Group 2 and Group 3.

[0058] In the method, additive and medium for producing gelatin according to the present invention, it is further preferred that the components of the at least two groups are: (1) glycine or serine, pyruvic acid or a salt thereof, and glutathione, (2) glycine, and pyruvic acid or a salt thereof, (3) pyruvic acid or a salt thereof, and glutathione, (4) pyruvic acid or a salt thereof, and arginine, (5) glycine and glutathione, or (6) serine and lysine, Either:

[0059] Preferably, at least two groups of components mentioned above are added to the medium in portions or successively. The glutathione is preferably reduced glutathione. As pyruvic acid or a salt thereof, sodium pyruvate is preferred.

[0060] In the method for producing gelatin and the medium according to the present invention, the preferred ranges of the total amount of each component added are as follows: Preferably, the total amount of glycine added is 1.0 g or less, or the total amount of serine added is 1.0 g or less, per liter of medium. Preferably, the total amount of pyruvic acid or a salt thereof added is 4.6 g or less per liter of medium. Preferably, the total amount of glutathione added is 6.2 g or less per liter of medium. Preferably, the total amount of arginine added is 0.7 g or less, or the total amount of lysine added is 2.3 g or less, per liter of medium.

[0061] More preferably, the total amount of glycine added is 0.8 g or less, or the total amount of serine added is 0.5 g or less, per 1 liter of medium. Even more preferably, the total amount of glycine added is 0.7 g or less, or the total amount of serine added is 0.3 g or less, per 1 liter of medium. Even more preferably, the total amount of glycine added is 0.3 g or less, or the total amount of serine added is 0.15 g or less, per 1 liter of medium. Particularly preferably, the total amount of glycine added is 0.15 g or less, or the total amount of serine added is 0.1 g or less, per 1 liter of medium.

[0062] More preferably, the total amount of pyruvic acid or its salts added per 1 liter of medium is 4.0 g or less. Even more preferably, the total amount of pyruvic acid or its salts added per 1 liter of medium is 3.5 g or less. Even more preferably, the total amount of pyruvic acid or its salts added per 1 liter of medium is 3.0 g or less. Particularly preferably, the total amount of pyruvic acid or its salts added per 1 liter of medium is 1.0 g or less.

[0063] More preferably, the total amount of glutathione added per 1 liter of medium is 5.0 g or less. Even more preferably, the total amount of glutathione added per 1 liter of medium is 4.0 g or less. Even more preferably, the total amount of glutathione added per 1 liter of medium is 3.0 g or less. Particularly preferably, the total amount of glutathione added per 1 liter of medium is 1.0 g or less.

[0064] More preferably, the total amount of arginine added is 0.5g or less, or the total amount of lysine added is 2.0g or less per 1 liter of medium. Even more preferably, the total amount of arginine added is 0.3g or less, or the total amount of lysine added is 1.0g or less per 1 liter of medium. Even more preferably, the total amount of arginine added is 0.2g or less, or the total amount of lysine added is 0.2g or less per 1 liter of medium. Particularly preferably, the total amount of arginine added is 0.15g or less, or the total amount of lysine added is 0.15g or less per 1 liter of medium.

[0065] When serine, pyruvic acid or a salt thereof, and glutathione are combined, the total amount of each of serine, pyruvic acid or a salt thereof, and glutathione to be added per liter of medium is preferably 0.5 g or less, 4.0 g or less, or 5.0 g or less, more preferably 0.3 g or less, 3.5 g or less, or 4.0 g or less, and even more preferably 0.15 g or less, 3.5 g or less, or 3.5 g or less.

[0066] When serine and lysine are combined, the total amount of serine and lysine to be added per liter of medium is preferably 0.5 g or less, 2.0 g or less, more preferably 0.3 g or less, 1.0 g or less, even more preferably 0.15 g or less, 0.2 g or less, and particularly preferably 0.1 g or less, 0.15 g or less.

[0067] When glycine, pyruvic acid or a salt thereof, and glutathione are combined, the total amount of each of glycine, pyruvic acid or a salt thereof, and glutathione to be added per liter of medium is preferably 0.8 g or less, 4.0 g or less, or 4.0 g or less, more preferably 0.7 g or less, 3.5 g or less, or 3.5 g or less, even more preferably 0.3 g or less, 3.5 g or less, or 3.5 g or less, and particularly preferably 0.15 g or less, 3.5 g or less, or 3.5 g or less.

[0068] When glycine, pyruvic acid or a salt thereof are combined, the total amount of each of glycine, pyruvic acid or a salt thereof to be added per liter of medium is preferably 0.8 g or less, 4.0 g or less, more preferably 0.7 g or less, 3.5 g or less, even more preferably 0.3 g or less, 3.5 g or less, and particularly preferably 0.15 g or less, 3.5 g or less.

[0069] When glycine and glutathione are combined, the total amount of glycine and glutathione added per liter of medium is preferably 0.8 g or less, 5.0 g or less, more preferably 0.7 g or less, 4.0 g or less, even more preferably 0.3 g or less, 3.0 g or less, and particularly preferably 0.15 g or less, 1.0 g or less.

[0070] The method for producing gelatin of the present invention comprises culturing a methylotrophic yeast having a gelatin gene. The yeast can be cultured by appropriately using a conventional yeast culture method, and the culture conditions can be appropriately set so as to be suitable for the growth of the yeast.

[0071] The medium used for the culture may be either a natural medium or a synthetic medium, so long as it contains a carbon source, a nitrogen source, minerals (inorganic substances), and, if necessary, trace nutrients required by the strain used (e.g., vitamins such as biotin).

[0072] The medium preferably contains methanol as a carbon source, but may contain a carbon source other than methanol, such as carbohydrates such as glucose, sucrose, maltose, fructose, mannose, trehalose, mannitol, sorbitol, starch, dextrin, and molasses, organic acids such as citric acid and succinic acid, and glycerol.

[0073] As the nitrogen source for the medium, for example, organic nitrogen sources such as corn steep liquor, soybean meal, or peptones, or inorganic nitrogen sources such as ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, ammonium phosphate, etc. Furthermore, nitrogen-containing natural products such as peptone, polypeptone, bactopeptone, meat extract, fish extract, yeast extract, corn steep liquor, soybean flour, soybean meal, dry yeast, casamino acid, soluble vegetable protein, etc. can also be used as the nitrogen source.

[0074] As minerals (inorganic substances) for the medium, for example, calcium salts, magnesium salts, potassium salts, sodium salts, phosphates, manganese salts, zinc salts, iron salts, copper salts, molybdenum salts, cobalt salts, etc. are appropriately used. Specifically, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, zinc sulfate, sodium chloride, potassium chloride, calcium chloride, etc. are used. Furthermore, phosphoric acid, sulfuric acid, potassium hydroxide, etc. are appropriately used.

[0075] The culture method is preferably a liquid culture method, and any of batch culture, fed-batch culture, continuous culture, and perfusion culture may be used.

[0076] The culture temperature and pH may be selected from conditions suitable for the growth of the genetically modified yeast. The temperature is preferably 20 to 40° C., more preferably 25 to 35° C. The pH is preferably 2 to 9, more preferably 3 to 7. The culture time is preferably 1 hour to 14 days, more preferably 6 hours to 10 days, and further preferably 12 hours to 7 days. The culture can be preferably carried out with shaking or aeration and stirring.

[0077] By culturing a methanol-utilizing yeast having a gelatin gene, gelatin can be accumulated in the host yeast or in the culture solution and recovered. Here, the culture solution means the culture supernatant as well as cultured cells, cultured bacteria, or cell or bacteria disruption. The method for producing gelatin according to the present invention includes a method of culturing a methanol-utilizing yeast having a gelatin gene and accumulating gelatin in the bacteria, or a method of secreting gelatin into the culture supernatant and accumulating gelatin, and preferably a method of secreting gelatin into the culture supernatant and accumulating gelatin.

[0078] As for the method of recovering gelatin, known purification methods can be appropriately combined and used. For example, methanol-utilizing yeast is cultured in a medium, and the culture liquid is centrifuged or filtered to remove the cells from the culture supernatant. The obtained culture supernatant can be subjected to salting out (ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (protein fraction precipitation method using acetone or ethanol, etc.), dialysis, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography, ultrafiltration, etc., either alone or in combination, to recover gelatin from the culture supernatant.

[0079] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. EXAMPLES

[0080] (1) Construction of yeast with methanol-inducible promoter A plasmid vector was prepared by linking a gene for a target protein (CBE3) and a zeocin resistance gene downstream of the methanol-inducible AOX1 promoter using pPICZα plasmid vector (Invitrogen). The nucleotide sequence of the target protein (CBE3) is shown in SEQ ID NO:2. The nucleotide sequence of the prepared plasmid vector is shown in SEQ ID NO:3.

[0081] The Pichia pastoris strain (X-33) transformed by electroporation with the above-mentioned plasmid vector was plated on a YPD agar medium plate containing the antibiotic zeocin (500 μg / mL) as a selection marker for screening, and transformed yeast strains were obtained from the grown colonies.

[0082] SEQ ID NO:2:

[0083] SEQ ID NO:3:

[0084] (2) Preparation of cell suspension The yeast prepared in (1) above was added to the YNB medium (w / o amino acid) shown in Table 1 and cultured for 20 to 24 hours at 30°C and 250 rpm in a Bio Shaker (TAITEC, BR-43FL). The culture solution was centrifuged for 5 minutes at 25°C and 300 g in a centrifuge (TOMY, MX-307), and the culture supernatant was completely removed. The pellet was suspended in the BMD medium (Buffered Minimal Medium Supplemented with 1% Glucose) shown in Table 1, and the turbidity of the suspension was adjusted to be in the range of 0.2 to 0.3 (OD 600 nm) using a turbidimeter (Biochrom, CO8000).

[0085] [Table 1]

[0086] (3) Seeding and culturing in Deep96-well plates The cell suspension prepared in (2) above was added to a Deep96-well plate (NEST, WNB503001) at 250 μL each. A gas exchangeable seal (Excel, BS-25) was attached to the plate, and the plate was incubated at 30°C and 1000 rpm in a plate thermoshaker (Allsheng, MB100-4A) for 64 to 66 hours. Immediately after that, various additives were prepared to the desired concentration using BMM2 solution (Buffered Minimal Medium Supplemented with 1% Methanol) shown in Table 2, and administered at 250 μL / well. Approximately 70 to 74 hours, 90 to 96 hours, and 114 to 120 hours after the start of culture, various additives were prepared to the desired concentration using BMM10 solution (Buffered Minimal Medium Supplemented with 5% Methanol) shown in Table 2, and administered at 50 μL / well. After about 138 to 144 hours, the culture medium in the wells was collected and placed in a centrifugal filter unit (Millipore, UFC30GV00), and centrifuged at 25° C. and 10,000 g for 5 minutes, after which the culture medium free of bacterial cells was collected.

[0087] Additive Concentrations The following concentrations of additives were administered alone or in combination: Lysine: 0.185, 0.9, 1.8, 3.7, 7.3, 29, 73mg / mL Arginine: 0.183, 0.185, 1.1, 4.4, 8.7, 35, 87mg / mL Glycine: 0.04, 0.08, 0.16, 0.32, 1, 1.6, 5, 16mg / mL Serine: 0.04, 0.08, 0.16, 0.32, 1.6, 5, 16mg / mL Sodium pyruvate: 0.1, 1, 4, 5, 10 mg / mL Reduced glutathione: 0.1, 1, 4, 5, 10mg / mL Oxidized glutathione: 0.1, 1, 10mg / mL

[0088] [Table 2]

[0089] (4) Quantification of secreted CBE3 The culture medium from which the bacteria had been removed was used as the measurement sample. Gel filtration chromatography was performed on 5 μL of the measurement sample under the following conditions, and a curve was obtained with the absorbance on the vertical axis and the molecular weight on the horizontal axis. The concentration was calculated from the area of ​​the molecular weight peak of CBE3 (51.6 kDa) from the obtained curve. (conditions) Column: Waters, ACQUITY UPLC Protein BEH SEC Column temperature: 30℃ Column size: inner diameter 4.6 mm, length 150 mm, particle size 1.7 μm ·Flow rate: 0.2mL / min ·Injection volume: 5μL Eluent: 0.1 mol / L sodium sulfate, 0.01 mol / L sodium dihydrogen phosphate, 1% SDS, pH 5.3 Rinse solution: Water Detection wavelength: 215nm

[0090] For each of the Comparative Examples and Examples, the total amount of additive added per liter of medium, the protein yield, and the rate of increase in protein yield from when no additive was added are shown in Tables 3 and 4 and Figures 1 and 2.

[0091] The total amount of additives added per liter of medium is, for example, the value calculated as follows in Comparative Example 2. Additive (lysine) concentration: 0.185 mg / mL Total amount of additives: 0.185mg / mL×(250μL+50μL×3)=0.074mg Total volume of medium: 250 μL (cell suspension) + 250 μL (BMM2 solution) + 50 μL x 3 = 0.65 mL Total amount of additives per liter of medium: 0.074mg / 0.65mL=0.114g / L

[0092] The rate of increase in protein yield from the case where no addition was made is, for example, in Comparative Example 2, a value calculated as follows. A = Protein yield for Comparative Example 2 (lysine added): 0.201 mg / mL B = Protein yield in Comparative Example 1 (no additives): 0.207 mg / mL Increase in protein yield from no addition = AB / B x 100 = -3%

[0093] As shown in Table 4, Group 1: at least one of glycine and serine; Group 2: Pyruvic acid or its salts Group 3: Glutathione, and Group 4: At least one of arginine and lysine By using at least two groups of ingredients selected from the following, advantageous synergistic effects are observed:

[0094] [Table 3] JPEG2025017882000004.jpg126170

[0095] [Table 4]

Claims

1. Group 1: At least one of glycine and serine Group 2: Pyruvic acid or its salts Group 3: glutathione, and Group 4: At least one of arginine and lysine and methanol to a medium, and culturing a methylotrophic yeast having a gelatin gene.

2. 2. The method of claim 1, wherein the at least two groups of components are group 1, group 2, and group 3 components.

3. The at least two groups of components are (1) glycine or serine, pyruvic acid or a salt thereof, and glutathione, (2) glycine, and pyruvic acid or a salt thereof; (3) pyruvic acid or a salt thereof, and glutathione, (4) pyruvic acid or a salt thereof, and arginine, (5) glycine and glutathione, or (6) serine and lysine, The method for producing gelatin according to claim 1, which is any one of the above.

4. 2. The method for producing gelatin according to claim 1, wherein the total amount of glycine added is 1.0 g or less, or the total amount of serine added is 1.0 g or less, per liter of medium.

5. 2. The method for producing gelatin according to claim 1, wherein the total amount of pyruvic acid or a salt thereof added is 4.6 g or less per liter of medium.

6. 2. The method for producing gelatin according to claim 1, wherein the total amount of glutathione added is 6.2 g or less per liter of medium.

7. 2. The method for producing gelatin according to claim 1, wherein the total amount of arginine added is 0.7 g or less or the total amount of lysine added is 2.3 g or less per liter of medium.

8. 8. The method for producing gelatin according to claim 1, wherein the methanol-inducible promoter is operably linked to the gelatin gene.

9. The method for producing gelatin according to any one of claims 1 to 7, wherein the gelatin has an amino acid sequence represented by the following formula (1): A - [(Gly - X - Y) ｎ ｍ - B Formula (1) In the formula, A represents one or more any amino acid residues, B represents one or more any amino acid residues, Gly represents a glycine residue, n Xs each independently represent any amino acid residue, and n Ys each independently represent any amino acid residue. m represents an integer of 2 to 10, n represents an integer of 3 to 100, and n Gly-X-Ys may be all or partly the same or different from each other.

10. The gelatin is (A) a polypeptide having the amino acid sequence shown in SEQ ID NO:1; (B) a polypeptide having an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO:1 and having bioaffinity; or (C) A polypeptide having an amino acid sequence in which one or several amino acid residues are deleted, substituted or added relative to the amino acid sequence shown in SEQ ID NO: 1, and having bioaffinity. The method for producing gelatin according to any one of claims 1 to 7,

11. Group 1: At least one of glycine and serine Group 2: Pyruvic acid or its salts Group 3: glutathione, and Group 4: At least one of arginine and lysine 8. An additive for use in the method for producing gelatin according to any one of claims 1 to 7, comprising at least two components of the group selected from:

12. 12. The additive of claim 11, wherein the at least two groups of components are Group 1, Group 2, and Group 3 components.

13. The at least two groups of components are (1) glycine or serine, pyruvic acid or a salt thereof, and glutathione, (2) glycine, and pyruvic acid or a salt thereof; (3) pyruvic acid or a salt thereof, and glutathione, (4) pyruvic acid or a salt thereof, and arginine, (5) glycine and glutathione, or (6) serine and lysine, The additive according to claim 11,

14. Group 1: At least one of glycine and serine Group 2: Pyruvic acid or its salts Group 3: glutathione, and Group 4: At least one of arginine and lysine at least two groups of components selected from: methanol; Nitrogen source; Minerals; and vitamin A medium for use in producing gelatin by a methylotrophic yeast, comprising:

15. 15. The medium of claim 14, wherein the at least two groups of components are Group 1, Group 2, and Group 3 components.

16. The at least two groups of components are (1) glycine or serine, pyruvic acid or a salt thereof, and glutathione, (2) glycine, and pyruvic acid or a salt thereof; (3) pyruvic acid or a salt thereof, and glutathione, (4) pyruvic acid or a salt thereof, and arginine, (5) glycine and glutathione, or (6) serine and lysine, The medium according to claim 14, which is any one of the above.

17. 17. The medium according to claim 14, wherein the total amount of glycine added is 1.0 g or less or the total amount of serine added is 1.0 g or less per liter of medium.

18. 17. The medium according to claim 14, wherein the total amount of pyruvic acid or a salt thereof added is 4.6 g or less per liter of the medium.

19. 17. The medium according to claim 14, wherein the total amount of glutathione added is 6.2 g or less per liter of medium.

20. 17. The medium according to claim 14, wherein the total amount of arginine added is 0.7 g or less or the total amount of lysine added is 2.3 g or less per liter of medium.

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