Method for producing recombinant protein

Abstract

A method for producing a recombinant protein, the method comprising: culturing, in a culture medium, a recombinant cell that expresses the recombinant protein; inducing the expression of a gene encoding the recombinant protein; and collecting the recombinant protein, wherein the recombinant protein has a glycine residue content of 25% or more, and the recombinant cell contains at least one modification selected from the group consisting of (A) and (B) below. (A) A modification that increases threonine synthesis, and (B) a modification that increases synthesis from threonine to glycine.

Description

Methods for producing recombinant proteins

[0001] This invention relates to a method for producing recombinant proteins.

[0002] Glycine-rich proteins such as collagen, elastin, and fibroin are characterized by their high content of glycine residues. These proteins have proven useful in various industries, and there has been a particular need to establish effective mass production methods using recombinant protein production by microorganisms.

[0003] Under these circumstances, Non-Patent Document 1 discloses a technique that increases glycine production by activating the system for synthesizing glycine from serine using E. coli with an enhanced glycyl-tRNA pool, thereby increasing the productivity of glycine-rich proteins.

[0004] PNAS 2010, vol. 107, no. 32, 14059-14063.

[0005] However, in the technology described in Non-Patent Document 1, it was found that when the system for synthesizing glycine from serine is activated, the production of C1 units increases along with the increase in glycine production. However, because the demand for the produced C1 units is less than the demand for glycine, C1 units accumulate, which suppresses the rate of glycine production. It was also found that the accumulation of C1 units leads to the accumulation of organic acids such as formic acid, lowering the pH of the culture medium, thus requiring extra effort to control the pH.

[0006] The present invention aims to provide a method for producing glycine-rich proteins that can improve the productivity of glycine-rich proteins while suppressing the production of undesirable metabolic pathway products. Furthermore, the present invention aims to provide recombinant cells capable of producing glycine-rich proteins with high productivity while suppressing the production of undesirable metabolic pathway products.

[0007] To address the above challenges, the inventors conducted extensive research and discovered that by enhancing the synthesis pathway from threonine to glycine, it is possible to improve the productivity of glycine-rich proteins while suppressing the production of undesirable metabolic pathway products.

[0008] This invention was completed based on these findings and includes the following broad embodiments of the invention.

[0009] [Item 1] A method for producing a recombinant protein, comprising culturing recombinant cells expressing a recombinant protein in a culture medium, inducing the expression of a gene encoding the recombinant protein, and collecting the recombinant protein, wherein the glycine residue content in the recombinant protein is 25% or more, and the recombinant cells include at least one modification selected from the group consisting of (A) and (B) below: (A) a modification that increases threonine synthesis; (B) a modification that increases glycine synthesis from threonine. [Item 2] The method for producing a recombinant protein according to Item 1, wherein the recombinant cells are cells modified to increase threonine synthesis and to increase glycine synthesis from threonine. [Item 3] The method for producing a recombinant protein according to Item 1 or 2, wherein (A) includes a mutation in a gene encoding a protein having aspartate kinase activity. [Clause 4] The method for producing a recombinant protein according to any one of Clauses 1 to 3, wherein (B) is modified to overexpress a gene encoding a protein having L-threonine-3-dehydrogenase activity and a gene encoding a protein having 2-amino-3-oxobutanoic acid coenzyme A ligase activity. [Clause 5] The method for producing a recombinant protein according to any one of Clauses 1 to 4, wherein the recombinant protein is a structural protein. [Clause 6] The method for producing a recombinant protein according to any one of Clauses 1 to 5, wherein the recombinant protein is fibroin, collagen, or elastin, or a fusion protein thereof. [Clause 7] Recombinant cells for producing a recombinant protein, wherein the recombinant protein contains 25% or more of glycine residues, and the recombinant cells include at least one modification selected from the group consisting of (A) and (B) below: (A) a modification that increases threonine synthesis; (B) a modification that increases glycine synthesis from threonine. [Clause 8] The recombinant cell according to Clause 7, wherein the recombinant cell is modified to increase threonine synthesis and to increase glycine synthesis from threonine. [Clause 9] The recombinant cell according to Clause 7 or 8, wherein (A) comprises a mutation in a gene encoding a protein having aspartate kinase activity.[Clause 10] The recombinant cell according to any one of Clauses 7 to 9, wherein (B) is modified to overexpress a gene encoding a protein having L-threonine-3-dehydrogenase activity and a gene encoding a protein having 2-amino-3-oxobutanoic acid coenzyme A ligase activity. [Clause 11] The recombinant cell according to any one of Clauses 7 to 10, wherein the recombinant protein is a structural protein. [Clause 12] The recombinant cell according to any one of Clauses 7 to 11, wherein the recombinant protein is fibroin, collagen, or elastin, or a fusion protein thereof.

[0010] According to the present invention, it is possible to improve the productivity of glycine-rich proteins while suppressing the production of undesirable metabolic pathway products.

[0011] In this specification, the singular form (a, an, the, etc.) includes both singular and plural forms unless otherwise explicitly stated or the context clearly contradicts it. In this specification, "comprise" is a concept that also includes "consistently essentially of" and "consistently of".

[0012] The present invention provides a method for producing recombinant proteins. The method of the present invention comprises culturing recombinant cells expressing recombinant proteins in a culture medium, inducing the expression of a gene encoding the recombinant protein, and collecting the recombinant protein. Furthermore, the glycine residue content in the recombinant protein is 25% or more. In addition, the recombinant cells include at least one modification selected from the group consisting of (A) and (B) below: (A) a modification that increases threonine synthesis; (B) a modification that increases glycine synthesis from threonine.

[0013] 1. Recombinant Proteins The recombinant proteins produced in this invention are not particularly limited as long as they contain 25% or more glycine residues. The glycine residue content may be 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 45% or more, or 50% or more. Furthermore, there is no particular upper limit to the glycine residue content, but examples include 99% or less, 90% or less, or 80% or less. Recombinant proteins may be, for example, proteins derived from microorganisms, plants, animals, viruses, or even proteins with artificially designed amino acid sequences. Proteins may be monomeric proteins or polymeric proteins. Proteins may be secreted proteins or non-secreted proteins. Furthermore, the term "protein" includes forms called peptides, such as oligopeptides and polypeptides. In this specification, "glycine residue content" is the value expressed by the following formula: Glycine residue content = (Number of glycine residues in recombinant protein / Total number of amino acid residues in recombinant protein) × 100 (%)

[0014] The number of amino acid residues in the recombinant protein according to this embodiment is not particularly limited, but may be, for example, 50 or more. Alternatively, the number of amino acid residues may be, for example, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, or 500 or more. The number of amino acid residues may also be, for example, 5000 or less, 4500 or less, 4000 or less, 3500 or less, 3000 or less, 2500 or less, 2000 or less, 1500 or less, or 1000 or less. The fewer the number of amino acid residues, the higher the solubility in the solvent tends to be. The preferred number of amino acid residues in a protein is, for example, 100-5000, 150-4500, 200-4000, 250-3500, 300-3000, 350-2500, 400-2000, 450-1500, or 500-1000.

[0015] In this specification, "amino acid residue" includes natural amino acids (Gly, Ala, Met, Ser, Thr, Cys, Met, Asp, Asn, Glu, Gln, Leu, Ile, Val, His, Lys, Arg, Phe, Tyr, Trp, etc.) and non-natural amino acids (e.g., DOPA, selenomethionine, homotyrosine, etc.).

[0016] The molecular weight of the recombinant protein according to this embodiment is not particularly limited, but may be, for example, 2 kDa to 500 kDa. Alternatively, the molecular weight may be, for example, 2 kDa or more, 3 kDa or more, 4 kDa or more, 5 kDa or more, 6 kDa or more, 7 kDa or more, 8 kDa or more, 9 kDa or more, 10 kDa or more, 20 kDa or more, 30 kDa or more, 40 kDa or more, 50 kDa or more, 60 kDa or more, 70 kDa or more, 80 kDa or more, 90 kDa or more, or 100 kDa or more, and may also be 500 kDa or less, 400 kDa or less, less than 360 kDa, 300 kDa or less, or 200 kDa or less. The molecular weight may be, for example, 2 kDa to 500 kDa, 3 kDa to 500 kDa, 4 kDa to 500 kDa, 5 kDa to 500 kDa, 6 kDa to 500 kDa, 7 kDa to 500 kDa, 8 kDa to 500 kDa, 9 kDa to 500 kDa, 10 kDa to 500 kDa, 20 kDa to 400 kDa, 30 kDa to 360 kDa, 40 kDa to 360 kDa, 50 kDa to 360 kDa, 60 kDa to 300 kDa, 70 kDa to 300 kDa, 80 kDa to 300 kDa, 90 kDa to 200 kDa, or 100 kDa to 200 kDa.

[0017] The recombinant protein according to this embodiment may have a repeat sequence. That is, the recombinant protein according to this embodiment may have multiple amino acid sequences (repeat sequence units) with high sequence identity within the recombinant protein. The number of amino acid residues in a repeat sequence unit may be 6 to 200. The total number of glycine residues, serine residues, glutamine residues, and alanine residues relative to the total number of amino acid residues in the repeat sequence unit may be 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, or 70% or more. Furthermore, the sequence identity between repeat sequence units may be, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The recombinant protein according to this embodiment may have at least two repeat sequence units.

[0018] The recombinant protein according to this embodiment may have an amino acid sequence including a domain sequence represented by formula 1: REP1-REP2. Such a recombinant protein may contain, for example, 2 or more, 4 or more, 8 or more, 10 or more, 20 or more, 50 or more, 100 or more, or 200 or more of the domain sequence. The upper limit may be 300 or less. REP1 in formula 1 may be an amino acid sequence consisting of 2 to 27 amino acid residues containing at least one alanine, and may be an integer of 2 to 20, 2 to 16, or 2 to 12. REP2 may be an amino acid sequence consisting of 10 to 200 amino acid residues. Multiple REP1s may have the same amino acid sequence or be different amino acid sequences. Multiple REP2s may have the same amino acid sequence or be different amino acid sequences. In addition, further amino acid sequences (N-terminal sequence and C-terminal sequence) may be added to either the N-terminal side and the C-terminal side or both of each domain sequence. The N-terminal and C-terminal sequences are not limited to those shown, but do not need to contain repeating sequences. Furthermore, domain sequences may appear consecutively, and additional N-terminal and / or C-terminal sequences may be added to either the N-terminal or C-terminal end, or both, of multiple consecutive domain sequences. The recombinant protein may contain a tag sequence (e.g., a His tag, serine-rich sequence, etc.) at either the N-terminus or C-terminus, or both. This enables isolation, immobilization, detection, and visualization of the recombinant protein. Additionally, short-chain peptide sequences that promote self-assembly may be added to the terminal or internal structure of the recombinant protein.

[0019] When forming the recombinant protein according to this embodiment, amino acids with relatively small side chains are more likely to form hydrogen bonds, making it easier to obtain a molded product with higher strength. In addition, since alanine and glycine residues are nonpolar amino acids with side chains, they are arranged to face inward during the folding process in polypeptide production, making it easier to adopt an α-helix structure or a β-sheet structure. Therefore, it is desirable that the recombinant protein produced in the present invention has a glycine residue content of 25% or more, as well as a high proportion of alanine residues. From the viewpoint of obtaining a molded product with superior strength, the alanine residue content may be, for example, 10 to 40%, and may be 12 to 40%, 15 to 40%, 10 to 30%, 12 to 30%, or 15 to 30%. The ratio of alanine residues to the total number of amino acid residues in REP1 should be 40% or more, but may also be 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning that it consists only of alanine residues).

[0020] REP2 in Formula 1 may be a glycine-rich region having a glycine residue content of at least 30%, and preferably has a glycine residue content of 32% or more, 35% or more, 38% or more, or 40% or more.

[0021] In the recombinant protein according to this embodiment, each repeat sequence unit may include a domain sequence represented by formula 1: REP1-REP2. Because the domain sequence is included in the repeat sequence unit, the recombinant protein according to this embodiment repeatedly has these secondary structures. Therefore, when the recombinant protein is made into a molded article such as a fiber, film, or resin, it is expected that these secondary structures will exhibit high strength.

[0022] The recombinant protein according to this embodiment may have a different amino acid sequence from that of a naturally occurring protein. Alternatively, it may be a protein whose amino acid sequence has been modified based on the amino acid sequence of a naturally occurring protein (for example, a protein whose amino acid sequence has been modified by altering the gene sequence of a cloned naturally occurring protein).

[0023] The recombinant protein according to this embodiment may be a structural protein. In this specification, "structural protein" refers to a protein in which amino acids are linked together via peptide bonds and which exhibits mechanical properties (strength, elasticity, toughness, shape retention, etc.) based on the primary structure, secondary structure, or intermolecular interactions of its amino acid sequence. Furthermore, a structural protein is a type of protein that can be used industrially, and means a protein involved in the structure of living organisms, a protein that constitutes a structure produced by living organisms, or a protein derived from such a structure. Moreover, a structural protein is a protein that self-aggregates under certain conditions to form structures such as fibers, films, resins, gels, micelles, and nanoparticles, and can be said to be a protein that forms the backbone of living organisms, materials, etc., by repeatedly having a characteristic amino acid sequence or a motif consisting of a number of amino acid residues.

[0024] In this specification, structural proteins may be natural proteins or artificial proteins. That is, structural proteins may be of biological origin or artificially designed, and may include natural proteins (e.g., silk fibroin, spirolin, collagen, elastin, resilin, keratin, reflectin, etc.) and their modifiers or reconstituted sequences containing their structural motifs.

[0025] In this specification, "artificial protein" means a protein that is artificially produced, and includes recombinant proteins and synthetic proteins. An artificial protein may have a domain sequence that differs from the amino acid sequence of a naturally occurring protein. Furthermore, an "artificial protein" may be one whose amino acid sequence is modified based on the amino acid sequence of a naturally occurring protein (for example, by modifying the gene sequence of a cloned naturally occurring protein), or it may be one that is artificially designed and synthesized without relying on a naturally occurring protein (for example, one that has a desired amino acid sequence by chemically synthesizing nucleic acid encoding a designed amino acid sequence). Unlike natural proteins, artificial proteins allow for the free design of their amino acid sequences. Therefore, when using such artificial proteins as molding materials or molded products, the function, characteristics, and physical properties of the molding material or molded product can be arbitrarily controlled by appropriately designing the amino acid sequence of the artificial protein. In addition, because uniform molecular design is always possible, it is possible to stably obtain proteins with high homology to the target protein that are suitable for the purpose. Thus, the quality of molding materials or molded products obtained using artificial proteins can be advantageously stabilized. From this perspective, artificial structural proteins (i.e., structural proteins that are also artificial proteins) are advantageously used as artificial proteins.

[0026] Furthermore, the term "structural proteins" in this specification broadly includes a group of high-molecular-weight proteins with repeating motifs that have evolved to support the mechanical and physical properties of biological structures, materials, and the like.

[0027] These structural proteins include the following various groups 1 to 6 according to their functional classification. 1. Fiber-forming structural protein group: Examples: silk fibroin, spiroin, collagen, flagellin, etc. It has high strength and high rigidity and forms regular structures such as β-sheets and triple helices. 2. Elastic structural protein group: Examples: elastin, resilin, tenascin, fibronectin, etc. It has a random coil or β-turn structure due to repetitive sequences and is responsible for reversible stretching, energy absorption, etc. 3. Outer shell / protective structural protein group: Examples: keratin, chitin-binding protein, CP-LCP, scalenin, etc. It forms a highly durable outer skin / exoskeleton structure by means of cysteine cross-linking, hydrophobic interactions, etc. 4. Skeletal structural protein group: Examples: actin, tubulin, tropomyosin, etc. It forms a cell skeleton, a support structure, etc. and contributes to tension and shape maintenance. 5. Optical structural protein group: Examples: reflectin, g limerin, etc. It controls the reflection, interference, and scattering characteristics of light by self-assembly and is responsible for color change and camouflage of organisms. 6. Adhesive / composite structural protein group: Examples: spigrin, adhesin, fibrin, etc. It is responsible for biological adhesion and formation of composite structures by means of intermolecular cross-linking, non-covalent bond networks, etc.

[0028] Each of the above groups includes natural proteins themselves, or artificial design sequences, fusion-type sequences (hybrid motifs), variants, and derivatives based on them.

[0029] In addition, the structural protein may have sequence characteristics such as repetitive sequences, periodic arrangements of hydrophobic / hydrophilic residues, the ability to form β-sheet / coiled-coil structures, and self-assembly ability.

[0030] In one embodiment, the structural protein may have the following primary structural features 1 to 5. 1. Presence of repetitive motifs: In the structural protein, it is preferable that a characteristic motif consisting of about 2 to 50 residues is repeated at least twice or more. The motif can form a β-sheet, β-turn, random coil, or coiled-coil structure. Examples of the repetitive motif include, for example, glycine-alanine repeat (Gly-Ala-Gly-Ala-Gly-Ser (SEQ ID NO: 3), etc.), proline-glycine repeat (Gly-Pro-Gly-Gly-X (X is any amino acid) (SEQ ID NO: 4), etc.), β - zipper motif (Val-Gln-Ile-Val-Tyr-Lys (SEQ ID NO: 5), Leu-Asn-Ile-Tyr-Gln-Tyr (SEQ ID NO: 6), Asn-Asn-Gln-Gln-Asn-Tyr (SEQ ID NO: 7), etc.), collagen type (Gly-X-Y (X / Y = Pro, Hyp, etc.), etc.).

[0031] 2. Interposition of linker sequences: In the structural protein, a linker that imparts flexibility (such as Gly-Ser, Ala-Gly, Gly-Gly-Gly-Ser (SEQ ID NO: 8), etc.) may be arranged between the repetitive units.

[0032] 3. Periodic distribution of hydrophobic and hydrophilic residues: In the structural protein, hydrophobic residues (such as Ala, Val, Leu, Ile, Phe, etc.) and hydrophilic residues (such as Ser, Thr, Gln, Asn, etc.) are periodically arranged, which can induce phase separation and secondary structure formation.

[0033] The structural protein according to this embodiment can be classified into a so-called hydrophobic structural protein having hydrophobicity and a so-called hydrophilic structural protein having hydrophilicity, for example, depending on the balance of the numbers of hydrophobic and hydrophilic residues in the amino acid sequence. When the structural protein is a hydrophobic structural protein, when a molded body such as a fiber, film, gel, resin (heat and pressure molded body), etc. is manufactured by a known method using the structural protein as a raw material, the water resistance of the manufactured molded body is improved. For example, when the molded body is used as a general-purpose industrial material, the service life can be advantageously extended.

[0034] The hydrophobicity of a structural protein can be estimated using the average HI (Hydrophisity Index) value of each amino acid constituting the structural protein as an indicator. In this specification, the average HI value of a hydrophobic structural protein may be greater than 0 when considering the entire length of the amino acid sequence of the hydrophobic structural protein, and the average HI may be, for example, 0.00 or more, 0.10 or more, 0.20 or more, 0.22 or more, 0.25 or more, 0.30 or more, 0.35 or more, 0.40 or more, 0.45 or more, 0.50 or more, 0.55 or more, 0.60 or more, 0.65 or more, or 0.70 or more. Furthermore, although there is no particular upper limit, it may be, for example, 1.00 or less, or 0.7 or less.

[0035] The average HI value of a hydrophobic structural protein is determined using known hydrophobic indices of amino acid residues according to known methods. Known hydrophobic indices of amino acid residues are shown in Table 1 below. For example, the degree of hydrophobicity may be calculated according to the method described in Kyte J, Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein", J. Mol. Biol., 157, pp. 105-132.

[0036]

[0037] 4. Charge distribution and arrangement of polar residues: Structural proteins are preferably neutral to weakly negatively charged overall, and hydration and aggregation behavior can be controlled by surface exposure of polar residues.

[0038] 5. Substructures responsible for self-assembly: Structural proteins have, for example, β-sheet-forming regions, hydrophobic domains, aromatic stacking sites, etc., and are capable of self-assembly or higher-order structure formation through external stimuli (pH, ionic strength, phosphorylation, etc.).

[0039] In one embodiment, a structural protein having the following amino acid sequence is exemplified, but the present invention is not limited to these. • (Gly-Ala-Gly-Ala-Gly-Ser)n (n = 3-50) • (Gly-Pro-Gly-Gly-X)n (X is any natural amino acid) • ((Leu-Asn-Ile-Tyr-Gln-Tyr)-Gly-Ser (Sequence ID 9))n (Amyloid motif fusion type) • ((Gly-Ala-Gly-Ala-Gly-Ser)-(Gly-Pro-Gly-Gly-X) (Sequence ID 10))n (Hybrid of silk motif and elastin motif) • ((Gly-Ala-Gly-Ala-Gly-Ser)-(Val-Gln-Ile-Val-Tyr-Lys) (Sequence ID 11))n (Example of fusion of silk motif and amyloid motif) • (Gly-XY)n (X / Y = Pro, Hyp: Collagen-type triple helix forming sequence)

[0040] The above arrangement can form β-sheets, β-turns, or random coil structures through self-assembly or external processes (solvent conditions, stretching, drying, etc.). These secondary structures can further orient and stack intermolecularly, forming fibril structures, nanoscale separation of crystalline and amorphous phases, and hierarchical aggregates, thereby exhibiting unique mechanical properties.

[0041] Furthermore, in one embodiment, the amino acid sequence of the structural protein may include a modified form (substitution, insertion, deletion, chemical modification) of a naturally derived motif. In addition, a tag (His tag, serine-rich sequence, etc.) may be attached to the C-terminus or N-terminus of the structural protein. The amino acid residues may include natural or non-natural amino acids (e.g., DOPA, selenomethionine, homotyrosine, etc.). Furthermore, a short-chain peptide sequence that promotes self-assembly may be added to the terminal or internal part of the sequence.

[0042] The number of amino acid residues in a structural protein is not particularly limited, but may be, for example, 5 or more. Furthermore, the number of amino acid residues may be, for example, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 400 or more, or 500 or more. The number of amino acid residues may also be, for example, 5000 or less, 2500 or less, 1000 or less, 750 or less, 500 or less, 250 or less, 1000 or less, 500 or less, 100 or less, or 50 or less. The fewer the number of amino acid residues, the higher the solubility in the solvent tends to be. Preferred numbers of amino acid residues for a protein are, for example, 5 to 5000, 10 to 4500, 15 to 4000, 20 to 3500, 20 to 3000, 20 to 2500, 30 to 2000, 50 to 1500, or 100 to 1000.

[0043] Furthermore, the molecular weight of the structural protein is not particularly limited, but may be, for example, 1 kDa to 500 kDa. The molecular weight may also be, for example, 1 kDa or more, 2 kDa or more, 3 kDa or more, 4 kDa or more, 5 kDa or more, 6 kDa or more, 7 kDa or more, 8 kDa or more, 9 kDa or more, 10 kDa or more, 20 kDa or more, 30 kDa or more, 40 kDa or more, 50 kDa or more, 60 kDa or more, 70 kDa or more, 80 kDa or more, 90 kDa or more, or 100 kDa or more, and may also be 500 kDa or less, 400 kDa or less, less than 360 kDa, 300 kDa or less, or 200 kDa or less. The molecular weight may be, for example, 1 kDa to 500 kDa, 2 kDa to 500 kDa, 3 kDa to 500 kDa, 4 kDa to 500 kDa, 5 kDa to 500 kDa, 6 kDa to 500 kDa, 7 kDa to 500 kDa, 8 kDa to 500 kDa, 9 kDa to 500 kDa, 2 kDa to 400 kDa, 2 kDa to 200 kDa, 2 kDa to 100 kDa, 2 kDa to 50 kDa, 2 kDa to 30 kDa, 3 kDa to 300 kDa, 3 kDa to 200 kDa, 3 kDa to 100 kDa, or 10 kDa to 300 kDa, 10 kDa to 200 kDa. The lower the molecular weight of a structural protein, the higher its solubility in the solvent tends to be. Preferred amino acid residue counts for structural proteins are, for example, 1 kDa to 50 kDa and 2 kDa to 10 kDa.

[0044] In addition, the artificial structural proteins among the structural proteins in this specification include structural protein derivatives such as block copolymers. These structural protein derivatives may be, for example, block copolymers comprising a first segment containing a polypeptide chain and a second segment bound to the first segment.

[0045] The polypeptide chain constituting the first segment may be, for example, any of the structural proteins described in detail earlier.

[0046] The second segment may be a molecular chain having a plasticizing function for polypeptide chains. A molecular chain having a plasticizing function for polypeptide chains means a molecular chain that enhances the flexibility of the fiber compared to a fiber containing only polypeptide chains. A block copolymer containing a second segment containing a molecular chain having a plasticizing function for polypeptide chains may have high flexibility suitable for fabrics used in clothing, etc. In addition, a block copolymer containing a second segment containing a molecular chain having a plasticizing function for polypeptide chains may be biodegradable.

[0047] The second segment may be a molecular chain containing polyoxyalkylene, polyester, polycarbonate, polyamide, polyol, or modified polysaccharide chain. These molecular chains may have a plasticizing function for polypeptide chains.

[0048] Examples of polyoxyalkylenes that may constitute the second segment include polyoxyethylene, polyoxypropylene, and polyoxytetramethylene. Examples of polyesters that may constitute the second segment include polylactic acid, poly(3-hydroxybutanoic acid), polyhydroxybutanoic acid / hydroxyvaleryl acid copolymer, polyhydroxybutanoic acid / 4-hydroxybutanoic acid copolymer, polyhydroxybutanoic acid / hydroxyhexanoic acid copolymer, polytrimethylene terephthalate, butanediol / long-chain dicarboxylic acid copolymer, polyethylene terephthalate, polybutylene succinate, polybutylene succinate adipate copolymer, polybutylene adipate terephthalate copolymer, polycaprolactone, and polytrimethylene frangipanecarboxylate. Polycarbonates that may constitute the second segment may contain constituent units derived from aliphatic diols, examples of which include 1,6-hexanediol polycarbonate, 1,5-pentanediol polycarbonate, and 1,10-decanediol carbonate. Examples of polyamides that can constitute the second segment include nylon 3, nylon 4, nylon 5, nylon 6, nylon 11, and nylon 610. Examples of polyols that can constitute the second segment include polyvinyl alcohol and ethylene-vinyl alcohol copolymers. Examples of modified polysaccharides that can constitute the second segment include chemically modified polysaccharides such as cellulose, starch, chitin, and chitosan. Specific examples of modified polysaccharides include cellulose acetate, ethylcellulose, starch acetate, hydroxypropylated starch, carboxymethyl chitin, and carboxymethyl chitosan.

[0049] Furthermore, artificial structural proteins also include chemically modified structural proteins. That is, for example, at least some or all of the lysine residues, serine residues, threonine residues, tyrosine residues, or cysteine ​​residues in a structural protein may be modified to confer specific functions or characteristics.

[0050] In one embodiment, examples of the structural protein in the invention include fibroin, collagen, elastin, keratin, resilin, and fusion proteins containing these. Specific examples of the fusion protein include silk elastin, silk casein, collagen elastin, collagen silk, and amyloid silk.

[0051] In this specification, fibroin includes naturally-derived fibroin and modified fibroin. In this specification, "naturally-derived fibroin" means fibroin having the same amino acid sequence as naturally-derived fibroin, and "modified fibroin" means fibroin having an amino acid sequence different from that of naturally-derived fibroin. Examples of fibroin include spider silk fibroin, silkworm silk fibroin, hornet silk fibroin, and the like.

[0052] The fibroin according to this embodiment may be, for example, a protein containing a domain sequence represented by Formula 2: [(A) n motif - REP3] ｍ , or Formula 3: [(A) n motif - REP3] ｍ -(A) ｎ motif. Here, the "domain sequence" is an amino acid sequence that gives rise to a crystalline region (typically corresponding to the (A) ｎ motif of the amino acid sequence, but not limited thereto) and an amorphous region (typically corresponding to REP3 of the amino acid sequence, but not limited thereto), and means an amino acid sequence represented by Formula 2: [(A) ｎ motif - REP" ｍ , or Formula 3: [(A) n motif - REP3] ｍ -(A) ｎ motif.

[0053] Here, the (A) ｎ motif represents an amino acid sequence composed of 2 to 27 amino acid residues. The (A) ｎThe motif is preferably an amino acid sequence consisting of 2 to 20 amino acid residues, more preferably an amino acid sequence consisting of 2 to 16 amino acid residues, and even more preferably an amino acid sequence consisting of 2 to 12 amino acid residues.

[0054] (A) ｎ The motif is (A) ｎ The total number of alanine, serine, threonine, and valine residues in the motif should be 40% or more, but preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, particularly preferably 80% or more, and most preferably 100% (meaning that it is composed only of one or more amino acid residues selected from alanine, serine, threonine, and valine residues). (A) ｎ The motif is (A) ｎ The number of alanine residues relative to the total number of amino acid residues in the motif may be 40% or more, 60% or more, 80% or more, 90% or more, or even 100% (meaning composed solely of alanine residues). Multiple alanine residues exist in the domain sequence (A). ｎ The motif is preferably composed of at least one alanine residue. Composed of only alanine residues means (A) ｎ The motif is (A) ｎ This means having an amino acid sequence represented by (A represents an alanine residue, and n represents an integer from 2 to 27, preferably from 2 to 20, and more preferably from 2 to 16).

[0055] Multiple (A) present in the fibroin according to this embodiment ｎ The motifs may have the same amino acid sequence or different amino acid sequences.

[0056] Furthermore, REP3 exhibits an amino acid sequence consisting of 10 to 200 amino acid residues.

[0057] In one embodiment, the total number of glycine residues, serine residues, glutamine residues, and alanine residues relative to the total number of amino acid residues in REP3 may be 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, or 70% or more.

[0058] The multiple REPs present in the fibroin according to this embodiment may have the same amino acid sequence or different amino acid sequences.

[0059] In equations 2 and 3, "m" is [(A)] ｎ This refers to the number of repeats in the amino acid sequence indicated by [motif-REP3], and represents an integer between 2 and 300.

[0060] Specific examples of modified fibroins according to this embodiment include, for example, a modified fibroin derived from the large spindle bookmark protein produced in the large bottle gland of spiders, as described in International Publication No. 2019 / 194263 (first modified fibroin), a modified fibroin having a domain sequence with reduced glycine residue content (second modified fibroin), a modified fibroin having a domain sequence with reduced (A)n motif content (third modified fibroin), a modified fibroin with reduced glycine residue content and (A)n motif content (fourth modified fibroin), a modified fibroin having a domain sequence containing a region with locally high hydrophobicity (fifth modified fibroin), and a modified fibroin having a domain sequence with reduced glutamine residue content (sixth modified fibroin). The definitions of each of the first to sixth modified fibroins are incorporated herein by reference to the contents described in International Publication No. 2019 / 194263.

[0061] For example, as collagen, formula 4: [REP4] ｐExamples of proteins containing the domain sequence represented by (wherein formula 4, p is an integer from 5 to 300; REP4 represents an amino acid sequence consisting of Gly-X-Y, where X and Y represent any amino acid residues other than Gly; multiple REP2 sequences may have the same amino acid sequence or different amino acid sequences) are given.

[0062] Examples of elastin include proteins with amino acid sequences such as NCBl Genebank accession numbers AAC98395 (human), I47076 (sheep), and NP786966 (bovine).

[0063] 2. Recombinant Cells The recombinant cells according to this embodiment express recombinant proteins. The recombinant cells according to this embodiment may include, for example, a nucleic acid sequence encoding a recombinant protein and one or more regulatory sequences operably linked to the nucleic acid sequence. The recombinant cells according to this embodiment may contain one expression cassette, or multiple (for example, two, three, four, or five) expression cassettes.

[0064] The method for producing nucleic acids encoding recombinant proteins is not particularly limited. For example, nucleic acids can be produced by using genes encoding recombinant proteins such as natural fibroin, amplifying and cloning them by polymerase chain reaction (PCR), and then modifying them using genetic engineering techniques, or by chemical synthesis. The chemical synthesis method of nucleic acids is also not particularly limited. For example, genes can be chemically synthesized by linking oligonucleotides automatically synthesized using AKTA oligopilot plus 10 / 100 (GE Healthcare Japan Corporation), etc., based on amino acid sequence information of proteins obtained from the NCBI web database, etc., using PCR or the like. In this case, to facilitate the purification and / or confirmation of recombinant proteins, nucleic acids encoding structural proteins consisting of amino acid sequences in which an amino acid sequence consisting of a start codon and a His10 tag is added to the N-terminus of the above amino acid sequence may be synthesized.

[0065] Regulatory sequences are sequences that control the expression of recombinant proteins in the host (e.g., promoters, enhancers, ribosome-binding sequences, transcription termination sequences, etc.) and can be appropriately selected depending on the type of host. Regulatory sequences may be exogenous or endogenous (host-derived regulatory sequences).

[0066] Recombinant cells containing a recombinant protein expression cassette can be obtained, for example, by transforming host cells with an expression vector containing at least a nucleic acid sequence encoding a recombinant protein. The expression vector may also contain a recombinant protein expression cassette. Recombinant cells according to this embodiment may have the recombinant protein expression cassette outside of genomic DNA, or the recombinant protein expression cassette may be incorporated into genomic DNA, but it is preferable that the recombinant protein expression cassette is incorporated into genomic DNA.

[0067] The type of expression vector can be appropriately selected depending on the host type, including plasmid vectors, viral vectors, cosmid vectors, fosmid vectors, and artificial chromosome vectors. Preferably, the expression vector is one that can autonomously replicate in host cells or be incorporated into the host chromosome, and contains a promoter at a position where nucleic acids encoding recombinant structural proteins can be transcribed.

[0068] Any eukaryote, such as prokaryotes, yeasts, filamentous fungi, insect cells, animal cells, and plant cells, can be used as the host. Among these, from the viewpoint of rapid growth and reduction of culture costs, the host cell is preferably a prokaryotic cell such as a bacterium. The host cell may be a cocci, a spiral bacterium, or a rod, but a rod is preferred.

[0069] Examples of prokaryotic hosts include bacteria belonging to the genera Escherichia (e.g., Escherichia coli), Brevibacillus (e.g., Brevibacillus agri), Serratia (e.g., Serratia liceufaciens), Bacillus (e.g., Bacillus sacillaus), Microbacterium (e.g., Microbacterium ammoniaphyllum), Brevibacterium (e.g., Brevibacterium divaricataum), Corynebacterium (e.g., Corynebacterium ammoniagenes), and Pseudomonas (e.g., Pseudomonas putida). Among these, Escherichia is preferred, and Escherichia coli is more preferred.

[0070] When using prokaryotes as hosts, examples of vectors for introducing nucleic acids encoding structural proteins include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, and pNCO2 (Japanese Patent Publication No. 2002-238569).

[0071] Examples of eukaryotic hosts include yeasts and filamentous fungi (molds, etc.). Examples of yeasts include those belonging to the genera Saccharomyces, Pichia, and Schizosaccharomyces. Examples of filamentous fungi include those belonging to the genera Aspergillus, Penicillium, and Trichoderma.

[0072] When using eukaryotes as hosts, examples of vectors for introducing nucleic acids encoding structural proteins include YEp13 (ATCC37115) and YEp24 (ATCC37051).

[0073] Any method for introducing DNA into the host cells can be used as a transformation method for host cells. For example, methods using calcium ions (Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)), electroporation, spheroplast, protoplast, lithium acetate, and competent methods can be used.

[0074] In the present invention, recombinant cells are further modified to enhance the threonine-glycine synthesis pathway. If two or more modifications are made, the order is not particularly limited. That is, a recombinant protein expression cassette may be incorporated into host cells modified to enhance the threonine-glycine synthesis pathway, or host cells having a recombinant protein expression cassette may be modified to enhance the threonine-glycine synthesis pathway.

[0075] Modifications that enhance the threonine-glycine synthesis pathway include, for example: (A) Modifications that increase threonine synthesis. (B) Modifications that increase glycine synthesis from threonine.

[0076] Modifications that enhance the threonine-glycine synthesis pathway include at least one modification selected from the group consisting of (A) and (B) above. Preferably, the modification that enhances the threonine-glycine synthesis pathway includes both modification (A) and modification (B) ((A) and (B)).

[0077] (A) Modifications that increase threonine synthesis include, for example, modifications of regulatory factors in the threonine synthesis pathway. By modifying regulatory factors such as regulators and enzymes in the threonine synthesis pathway through substitution, deletion, insertion, addition, mutation, or artificial expression regulation, the metabolic flow and production amount can be controlled. Artificial expression regulation means inducing, reducing, or suppressing the expression of nucleic acids or genes to induce, reduce, or suppress the production of proteins or polypeptides, respectively. The expression levels of regulatory factors in the threonine synthesis pathway can be modified by incorporating the regulatory factors into an expression cassette and introducing them into the cell. Furthermore, the expression levels of regulatory factors in the threonine synthesis pathway can also be modified by adding enhancers or other regulatory sequences to the sequence of morphogenetic regulatory factors. Other modifications may also be included, or combinations thereof.

[0078] The modified threonine synthesis pathway regulator may be a "mutant threonine synthesis pathway regulator." A "mutant threonine synthesis pathway regulator" means a threonine synthesis pathway regulator having an amino acid sequence that corresponds to the substitution, deletion, insertion, and / or addition of one or more amino acid residues compared to the amino acid sequence of the wild-type threonine synthesis pathway regulator. A mutant threonine synthesis pathway regulator also includes cases where the wild-type threonine synthesis pathway regulator is completely deleted (for example, the gene encoding the threonine synthesis pathway regulator has been removed from chromosomal DNA, or the gene encoding the threonine synthesis pathway regulator is not expressed and therefore not expressed as a protein). The mutant threonine synthesis pathway regulator preferably consists of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of the wild-type threonine synthesis pathway regulator, more preferably having 95% or more sequence identity, and even more preferably having 99% or more sequence identity. The regulatory factors of the mutant threonine synthesis pathway may be those in which some or all of the biological activity of the regulatory factors of the wild-type threonine synthesis pathway is lost.

[0079] Cells containing regulatory factors for the mutant threonine synthesis pathway can be obtained, for example, by screening naturally occurring cells, by inducing mutations through drug treatment such as EMS (Ethyl Methanesulfonate) and / or UV irradiation, or by obtaining cells containing regulatory factors for the mutant threonine synthesis pathway through genetic engineering techniques.

[0080] Methods utilizing genetic engineering techniques include, for example, methods for introducing mutations randomly and methods for introducing mutations in a site-specific manner. For the former method of introducing mutations randomly, for example, a random mutation introduction kit (BD Diversify PCR Random Mutagenesis (manufactured by CLONTECH)) may be used. For the latter method of introducing mutations in a site-specific manner, for example, a site-specific mutation introduction kit (Mutan-K (manufactured by Takara Bio Inc.)) may be used.

[0081] Among these methods, it is preferable to obtain cells containing regulatory factors for the mutant threonine synthesis pathway using genetic engineering techniques, but this method is not the only option.

[0082] (A) Modifications that increase threonine synthesis include, for example, modifications to enzymes in the threonine synthesis pathway. Modifications to enzymes in the threonine synthesis pathway include insensitive mutations that suppress feedback inhibition by the amino acid product, and can be obtained by indicating resistance to threonine analogs such as AMV (α-amino-β-hydroxyvaleric acid) and lysine analogs such as AEC (S-(2-aminoethylcysteine)). Mutations to aspartate kinase and homoserine dehydrogenase are known examples of such mutations, and similar results can be obtained by artificially introducing genes with these known mutations into production strains. Examples include the bifunctional aspartate kinase-homoserine dehydrogenase gene (Escherichia coli thrA gene and its ortholog) and the monofunctional aspartate kinase (Escherichia coli lysC gene and its ortholog). Specific examples of enzyme gene modifications include thrA(S345F), thrA433, lysC(T342I), and their orthologs with the corresponding mutations mentioned above.

[0083] Furthermore, threonine synthesis can be further increased by overexpressing a modified gene that increases threonine synthesis. The details of gene overexpression will be discussed later.

[0084] (B) Modifications that increase glycine synthesis from threonine include, for example, modifications that overexpress genes encoding proteins having L-threonine-3-dehydrogenase activity and genes encoding proteins having 2-amino-3-oxobutanoic acid coenzyme A ligase activity, or modifications that overexpress genes encoding L-threonine aldolase.

[0085] Examples of proteins possessing L-threonine-3-dehydrogenase activity include those that catalyze the NAD+-dependent reduction of L-threonine to L-2-amino-3-oxobutanoic acid. Examples of genes encoding proteins possessing L-threonine-3-dehydrogenase activity include the E. coli tdh gene and its ortholog.

[0086] Proteins possessing 2-amino-3-oxobutanoic acid coenzyme A ligase activity include those that catalyze the cleavage of 2-amino-3-oxobutanoic acid into glycine and acetyl coenzyme A. Genes encoding proteins possessing 2-amino-3-oxobutanoic acid coenzyme A ligase activity include, for example, the E. coli kbl gene and its ortholog.

[0087] Examples of genes encoding L-threonine aldolase include the E. coli ltaE gene and its ortholog.

[0088] In this specification, “overexpression of a gene” means that the expression level (expression amount) of the gene is higher than that of an unmodified strain. The increase in the expression level of the gene can be measured, for example, as an increase in the expression level of the gene per cell (which may be the average expression level of the gene per cell). Bacteria can be modified so that the expression level of the gene per cell increases to, for example, 150% or more, 200% or more, or 300% or more of the expression level of an unmodified bacterial strain.

[0089] Methods that can be used to enhance gene expression include, but are not limited to, increasing the copy number of a gene, for example, the copy number of the gene in a bacterial chromosome and / or the copy number of the gene in an autonomously replicating plasmid held in the bacterium. The copy number of the gene can be increased, for example, by introducing the gene into a bacterial chromosome and / or by introducing an autonomously replicating plasmid containing the gene into the bacterium. Such modifications can be carried out by genetic engineering techniques well known to those skilled in the art.

[0090] Genes can also be introduced into bacterial chromosomal DNA, for example, by homologous recombination. Only one copy of the gene may be introduced, or two or more copies may be introduced. For example, multiple copies of a gene can be introduced into chromosomal DNA by performing homologous recombination using a sequence that has multiple copies in the chromosomal DNA. Examples of sequences with multiple copies in chromosomal DNA include, but are not limited to, repetitive DNA and inverted repeats located at the ends of transposable elements. Furthermore, multiple copies of the gene can be introduced into chromosomal DNA by incorporating the gene into a transposon and transferring it.

[0091] Another method that can be used to enhance gene expression is to increase the expression level of the gene by modifying its regulatory region. The regulatory region of a gene can be modified, for example, by introducing a wild-type and / or modified foreign regulatory region into the gene's original regulatory region. The “regulatory region” is also called the “regulatory sequence.” Examples of regulatory regions include promoters, enhancers, attenuators and termination signals, anti-termination signals, ribosome binding sites, and other regulatory elements (e.g., regions to which repressors or inducers bind, and / or, for example, binding sites for transcription and translation regulatory proteins in transcribed mRNA). Modification of the gene’s regulatory region may be combined with an increase in the gene’s copy number.

[0092] Examples of promoters suitable for enhancing gene expression include potent promoters that are stronger than the gene's innate promoter. For example, the J23119 promoter, T7 promoter, lac promoter, trp promoter, trc promoter, tac promoter, and the PR or PL promoter of lambda phage are all known as potent promoters.

[0093] The copy number of a gene, and the presence or absence of a gene, can be measured, for example, by restricting chromosomal DNA and then performing Southern blotting or fluorescence in situ hybridization (FISH) using a gene sequence-based probe. The level of gene expression can be determined by measuring the amount of mRNA transcribed from the gene using various well-known methods such as Northern blotting or quantitative RT-PCR. The amount of protein encoded by a gene can be measured by SDS-PAGE and subsequent known methods such as immunoblotting (Western blotting) or mass spectrometry of the protein sample.

[0094] In one embodiment, recombinant cells may include one or more modifications other than those that enhance the threonine-glycine synthesis pathway. Such additional modifications include, for example, modifications that increase tRNA-Gly.

[0095] 3. Method of the Present Invention The present invention is a method for producing recombinant protein, comprising culturing recombinant cells expressing recombinant protein in a culture medium, inducing the expression of a gene encoding the recombinant protein, and collecting the recombinant protein, wherein the synthesis pathway from threonine to glycine is enhanced in the recombinant cells.

[0096] The protein production medium for culturing recombinant cells is not particularly limited and can be selected from known natural or synthetic media depending on the type of recombinant cell. For example, a liquid medium containing, optionally, a carbon source, nitrogen source, phosphate source, sulfur source, vitamins, minerals, nutrients required by nutritional requirements, and other various organic and inorganic components can be used as the protein production medium. The types and concentrations of the medium components may be appropriately determined by those skilled in the art.

[0097] Protein production media preferably contain naturally derived components. Naturally derived components refer to components such as natural products (e.g., yeast) themselves, or extracts from natural products (e.g., Yeast Extract). The types of components and their respective amounts are usually not fully specified. Naturally derived components include, for example, at least one selected from the group consisting of vitamins, low molecular weight peptides (e.g., peptides with 2 to 20 amino acid residues), and amino acids.

[0098] Examples of carbon sources include sugars such as glucose, sucrose, lactose, galactose, fructose, and hydrolyzed starch; alcohols such as glycerol and sorbitol; and organic acids such as fumaric acid, citric acid, and succinic acid.

[0099] The carbon source may be one type or a mixture of two or more carbon sources in any ratio. The concentration of the carbon source in the protein production medium may be about 0.1 w / v% to 50 w / v%, preferably about 0.5 w / v% to 40 w / v%, more preferably about 1 w / v% to 30 w / v%, and particularly preferably about 5 w / v% to 20 w / v%. In this embodiment, it is preferable to use glycerol or glucose as the carbon source, and glycerol or glucose may be mixed with other carbon sources in any ratio. The ratio of glycerol or glucose in the carbon source is preferably 10% by weight or more, more preferably 50% by weight or more, and particularly preferably 70% by weight or more. The preferred initial concentration of the carbon source at the start of culture is as described above, but the carbon source may be added as appropriate according to the consumption of the carbon source during culture.

[0100] Examples of nitrogen sources include inorganic nitrogen salts such as nitrates, ammonium salts, ammonia gas, and ammonia water, as well as organic nitrogen sources such as amino acids, peptones, extracts, and corn steep liquor (CSL), a by-product of the corn starch manufacturing industry. Examples of peptones include casein peptone, meat peptone, cardiac muscle peptone, gelatin peptone, or soybean peptone. Examples of extracts include meat extract, yeast extract, and heart infusion. For nitrogen sources containing amino acids or peptides, a higher content of lower molecular weight peptides and amino acids is preferable.

[0101] Examples of phosphate sources include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate.

[0102] Examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione.

[0103] Examples of vitamins include biotin, choline chloride, cyanocobalamin, folic acid, inositol, nicotinic acid, 4-aminobenzoic acid, pantothenic acid, pyridoxine, riboflavin, thianmine, and thymdin. Sources of these vitamins include various extracts such as malt extract, potato extract, and tomato juice.

[0104] In addition to phosphorus, other minerals include sulfur, potassium, calcium, magnesium, iron, and sodium.

[0105] Culturing can be carried out aerobically, for example, by aeration culture or shaking culture. The oxygen concentration may be controlled to, for example, 5-50% of the saturated dissolved oxygen concentration, preferably 20-40% of the saturated dissolved oxygen concentration. The culture temperature may be, for example, 20-45°C, 25-40°C, or 30-37°C. The pH during cultivation may be, for example, 5-9. Inorganic or organic acidic or alkaline substances, such as calcium carbonate, ammonia gas, or ammonia water, can be used to adjust the pH. Culturing can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. The culture medium at the start of cultivation is also called the "initial medium." In fed-batch culture or continuous culture, the culture medium supplied to the culture system (fermenter) is also called the "fed-batch medium." In fed-batch culture or continuous culture, the act of supplying the fed-batch medium to the culture system is also called "fed-batch." Furthermore, cultivation may be carried out separately in pre-culturing and main culturing. Pre-culture may be performed using, for example, plate culture or liquid culture medium.

[0106] Recombinant protein expression can be induced according to the configuration of the expression system adopted. For example, when using the lac promoter, trc promoter, or tac promoter, isopropyl-β-D-thiogalactopyranoside (IPTG) or lactose can be added to the culture medium; when using the trp promoter, 3-β-indoleacrylic acid (IAA) can be added; when using the araBAD promoter, L-arabinose can be added; when using the teta promoter, anhydrotetracycline can be added; when using the rhaPBAD promoter, L-rhamnose can be added; and when using the proU promoter, NaCl can be added. Recombinant protein expression can be induced by depriving the culture medium of tryptophan, for example, when using the trp promoter. Furthermore, when using the cspA promoter, recombinant protein expression can be induced by lowering the temperature of the culture medium (for example, to about 15°C). Furthermore, when using the λPL promoter or λPR promoter, recombinant protein expression can be induced by raising the temperature of the culture medium (for example, to 42°C). Also, when using the phoA promoter or pstS promoter, recombinant protein expression can be induced by depleting phosphate in the culture medium. Furthermore, when using the T3 promoter, T7 promoter, or SP6 promoter, recombinant protein expression can be induced by appropriately inducing the expression of the corresponding RNA polymerase. Also, when using the above promoters modified as appropriate, the expression induction conditions can be appropriately selected. Depending on the configuration of the expression system, two or more expression induction conditions may be used in combination.

[0107] The time required to induce recombinant protein expression should be adjusted according to the host and type of recombinant protein used, and should continue until the set production volume is reached. Since the production rate varies depending on culture conditions such as the temperature of the culture medium, it is not necessary to uniquely determine the time required to induce recombinant protein expression. The time required to induce recombinant protein expression may be set in accordance with the progress of the subsequent process of recombinant protein separation and purification. Furthermore, in industrial production, it is preferable to set the time required to induce recombinant protein expression in a way that does not affect the proliferation of recombinant cells and the transport of the proliferated recombinant cells, which are carried out in parallel.

[0108] In the method of the present invention, the threonine-glycine synthesis pathway is enhanced in recombinant cells. "Enhanced threonine-glycine synthesis pathway" means that the amount of glycine synthesized from threonine is higher compared to control conditions. In this specification, "control conditions" means conditions in which the threonine-glycine synthesis pathway is not enhanced. Examples of "control conditions" include culturing recombinant cells that have not been modified to enhance the threonine-glycine synthesis pathway (i.e., recombinant proteins are expressed, but recombinant cells have not been modified to enhance the threonine-glycine synthesis pathway). In one embodiment, an example of "control conditions" is culturing recombinant cells that do not have any of the modifications described in (A) to (B) above, and producing recombinant proteins.

[0109] In the method of the present invention, the production of recombinant protein is improved compared to control conditions. "Improved production of recombinant protein compared to control conditions" means that the expression level of recombinant protein is higher compared to control conditions. Known methods such as Western blotting can be used to measure the expression level of recombinant protein. Preferably, the expression level of recombinant protein is 1.1 times or more of the same parameter under control conditions, more preferably 1.2 times or more, even more preferably 1.3 times or more, particularly preferably 1.4 times or more, and most preferably 1.5 times or more.

[0110] Recombinant proteins produced by recombinant cells can be collected using methods commonly used for protein isolation and purification. For example, if recombinant proteins are expressed in a lysed state within cells, after culturing, the recombinant cells can be recovered by centrifugation, suspended in an aqueous buffer, and then disrupted using an ultrasonic disruptor, French press, Manton Gaurine homogenizer, and Dynomil to obtain a cell-free extract. From the supernatant obtained by centrifugation of the cell-free extract, a purified sample can be obtained using methods commonly used for protein isolation and purification, such as solvent extraction, salting out with ammonium sulfate, desalting, precipitation with organic solvents, anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, and electrophoresis methods such as isoelectric focusing, either alone or in combination.

[0111] Furthermore, if recombinant protein is expressed in the form of an insoluble form within the cell, the recombinant cells are similarly collected, then disrupted and centrifuged to recover the insoluble recombinant protein as a precipitate fraction. The recovered insoluble recombinant protein can be solubilized with a protein denaturant. After this procedure, a purified sample of the recombinant protein can be obtained by the same isolation and purification method as described above.

[0112] When recombinant proteins, or derivatives of recombinant proteins with added sugar chains, are secreted extracellularly, the recombinant proteins or their derivatives can be recovered from the culture supernatant. Specifically, the culture supernatant can be obtained by processing the culture by methods such as centrifugation, and a purified sample can be obtained from the culture supernatant using the same isolation and purification method as described above.

[0113] The present invention will be described in detail below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0114] Example 1: Confirmation of the effect of increased expression level of modified aspartate kinase (recombinant protein) Based on the nucleotide sequence and amino acid sequence of fibroin derived from Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415), a modified fibroin (hereinafter also referred to as "PRT966") having the amino acid sequence shown in SEQ ID NO: 1 was designed. The amino acid sequence shown in SEQ ID NO: 1 has an amino acid sequence in which amino acid residues have been substituted, inserted, and deleted from the amino acid sequence of Nephila clavipes-derived fibroin in order to improve productivity, and furthermore, the amino acid sequence shown in SEQ ID NO: 2 (tag sequence and hinge sequence) has been added to the N-terminus. The glycine residue content of PRT966 is 31%.

[0115] Next, a nucleic acid encoding PRT966 was synthesized. An NdeI site was added to the 5' end of this nucleic acid, and an EcoRI site was added downstream of the stop codon. This nucleic acid was cloned into a cloning vector (pUC118). Subsequently, the nucleic acid was cleaved by restriction enzyme treatment with NdeI and EcoRI, and then recombined into a pET-22b(+) vector to obtain the pET-22(+) / PRT966 vector.

[0116] Furthermore, this plasmid was cut with BglII and BspEI (partial) and inserted into the BamHI - SmaI site of the pAH143 plasmid to obtain the pAH143 - PRT966 plasmid.

[0117] (Integration of a modified fibroin expression cassette into Escherichia coli genomic DNA) Using Escherichia coli BL21 (DE3) strain as the host, the pAH143-PRT966 plasmid was inserted into the attHK022 site using the method described in the literature (Haldimann A. et al., J Bacteriol. 2001 Nov;183(21):6384-93. doi: 10.1128 / JB.183.21.6384-6393.2001.) to obtain the BL-966 (base) strain.

[0118] (Preparation of thrA plasmid) A mutant thrA gene with a 1034th C replaced by a T was first introduced into the T5-Maccabee Purple plasmid (ATUM), and then the region containing the T5 promoter was introduced into the pAH162 plasmid to create pAH162-thrA plasmid. * A plasmid was obtained. Subsequently, the T5 promoter in the plasmid was modified to the J23119 promoter to produce pAH162-119-thrA * A plasmid was obtained. Although J23119 is a constitutively expressed promoter, this plasmid incorporates a lacO sequence near the original T5 promoter, so this construct is lactose (IPTG) induced.

[0119] (Integration of plasmid into genome) Plasmid pAH162-119-thrA* was incorporated into the attPhi80 site of the BL-966(base) strain prepared above, using the method described in the reference (Haldimann A. et al., J Bacteriol. 2001 Nov;183(21):6384-93. doi: 10.1128 / JB.183.21.6384-6393.2001.), and BL-966(119-thrA * He acquired shares.

[0120] (Culturing of expression strains) The strains used for expression were cultured overnight in LB medium, and 1 / 300 vol was inoculated into the expression medium and cultured at 37°C with aeration. After 16 hours, ENPRESSO reagent A at a final concentration of 0.1 mM IPTG, 3 u / ml was added, and sampling was performed after another 24 hours.

[0121] The expression medium described above is M9 medium with 25 mM ammonium chloride, 0.03% glucose and 0.5% EnPump200 (EnPresso) as carbon sources, and 0.02% yeast extract (Difco) as a nitrogen source.

[0122] (Analysis of PRT966 expression levels) After sampling the culture medium, cells were collected by centrifugation. Then, they were dissolved in five times the original volume of SDS-PAGE sample buffer, and 10 μL was separated on a 10% polyacrylamide gel. After staining with InVisio® His-Tag In-Gel Stain (Thermo Fisher Scientific), the fluorescence band was quantified using the ChemiDoc® Touch imaging system (BIO-RAD) to confirm the expression level of PRT966. BL-966(119-thrA) was defined as the fluorescence intensity of the BL-966(base) strain as 1. * The results for the stock are shown in Table 2.

[0123]

[0124] As shown in Table 2, the expression level of PRT966 was increased in the BL-966(119-thrA*) strain compared to the BL-966 strain. This result suggests that recombinant cells with mutations in proteins possessing aspartate kinase activity increase the production of glycine-rich proteins compared to control conditions lacking such modifications.

[0125] Example 2: Modification to increase the synthesis of glycine from threonine (construction of tdh-kbl plasmid) The above pAH162-119-thrA * Based on the plasmid, the kbl-tdh gene region amplified by PCR from the BL21(DE3) strain genomic DNA was converted to thrA * The pAH162-119-kbl-tdh plasmid was constructed by substitution. Subsequently, the plasmid was incorporated into the genome and the expression strain was cultured using the same method as in Example 1 to obtain the BL-966(119-kbl-tdh) strain.

[0126] (Analysis of PRT966 expression levels) After sampling the culture medium, cells were collected by centrifugation. Then, they were dissolved in five times the original volume of SDS-PAGE sample buffer, and 10 μL was separated on a 10% polyacrylamide gel. After staining with InVisio® His-Tag In-Gel Stain (Thermo Fisher Scientific), the fluorescence band was quantified using the ChemiDoc® Touch imaging system (BIO-RAD) to confirm the expression level of PRT966. The results for the BL-966(119-kbl-tdh) strain, with the fluorescence intensity of the BL-966(base) strain set to 1, are shown in Table 3.

[0127]

[0128] As shown in Table 3, the expression level of PRT966 was increased in the BL-966 (119-kbl-tdh) strain compared to the BL-966 strain. This result suggests that recombinant cells modified to overexpress genes encoding a protein with L-threonine-3-dehydrogenase activity and a protein encoding a protein with 2-amino-3-oxobutanoic acid coenzyme A ligase activity increase the production of glycine-rich proteins compared to control conditions lacking such modifications.

[0129] Example 3: Modifications that increase both threonine synthesis and threonine-to-glycine synthesis (thrA * -tdh-kbl plasmid construction) Based on the above pAH162-119-thrA* plasmid, the kbl-tdh gene region amplified by PCR from BL21(DE3) strain genomic DNA was converted to thrA *The pAH162-119-thrA*-kbl-tdh plasmid was constructed by inserting it into the 3' end. Furthermore, the pAH162-T7-thrA*-kbl-tdh plasmid was constructed by replacing the J23119 promoter with the T7 promoter based on the pAH162-119-thrA*-kbl-tdh plasmid. Subsequently, the plasmid was incorporated into the genome and the expression strain was cultured using the same method as in Example 1, resulting in BL-966(119-thrA * -kbl-tdh) strain and BL-966(T7-thrA * -kbl-tdh) strain obtained

[0130] (Analysis of PRT966 expression levels) After sampling the culture medium, cells were collected by centrifugation. Then, they were dissolved in five times the original volume of SDS-PAGE sample buffer, and 10 μL was separated on a 10% polyacrylamide gel. After staining with InVisio® His-Tag In-Gel Stain (Thermo Fisher Scientific), the fluorescence band was quantified using the ChemiDoc® Touch imaging system (BIO-RAD) to confirm the expression level of PRT966. BL-966(119-thrA) was defined as the fluorescence intensity of the BL-966(base) strain as 1. * -kbl-tdh) strain and BL-966(T7-thrA * The results for the -kbl-tdh) strain are shown in Table 4.

[0131]

[0132] As shown in Table 4, in the BL-966(119-thrA*-kbl-tdh) and BL-966(T7-thrA*-kbl-tdh) strains, the expression level of PRT966 was increased compared to the parent strain (BL-966 strain), regardless of the promoter used. These results suggest that recombinant cells with mutations in the gene encoding a protein with aspartate kinase activity, and overexpressing genes encoding a protein with L-threonine-3-dehydrogenase activity and a protein with 2-amino-3-oxobutanoic acid coenzyme A ligase activity, increase the production of glycine-rich proteins compared to control conditions lacking such modifications.

[0133] Example 4: Confirmation of productivity during high-density culture and the accumulation of undesirable substances (formic acid, a metabolite, and ammonia due to neutralization) The glycine cleavage system (GCV) is a system composed of multiple enzymes involved in the degradation of glycine. (Preparation of BL-966 (119-thrA*-kbl-tdh, ΔGCV) strain) Genomic DNA of the BL21 (DE3) strain was amplified by PCR and incorporated into the pKOV plasmid (https: / / arep.med.harvard.edu / labgc / pko3.html) to obtain the pKOV-GCV plasmid. The gcvT, gcvH, and gcvP gene regions in pKOV-GCV were deleted by PCR to obtain the pKOV-ΔaGCV plasmid. Subsequently, the pKOV-ΔGCV plasmid was introduced into the BL-966(119-thrA*-kbl-tdh) strain by transformation, and the gcvT, gcvH, and gcvP genes were deleted from the genome according to the instructions at (https: / / arep.med.harvard.edu / labgc / pko3.html) to obtain BL-966(119-thrA*-kbl-tdh, ΔGCV).

[0134] (Analysis of PRT966 expression levels) Three strains, BL-966(base), BL-966(119-thrA*-kbl-tdh), and BL-966(119-thrA*-kbl-tdh, ΔGCV), were cultured using the following method, and the expression levels of modified fibroin were analyzed. Frozen stocks of the test strains were added to 25 mL of pre-culture medium (seed culture medium in Table 5) to a concentration of 0.4 vol%. The culture medium temperature was maintained at 37°C, and flask culture was performed until the OD600 reached 5 or higher (approximately 8 hours) to obtain seed culture.

[0135]

[0136] The seed culture solution was added to a jar fermenter containing 1200 mL of protein production medium (production medium shown in Table 6) to achieve an OD600 of 0.05. The culture medium temperature was maintained at 37°C, and the pH was controlled to a constant 6.7. The dissolved oxygen concentration in the culture medium was also maintained at 2 mg / L.

[0137]

[0138] Immediately after the glucose in the protein production medium was completely consumed, the feed solution (fed-batch substrate solution in Table 7) was added at a rate of 40.8 g / hour. The culture medium temperature was maintained at 37°C, and the pH was controlled to a constant 6.9 by adding 25% aqueous ammonia. The dissolved oxygen concentration in the culture medium was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was performed for approximately 8.5 hours. Subsequently, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture medium to a final concentration of 0.1 mM to induce the expression of modified fibroin. When IPTG was added, the temperature was lowered to 30°C, and the feed solution was changed to 21.6 g / hour. The culture medium was sampled at 28 and 32 hours after induction, and the target protein, formic acid, and ammonia concentrations were quantified.

[0139]

[0140] The formic acid and ammonia concentrations in the supernatant were quantified enzymatically using the Roche CEDEX Bio HT Analyser, employing the Format Bio HT kit and the NH3 Bio HT kit, respectively. Table 8 shows the results for production volume under high-density culture and formic acid and ammonia concentrations in the culture medium. The target protein production volumes of the BL-966(119-thrA*-kbl-tdh) and BL-966(119-thrA*-kbl-tdh, ΔGCV) strains are shown, with the fluorescence intensity of the BL-966(base) strain at 32 h set to 1.

[0141] As shown in Table 8, in high-density culture for recombinant protein production, the expression level of PRT966 increased in the BL-966(119-thrA*-kbl-tdh) strain compared to the control. Furthermore, since the BL-966(119-thrA*-kbl-tdh,ΔGCV) strain enhances the glycine production pathway and suppresses the glycine degradation pathway, it was expected that the expression level of PRT966 would increase even more. Unexpectedly, however, the protein expression level in the BL-966(119-thrA*-kbl-tdh) strain was higher than that of the BL-966(119-thrA*-kbl-tdh,ΔGCV) strain.

[0142] Furthermore, the BL-966(119-thrA*-kbl-tdh) strain showed significantly lower formic acid and ammonia concentrations compared to the control and the BL-966(119-thrA*-kbl-tdh,ΔGCV) strain. This suggests that the BL-966(119-thrA*-kbl-tdh) strain can increase the production of glycine-rich proteins in high-density culture while suppressing the generation of undesirable metabolic pathway products.

Claims

1. A method for producing a recombinant protein, comprising culturing recombinant cells expressing a recombinant protein in a culture medium, inducing the expression of a gene encoding the recombinant protein, and collecting the recombinant protein, wherein the recombinant protein contains 25% or more glycine residues, and the recombinant cells include at least one modification selected from the group consisting of (A) and (B) below: (A) a modification that increases threonine synthesis; (B) a modification that increases glycine synthesis from threonine.

2. The manufacturing method according to claim 1, wherein the recombinant cells are modified to increase threonine synthesis and to increase glycine synthesis from threonine.

3. The method for producing a protein according to claim 1, wherein (A) comprises a mutation in a gene encoding a protein having aspartate kinase activity.

4. The method for producing the product according to claim 1, wherein (B) is modified to overexpress a gene encoding a protein having L-threonine-3-dehydrogenase activity and a gene encoding a protein having 2-amino-3-oxobutanoic acid coenzyme A ligase activity.

5. The method for producing a recombinant protein according to claim 1, wherein the recombinant protein is a structural protein.

6. The method for producing a recombinant protein according to claim 1, wherein the recombinant protein is fibroin, collagen, or elastin, or a fusion protein thereof.

7. Recombinant cells for producing recombinant proteins, wherein the recombinant protein contains 25% or more glycine residues, and the recombinant cells include at least one modification selected from the group consisting of (A) and (B) below: (A) a modification that increases threonine synthesis; (B) a modification that increases glycine synthesis from threonine.

8. The recombinant cell according to claim 7, wherein the recombinant cell is modified to increase threonine synthesis and to increase glycine synthesis from threonine.

9. The recombinant cell according to claim 7, wherein (A) comprises a mutation in a gene encoding a protein having aspartate kinase activity.

10. The recombinant cell according to claim 7, wherein (B) is modified to overexpress a gene encoding a protein having L-threonine-3-dehydrogenase activity and a gene encoding a protein having 2-amino-3-oxobutanoic acid coenzyme A ligase activity.

11. The recombinant cell according to claim 7, wherein the recombinant protein is a structural protein.

12. The recombinant cell according to claim 7, wherein the recombinant protein is fibroin, collagen, or elastin, or a fusion protein thereof.

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