Composition for preparing tagatose and method for preparing tagatose using the same

Abstract

The present application relates to variant polynucleotides that express fructose-4-epimerase or its variant enzyme at a significantly higher level than that of a non-mutated polynucleotide, as well as tagatose production compositions and tagatose production methods each using the variant polynucleotide.

Description

Technical Field

[0001] The present invention relates to a composition for preparing tagatose and a method for preparing tagatose using the composition. Background Art

[0002] Tagatose is a natural sweetener found in small quantities in foods such as milk, cheese, cocoa, and naturally sweet fruits like apples and oranges. Tagatose has a calorie count of 1.5 kcal / g, one-third that of sugar, and a glycemic index (GI) of 3, just 5% of sugar. However, tagatose has similar physical properties to sugar, offers multiple health benefits, and a similar sweetness to sugar. Therefore, it can be used in a variety of products as a sugar substitute that meets both health and taste requirements.

[0003] Traditionally known or commonly used methods for preparing tagatose include chemical methods (catalytic reactions) and biological methods (isomerase reactions) using galactose as the main raw material (PCT WO 2006 / 058092). However, since the price of lactose depends on the production, demand, and supply of raw milk and lactose in the international market, lactose, as the basic raw material of galactose, is used as the main raw material in traditional tagatose preparation methods, and its stable supply is limited. Therefore, there is a need for a tagatose production system using broad carbohydrates (sugar, glucose, fructose, etc.) as raw materials.

[0004] Therefore, a technology is currently being applied to produce tagatose using fructose as a raw material through a cell reaction based on Corynebacterium glutamicum. When fructose is added to cells that produce tagatose invertase, the fructose is converted into tagatose through the cell reaction to produce tagatose. To improve the efficiency of tagatose conversion, it is first necessary to increase the amount of tagatose invertase in the cells. To achieve this, a high expression system of tagatose invertase in the cells is absolutely necessary. Summary of the Invention

[0005] Technical issues

[0006] The present inventors conducted intensive research to increase the expression of fructose-4-epimerase (a tagatose converting enzyme), and as a result, discovered a mutant polynucleotide with significantly increased expression of fructose-4-epimerase, thereby completing the present invention.

[0007] Technical Solution

[0008] The present invention provides a mutant polynucleotide encoding a fructose-4-epimerase derived from Kosmotoga olearia; or a variant thereof.

[0009] The present invention provides a vector comprising a mutated polynucleotide.

[0010] The present invention provides a Corynebacterium sp. microorganism expressing Kosmotogaolearia-derived fructose-4-epimerase or a variant thereof, wherein the microorganism comprises a mutated polynucleotide or a vector comprising the mutated polynucleotide.

[0011] The present invention provides a method for producing fructose-4-epimerase, comprising culturing a microorganism of the genus Corynebacterium in a culture medium, wherein the microorganism comprises a mutated polynucleotide or a vector comprising the mutated polynucleotide.

[0012] The present invention provides a composition for producing tagatose, comprising a microorganism belonging to the genus Corynebacterium, the microorganism comprising a mutated polynucleotide or a vector comprising the mutated polynucleotide; or a culture of the microorganism.

[0013] The present invention provides a method for producing tagatose, comprising contacting a Corynebacterium microorganism; or a culture of the microorganism, with fructose, wherein the microorganism comprises a mutated polynucleotide or a vector comprising the mutated polynucleotide.

[0014] The present invention provides use of a Corynebacterium microorganism for producing tagatose, wherein the microorganism comprises a mutated polynucleotide or a vector comprising the mutated polynucleotide.

[0015] Beneficial effects

[0016] The mutated polynucleotide of the present invention has a significantly higher expression level of fructose-4-epimerase or its variant compared to the non-mutated polynucleotide, and thus, economic efficiency is higher when the enzyme is produced from a microorganism comprising the mutated polynucleotide. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the expression level of fructose-4-epimerase expressed by the mutant polynucleotide, wherein E is an empty plasmid, WT is #7_H4_KO (KNF4E), and variant is a #7_H4_KO (KNF4E) variant;

[0018] Figure 2 is the tagatose conversion rate of the fructose-4-epimerase expressed by the mutant polynucleotide;

[0019] Figure 3is the expression level of the fructose-4-epimerase variant expressed by the mutant polynucleotide, wherein 1 is blank, 2 is #7_H4_KO(KNF4E), 3 is #7_H4_KO(KNF4E)_variant, 4 is #7_H4_KO(KNF4E)_variant_3aa mutation (N97Y, T124W, N367V), and 5 is #7_H4_KO(KNF4E)_variant_1aa mutation (T124W);

[0020] Figure 4 is the tagatose conversion rate of the fructose-4-epimerase variant expressed by the mutated polynucleotide;

[0021] Figure 5 is the mutated polynucleotide expression level obtained by administering nucleotide mutations to the Thermotoga neapolitana-derived fructose-4-epimerase variant, wherein Con is an empty plasmid, WT is H4_TN_initial, variant 2 is H4_TN_variant_2, CJ is mUxaE, and variant 1 is H4_TN_variant_1.

[0022] Figure 6 is the tagatose conversion rate caused by the mutated polynucleotide obtained by applying nucleotide mutations to the fructose-4-epimerase variant derived from Thermotoga neoapollogenes. DETAILED DESCRIPTION

[0023] The present invention is described in detail below. At the same time, each description and embodiment disclosed in this summary of the present invention can also be applied to other descriptions and embodiments. That is, all combinations of the different elements disclosed in this summary of the present invention are within the scope of the present invention. Moreover, the scope of the present invention is not limited to the following specific description. In addition, those skilled in the art will understand or be able to ascertain many equivalents to the specific embodiments of the present invention described herein using only routine experimentation. Furthermore, such equivalents are understood to be within the scope of the present invention.

[0024] To achieve the object of the present invention, one aspect of the present invention provides a mutant polynucleotide encoding a Kosmotogaolearia-derived fructose-4-epimerase; or a variant thereof.

[0025] The mutated polynucleotide may be one in which any one or more amino acid encoding polynucleotides are modified in a polynucleotide encoding fructose-4-epimerase or a variant thereof, but is not limited thereto. In particular, the mutated polynucleotide may include any one or more of the following mutations: i) a polynucleotide encoding leucine (L) selected from CTC, CTG, CTT, and TTG; ii) a polynucleotide encoding isoleucine (I) selected from ATC and ATT; iii) a polynucleotide encoding threonine (T) selected from ACT, ACG, and ACC; iv) a polynucleotide encoding arginine (R) selected from CGT, CGC, and CGG; or v) a polynucleotide encoding glycine (G) selected from GGC, GGT, and GGA, and may include all mutations of i) to v), but is not limited thereto.

[0026] In addition, the mutated polynucleotide may also include any one or more of the following mutations: vi) a polynucleotide encoding valine (V) selected from GTA, GTC, GTG and GTT; vii) a polynucleotide encoding serine (S) selected from TCA, TCT, TCC and AGC; viii) a polynucleotide encoding alanine (A) selected from GCG, GCT, GCC and GCA; or ix) a polynucleotide encoding glutamine (Q) selected from CAG and CAA, but are not limited thereto.

[0027] More specifically, the mutated polynucleotide may include any one or more of the following mutations: i) a polynucleotide encoding leucine (L) is selected from CTC, CTG, CTT and TTG; ii) a polynucleotide encoding isoleucine (I) is selected from ATC and ATT; iii) a polynucleotide encoding threonine (T) is selected from ACT, ACG and ACC; iv) a polynucleotide encoding arginine (R) is selected from CGT, CGC and CGG; v) a polynucleotide encoding glycine (G) is selected from GGC, GGT and GGA; vi) a polynucleotide encoding valine (V) is selected from GTA, GTC, GTG and GTT; vii) a polynucleotide encoding serine (S) is selected from TCA, TCT, TCC and AGC; viii) a polynucleotide encoding alanine (A) is selected from GCG, GCT, GCC and GCA; or ix) a polynucleotide encoding glutamine (Q) is selected from CAG and CAA, but is not limited thereto.

[0028] The mutated polynucleotide can be introduced into a Corynebacterium microorganism and expressed therein. In particular, in a Corynebacterium microorganism into which the mutated polynucleotide has been introduced, the expression level of fructose 4-epimerase expressed in the microorganism or the amount of tagatose produced can be increased. More particularly, the expression level of fructose 4-epimerase and the amount of tagatose produced can be increased compared to a Corynebacterium microorganism into which the mutated polynucleotide has not been introduced, i.e., a Corynebacterium microorganism into which an unmutated polynucleotide has been introduced or a wild-type Corynebacterium microorganism.

[0029] Generally, each microorganism has a nucleic acid sequence encoding an amino acid. For example, in a wild-type protein derived from a microorganism of the genus Kosmotoga, the nucleic acid sequence of SEQ ID NO: 1 is translated into the amino acid sequence of SEQ ID NO: 2.

[0030] Therefore, in the present invention, the gene encoding the wild-type protein derived from a microorganism of the genus Kosmotoga is mutated so that translation is properly performed in a microorganism of the genus Corynebacterium. The mutation may be any one or more of the following, for example, selected from: TTT (polynucleotide encoding phenylalanine (F)) mutates to TTC and TTC mutates to TTT; selected from: TTG (polynucleotide encoding leucine (L)) mutates to CTC, CTT mutates to CTC, TTG mutates to CTG, CTT mutates to CTG, CTA mutates to CTG, TTG mutates to CTT, CTC mutates to CTT, TTA mutates to CTT, CTT mutates to TTG, CTG mutates to TTG, CTC mutates to TTG, CCT mutates to TTG, and TTA mutates to TTG; selected from: ATT (polynucleotide encoding isoleucine ( I) mutated into ATC, ATA mutated into ATC, ATC mutated into ATT and ATA mutated into ATT; selected from: GTT (polynucleotide encoding valine (V)) mutated into GTA, GTC mutated into GTA, GTT mutated into GTC, GTA mutated into GTC, GTA mutated into GTG, GTT mutated into GTG, GTA mutated into GTT; selected from: AGC (polynucleotide encoding serine (S)) mutated into TCA, TCT mutated into TCA, AGC mutated into TCT, AGC mutated into TCC, AGT mutated into TCC, TCA mutated into TCC, TCT mutated into TCC , TCA mutates to AGC and TCG mutates to AGC; selected from: TAT (polynucleotide encoding tyrosine (Y)) mutates to TAC, TAC mutates to TAT; selected from: CAT (polynucleotide encoding histidine (H)) mutates to CAC and CAC mutates to CAT; selected from: CAA (polynucleotide encoding glutamine (Q)) mutates to CAG; selected from: AAT (polynucleotide encoding asparagine (N)) mutates to AAC and AAC mutates to AAT; selected from: AAG (polynucleotide encoding lysine (K)) mutates to AAA and AAA mutates to AAG; selected from: GAT (polynucleotide encoding aspartic acid (D)) selected from the group consisting of: AGA (a polynucleotide encoding arginine (R)) mutated into CGT, AGG mutated into CGT, CGA mutated into CGT, AGA mutated into CGC, CGA mutated into CGC, CGG mutated into CGC, CGT mutated into CGC, AGA mutated into CGG, CGA mutated into CGG, and CGT mutated into CGG;Selected from: GGA (polynucleotide encoding glycine (G)) mutated to GGC, GGG mutated to GGC, GGT mutated to GGC, GGA mutated to GGT, GGC mutated to GGT, GGG mutated to GGT, GGC mutated to GGA, GGG mutated to GGA, GGT mutated to GGA; Selected from: CCA (polynucleotide encoding proline (P)) mutated to CCC, CCG mutated to CCC, CCT mutated to CCC, CCA mutated to CCT, CCC mutated to CCT, CCG mutated to CCT, CCC mutated to CC G, CCG mutates to CCA and CCT mutates to CCA; selected from: ACA (polynucleotide encoding threonine (T)) mutates to ACT, ACA mutates to ACG, ACT mutates to ACC and ACA mutates to ACC; or selected from: GCA (polynucleotide encoding alanine (A)) mutates to GCT, GCC mutates to GCT, GCG mutates to GCT, GCG mutates to GCC, GCA mutates to GCC, GCT mutates to GCC, GCC mutates to GCA, GCG mutates to GCA and GCT mutates to GCA, but not limited thereto. ;

[0031] As used herein, the term "polynucleotide" refers to a DNA or RNA chain of a certain length or longer that is a nucleotide polymer in which nucleotide monomers are covalently linked in the shape of a long chain.

[0032] As used herein, the term "mutated polynucleotide" refers to a polynucleotide that has been mutated by replacing any one or more nucleotides constituting the polynucleotide with other nucleotides. Mutated polynucleotides can be used interchangeably with terms such as mutant polynucleotides and polynucleotide mutant enzymes.

[0033] As used herein, the term "fructose 4-epimerase" refers to an enzyme that exhibits fructose 4-epimerization activity, which diastereomerizes the 4-carbon position of fructose, thereby converting fructose into tagatose. For the purposes of the present invention, fructose 4-epimerase can include any enzyme without limitation, as long as it uses fructose as a substrate and can produce tagatose, and can be used interchangeably with "D-fructose 4-epimerase." For example, the known database KEGG (Kyoto Encyclopedia of Genes and Genomes) includes tagatose diphosphate aldolase or tagatose diphosphate aldolase class II accessory protein EC 4.1.2.40, as long as it exhibits activity converting fructose as a substrate into tagatose. Tagatose diphosphate aldolase is known to be an enzyme that uses D-tagatose 1,6-diphosphate as a substrate to generate phosphoglycerone and D-glyceraldehyde 3-phosphate, as shown in the following [Scheme 1].

[0034] [Scheme 1]

[0035] D-Tagatose 1,6-diphosphate <=> phosphoglycerone + D-glyceraldehyde 3-phosphate

[0036] For example, tagatose-6-phosphate kinase (EC 2.7.1.144) can be included in the fructose-4-epimerase as long as it exhibits activity in converting fructose, a substrate, into tagatose. Tagatose-6-phosphate kinase is known to be an enzyme that uses ADP and D-tagatose 6-phosphate as substrates to generate ADP and D-tagatose 1,6-diphosphate, as shown in [Scheme 2] below.

[0037] [Scheme 2]

[0038] ATP + D-tagatose 6-phosphate <=> ADP + D-tagatose 1,6-diphosphate

[0039] Regarding the activity of fructose-4-epimerase, the conversion rate of fructose as a substrate to tagatose (conversion rate = tagatose weight / initial fructose weight × 100) may be 0.01% or more, particularly 0.1% or more, more particularly 0.3% or more. More particularly, the conversion rate may be in the range of 0.01% to 100%, or in the range of 0.1% to 50%, but is not limited thereto.

[0040] The polynucleotide encoding fructose-4-epimerase in the present invention may include the nucleic acid sequence of SEQ ID NO: 1, specifically, may essentially consist of the nucleic acid sequence of SEQ ID NO: 1, more specifically, may consist of the nucleic acid sequence of SEQ ID NO: 1, but is not limited thereto.

[0041] SEQ ID NO: 1 is a nucleic acid sequence encoding fructose-4-epimerase. The nucleic acid sequence of SEQ ID NO: 1 may include polynucleotides comprising nucleic acid sequences having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher homology or identity thereto. Obviously, polynucleotides comprising nucleic acid sequences in which some sequences are deleted, modified, substituted, or added are also encompassed by the nucleic acid sequence of SEQ ID NO: 1 of the present invention, as long as the nucleic acid sequence possesses such homology or identity and exhibits the efficacy corresponding to SEQ ID NO: 1.

[0042] In other words, even when described in the present invention as a "polynucleotide consisting of a nucleic acid sequence shown in a specific SEQ ID NO" or a "polynucleotide having a nucleic acid sequence shown in a specific SEQ ID NO," it is clear that a polynucleotide consisting of a nucleic acid sequence in which some sequences are deleted, modified, substituted, or added can also be used in the present invention, as long as it exhibits the same or corresponding activity as a polynucleotide consisting of the nucleic acid sequence of the corresponding SEQ ID NO: For example, it is clear that a "polynucleotide consisting of the nucleic acid sequence of SEQ ID NO: 1" can belong to the "polynucleotide consisting of the nucleic acid sequence of SEQ ID NO: 1" as long as it exhibits the same or corresponding activity as that of the polynucleotide.

[0043] The fructose-4-epimerase of the present invention may comprise the amino acid sequence of SEQ ID NO: 2, specifically may consist essentially of the amino acid sequence of SEQ ID NO: 2, more specifically may consist of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

[0044] SEQ ID NO: 2 is an amino acid sequence encoding fructose-4-epimerase. The amino acid sequence of SEQ ID NO: 2 may include polypeptides consisting of amino acid sequences having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher homology or identity thereto. Obviously, polypeptides consisting of amino acid sequences in which some sequences are deleted, modified, substituted, or added are also included within the scope of the amino acid sequence of SEQ ID NO: 2 of the present invention, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to SEQ ID NO: 2.

[0045] The fructose-4-epimerase variant derived from Kosmotoga olearia of the present invention may be a variant in which any one or more amino acids in the amino acid sequence of the fructose-4-epimerase derived from Kosmotoga olearia are substituted and exhibit fructose-4-epimerase activity, but is not limited thereto. The amino acid substitution may be a specific substitution of 1-20 amino acids, 1-15 amino acids, 1-12 amino acids, 1-10 amino acids, 1-9 amino acids, or 1-7 amino acids. For example, the amino acid substitution may include any one or more selected from the following substitutions: a) substitution of the amino acid corresponding to position 97 with tyrosine (Y), b) substitution of the amino acid corresponding to position 124 with tryptophan (W), and c) substitution of the amino acid corresponding to position 367 with valine (V), but is not limited thereto.

[0046] As used herein, the term "enzyme variant" refers to a protein in which the function or property of the protein is maintained, while one or more conservative amino acid substitutions and / or modifications are different from the sequence. The enzyme variant differs from the identified sequence by several amino acid substitutions, deletions or additions. Such enzyme variants can generally be identified as follows: one or more amino acids in the amino acid sequence of the modified protein are modified and the properties of the modified protein are evaluated. In other words, the ability of the enzyme variant can be increased, unchanged or reduced compared to the native protein. Some enzyme variants may include enzyme variants in which one or more parts, such as N-terminal leader sequences or transmembrane domains, are removed. Other enzyme variants may include enzyme variants that remove a portion from the mature protein N- and / or C-terminus. For the term "enzyme variant", modifications, modified proteins, modified polypeptides, mutants, mutant proteins, divergences, variants, etc. may be used, and enzyme variants are not limited thereto, as long as they are terms used for mutation meanings.

[0047] For the purpose of the present invention, in an enzyme variant, the activity of the modified protein may be increased compared to a naturally occurring wild-type or unmodified protein, but the enzyme variant is not limited thereto.

[0048] As used herein, the term "conservative substitution" refers to the substitution of an amino acid with another amino acid that exhibits similar structural and / or chemical properties. For example, an enzyme variant may have one or more conservative substitutions while still retaining one or more biological activities. Such amino acid substitutions generally occur based on similarities in residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity. For example, among charged amino acids, positively charged (basic) amino acids include arginine, lysine, and histidine, and negatively charged (acidic) amino acids include glutamic acid and aspartic acid. Among uncharged amino acids, non-polar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, polar or hydrophilic amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine, and aromatic amino acids among non-polar amino acids include phenylalanine, tryptophan, and tyrosine.

[0049] Enzyme variants may include amino acid deletions or additions that have minimal effect on the secondary structure and properties of the polypeptide. For example, a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminus of the protein that participates in co-translational or post-translational protein transfer. Polypeptides may be conjugated to other sequences or linkers to enable identification, purification, or synthesis of the polypeptide.

[0050] "Substitution with another amino acid" is not limited as long as the amino acid is different from the amino acid before substitution. In other words, substitution with another amino acid is not limited as long as the amino acid asparagine (N) at position 97 of the amino acid sequence corresponding to SEQ ID NO: 2 is substituted with an amino acid residue other than asparagine, the amino acid threonine (T) at position 124 is substituted with an amino acid residue other than threonine, or the amino acid asparagine (N) at position 367 is substituted with an amino acid residue other than asparagine. In the present invention, when it is expressed that "a specific amino acid is substituted", it is clear that the amino acid is substituted with an amino acid different from the amino acid before substitution, even if it is not separately specified that the amino acid is substituted with another amino acid.

[0051] The enzyme variant may be one in which one or more of the amino acids at positions 97, 124, and 367 corresponding to the amino acid sequence of SEQ ID NO: 2 are substituted with an amino acid different from that before substitution. Alternatively, the enzyme variant may have an uncharged amino acid and be substituted with an amino acid different from that before substitution, but is not limited thereto.

[0052] In particular, the enzyme variant may include any one or more substitutions selected from the group consisting of: in the amino acid sequence of SEQ ID NO: 2, a) substitution of the amino acid corresponding to position 97 with another amino acid, b) substitution of the amino acid corresponding to position 124 with another amino acid, and c) substitution of the amino acid corresponding to position 367 with another amino acid. More particularly, the enzyme variant may be one in which a) the amino acid corresponding to position 97 is substituted with tyrosine (Y), b) the amino acid corresponding to position 124 is substituted with tryptophan (W), and c) the amino acid corresponding to position 367 is substituted with valine (V).

[0053] The fructose-4-epimerase variant provided by the present invention may refer to an enzyme variant in which an amino acid at a specific position of the protein having the above-mentioned fructose-4-epimerization ability is substituted, thereby showing an increased fructose-4-epimerization ability and / or the stability of the fructose-4-epimerization ability compared to the protein before mutation.

[0054] The enzyme variant may include the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7, but is not limited thereto.

[0055] The enzyme variant may include substitution of the amino acid at position 97, 124 or 367 corresponding to SEQ ID NO: 2 with another amino acid, have at least 80%, 90%, 95%, 96%, 97%, 98% or 99% or more or 100% sequence homology to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7, and exhibit fructose-4-epimerization activity.

[0056] The enzyme variant may include an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7 or an amino acid sequence having 80% or more homology or identity with the amino acid sequence, and one or more fixed amino acids in the amino acid at position 97, 124 or 367 of the corresponding amino acid sequence, but is not limited thereto. In particular, the enzyme variant of the present invention may include a polypeptide having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% or more homology or identity with the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7. Obviously, the scope of the present invention also includes proteins having a certain amino acid sequence, in which, except for some sequence deletions, modifications, substitutions or additions corresponding to positions 97, 124 or 367, the amino acid sequence has such homology or identity and shows the efficacy of the corresponding protein.

[0057] The mutated polynucleotide of the present invention can be any one or more polynucleotides encoding a single amino acid mutated in the nucleic acid sequence of SEQ ID NO: 1 encoding fructose-4-epimerase, or a nucleic acid sequence encoding a fructose-4-epimerase variant, wherein any one or more amino acids in the amino acid sequence of fructose-4-epimerase are substituted, but is not limited thereto. In particular, the mutation may include one or more of the following mutations: i) the polynucleotide encoding leucine (L) is selected from CTC, CTG, CTT and TTG; ii) the polynucleotide encoding isoleucine (I) is selected from ATC and ATT; iii) the polynucleotide encoding valine (V) is selected from GTA, GTC, GTG and GTT; iv) the polynucleotide encoding serine (S) is selected from TCA, TCT, TCC and AGC; v) the polynucleotide encoding threonine (T) is selected from ACT, ACG and ACC; vi) the polynucleotide encoding alanine (A) is selected from GCT, GCC and GCA; vii) the polynucleotide encoding glutamine (Q) is selected from CAG; viii) the polynucleotide encoding arginine (R) is selected from CGT, CGC and CGG; or ix) the polynucleotide encoding glycine (G) is selected from GGC, GGT and GGA, but is not limited thereto.

[0058] The polynucleotide mutation is not particularly limited as long as it is carried out within a certain range in which only the codon is changed but the amino acid sequence encoding fructose-4-epimerase is unchanged. NO:1 nucleic acid sequence, i) a polynucleotide encoding leucine (L) at any one or more positions 6, 12, 36, 47, 82, 89, 94, 126, 128, 134, 145, 156, 157, 159, 172, 209, 219, 253, 278, 289, 295, 299, 306, 313, 320, 333, 352, 359, 362, 368, 378, 382, ​​389, 391, 407, 408, 415, 422, and 424 may be selected from CTC, CTG, CTT and TTG and mutations; ii) a polynucleotide encoding isoleucine (I) at any one or more positions 9, 17, 32, 48, 76, 80, 87, 88, 112, 115, 150, 178, 195, 227, 242, 257, 263, 288, 309, 379, 387, 392, 403, 413, 416 and 420 may be selected from ATC and ATT and mutations; iii) a polynucleotide encoding isoleucine (I) at any one or more positions 34, 35, 44, 45, 51, 62, 65, 154, 18 iv) a polynucleotide encoding threonine (T) at any one or more positions 0, 198, 206, 210, 254, 270, 274, 296, 317, 373, 380, 384, 390, 421 and 431, selected from ACT, ACG and ACC and mutations; iv) a polynucleotide encoding threonine (T) at any one or more positions 29, 70, 109, 116, 133, 153, 163, 173, 205, 225, 247, 249, 251, 300, 330, 348, 351, 354, 361, 363, 372, 404 v) the polynucleotide encoding arginine (R) at any one or more positions 16, 20, 43, 59, 60, 63, 81, 90, 91, 95, 101, 122, 139, 170, 179, 186, 187, 192, 218, 224, 234, 238, 267, 285, 292, 319, 345, and 406 may be selected from the group consisting of GGC, GGT, and GGA, and mutations;vi) encoding valine at any one or more of positions 10, 21, 24, 31, 46, 55, 73, 149, 175, 177, 182, 197, 200, 207, 226, 229, 230, 231, 235, 264, 282, 286, 291, 325, 327, 328, 371, 375, and 423 ( v) the polynucleotide encoding serine (S) at any one or more positions 11, 23, 26, 75, 104, 131, 168, 171, 194, 199, 269, 307, 308, 322, 357, 393 and 414 may be selected from the group consisting of TCA, TCT, TCC and AGC and mutations; vii) the polynucleotide encoding serine (S) at any one or more positions 11, 23, 26, 75, 104, 131, 168, 171, 194, 199, 269, 307, 308, 322, 357, 393 and 414 may be selected from the group consisting of TCA, TCT, TCC and AGC and mutations; viii) at position 27, 39, 50, 67, 77, 107, 111, 119, 121, 130, 135, 151, 155, 161, 164, 169, 188, 213, 222, 228, 250, 256, 275, 277, 287, 294, 298, 302, 305, 331, 353 The polynucleotide encoding alanine (A) at any one or more positions 7, 13, 15, 54, 57, 102, 106, 189, 201, 232, and 273 may be selected from the group consisting of CAG and CAA, and mutations thereof; and ix) the polynucleotide encoding glutamine (Q) at any one or more positions 7, 13, 15, 54, 57, 102, 106, 189, 201, 232, and 273 may be selected from the group consisting of CAG and CAA, and mutations thereof, but polynucleotide mutations are not limited thereto.

[0059] When any one or more amino acids selected from threonine (T), aspartic acid (D), serine (S) and glutamic acid (E) in the amino acid sequence constituting an enzyme are arranged consecutively to form two identical amino acids, the polynucleotide encoding the first amino acid and the polynucleotide encoding the second amino acid of the same amino acids may be different from each other, but are not limited thereto.

[0060] When any one or more amino acids selected from proline (P) and valine (V) in the amino acid sequence constituting the enzyme are arranged consecutively as three or more identical amino acids, the polynucleotides encoding at least two amino acids in the polynucleotides encoding the same amino acids may be identical to each other, but are not limited thereto.

[0061] The fructose-4-epimerase variant includes any one or more substitutions selected from the following: in the amino acid sequence of SEQ ID NO: 2, a) the amino acid corresponding to position 97 is substituted with tyrosine (Y), b) the amino acid corresponding to position 124 is substituted with tryptophan (W), and c) the amino acid corresponding to position 367 is substituted with valine (V), the polynucleotide encoding tyrosine (Y) at position 97 can be TAC, the polynucleotide encoding tryptophan (W) at position 124 can be TGG, and the polynucleotide encoding valine (V) at position 367 can be GTT, but the fructose-4-epimerase variant is not limited thereto.

[0062] The mutated polynucleotide may have any one or more nucleic acid sequences selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 6, but is not limited thereto.

[0063] The nucleic acid sequence of SEQ ID NO: 3 can encode the amino acid sequence of SEQ ID NO: 2 constituting a fructose-4-epimerase, the nucleic acid sequence of SEQ ID NO: 4 can encode the amino acid sequence of SEQ ID NO: 5 constituting a fructose-4-epimerase variant, and the nucleic acid sequence of SEQ ID NO: 6 can encode the amino acid sequence of SEQ ID NO: 7 constituting a fructose-4-epimerase variant, but are not limited thereto.

[0064] In particular, in the amino acid sequence of fructose-4-epimerase derived from Kosmotoga olearia (encoded by the nucleic acid sequence of SEQ ID NO: 3), the nucleic acid sequences of SEQ ID NO: 4 and SEQ ID NO: 6 can be substituted with appropriate bases to achieve any one or more amino acid substitutions selected from the following: a) substitution of the amino acid corresponding to position 97 with tyrosine (Y), b) substitution of the amino acid corresponding to position 124 with tryptophan (W), and c) substitution of the amino acid corresponding to position 367 with valine (V), but are not limited thereto. Substitution with an appropriate base can be, as described above, substitution of the polynucleotide encoding tyrosine (Y) at position 97 with TAC, substitution of the polynucleotide encoding tryptophan (W) at position 124 with TGG, and substitution of the polynucleotide encoding valine (V) at position 367 with GTT, but are not limited thereto.

[0065] The amino acid sequence of fructose-4-epimerase derived from Kosmotoga olearia encoded by the nucleic acid sequence of SEQ ID NO: 3 can be translated into the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

[0066] The mutant polynucleotides of the present invention are defined as having any one or more nucleic acid sequences selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 6, but do not exclude the inclusion of nonsense sequences before or after the nucleic acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 6, or any naturally occurring mutations or silent mutations thereof. It is clear to those skilled in the art that a polynucleotide corresponds to a mutant polynucleotide of the present invention, as long as it exhibits the same or corresponding activity as a polynucleotide comprising the nucleic acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 6. As a specific example, the mutant polynucleotides of the present invention may be composed of the nucleic acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 6, or a nucleic acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher homology or identity to such nucleic acid sequences. Obviously, polynucleotides composed of nucleic acid sequences in which some sequences are deleted, modified, substituted, or added are also included within the scope of the mutant polynucleotides of the present invention, as long as the nucleic acid sequence has such homology or identity and exhibits the efficacy of the corresponding mutant polynucleotide.

[0067] The mutated polynucleotide may include any polynucleotide sequence encoding a protein exhibiting fructose-4-epimerase activity, which is hybridized with a probe prepared from a known gene sequence, for example, under stringent conditions, to a sequence complementary to all or part of the nucleic acid sequence of the mutated polynucleotide, but is not limited thereto. "Stringent conditions" refer to conditions under which specific hybridization between polynucleotides can occur. These conditions are described in detail in the literature (J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York). Examples thereof include conditions in which genes that hybridize to each other have high homology or identity, i.e., genes have 70% or more, 80% or more, particularly 85% or more, particularly 90% or more, more particularly 95% or more, even more particularly 97% or more, and particularly 99% or more homology or identity, and genes having lower homology or identity than this do not hybridize to each other, or conditions in which washing is performed once, particularly 2-3 times, with a salt concentration and temperature corresponding to 60°C, 1X SSC and 0.1% SDS, particularly 60°C, 0.1X SSC and 0.1% SDS, more particularly 68°C, 0.1X SSC and 0.1% SDS, which are washing conditions for conventional southern hybridization.

[0068] Hybridization requires that the two polynucleotides have complementary sequences, although mismatches between bases are possible, depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, in DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Thus, the present invention also includes isolated polynucleotide fragments that are complementary to substantially similar polynucleotide sequences as well as entire sequences.

[0069] In particular, polynucleotides having homology or identity can be detected using hybridization conditions including at 55°C T m The hybridization step was performed under the above conditions. m The value may be 60° C., 63° C., or 65° C., but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.

[0070] The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementation, and parameters are well known in the art.

[0071] As used herein, the term "homology" or "identity" refers to the degree to which two given amino acid sequences or nucleic acid sequences are related to each other and can be expressed as a percentage. The terms "homology" and "identity" are often used interchangeably.

[0072] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and the default gap penalty determined by the program used can be used together. Basically, homologous or identical sequences can hybridize to each other under moderate or high stringency conditions to generally about 50%, 60%, 70%, 80% or 90% or more of the entire sequence or full-length sequence. For hybridization, polynucleotides also consider polynucleotides that contain degenerate codons instead of codons.

[0073] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using, for example, default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 and known computer algorithms such as the "FASTA" program. Alternatively, the Needleman–Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443–453), such as the Needleman algorithm implemented in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276–277) (version 5.0.0 or later), can be used to determine homology, similarity, or identity (including the GCG program packages (Devereux, J. et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J. Mol. Biol. 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ED., Academic Press, San Diego, 1994, and CARILLO et al., 2003). (1988) SIAM J Applied Math 48:1073). For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0074] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information, for example, using a GAP computer program such as Needleman et al. (1970), J Mol Biol. 48: 443, known for example in Smith and Waterman, Adv. Appl. Math (1981) 2: 482. In general, homology, similarity or identity can be defined as the value obtained by dividing the number of similarly aligned symbols (i.e., nucleotides or amino acids) in the GAP program by the total number of symbols in the shorter of the two sequences. The default parameters of the GAP program may include (1) a binary comparison matrix (containing a value of 1 for identity and a value of 0 for non-identity) and a weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or an EDNAFULL (EMBOSS version of NCBINUC 4.4) alternative matrix), as disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353–358 (1979); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps. Thus, as used herein, the terms "homology" or "identity" refer to the relatedness between sequences.

[0075] Another aspect of the present invention provides a vector comprising a mutated polynucleotide of the present invention.

[0076] The mutated polynucleotides are as described above.

[0077] As used herein, the term "vector" refers to a DNA product comprising a nucleic acid sequence encoding a polynucleotide of a target polypeptide, operably linked to appropriate control sequences that enable expression of the target polypeptide in a suitable host. Control sequences may include a promoter capable of initiating transcription, any operator sequence that controls such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences that control termination of transcription and translation. After transformation into a suitable host cell, the vector can replicate or function independently of the host genome and may itself integrate into the genome.

[0078] The vector may be in a form in which the mutant polynucleotide of the present invention is operably linked.

[0079] As used herein, the term "operably linked" generally refers to the operative linkage of a base expression control sequence and a nucleic acid sequence encoding a target protein to perform a function, thereby affecting the expression of the encoding nucleic acid sequence. Operable linkage with a vector can be established using genetic recombination techniques known in the art, and site-specific DNA cleavage and ligation can be established using cleavage and ligation enzymes known in the art.

[0080] The vector used in the present invention is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and phages in natural or recombinant states. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as phage vectors or cosmid vectors, and pHCP (Korean Patent Publication No. 10-2018-0092110), pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, etc. can be used as plasmid vectors. In particular, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0081] For example, the polynucleotide encoding the target polypeptide can be inserted into the chromosome by a vector for intracellular chromosome insertion. The insertion of the polynucleotide into the chromosome can be implemented by any method known in the art, such as homologous recombination, but is not limited thereto. The vector can also include a selection marker for determining whether the polynucleotide is inserted into the chromosome. The selection marker is used to select cells transformed with the vector, i.e., to confirm whether the target nucleic acid molecule is inserted, and a marker for imparting selective phenotypes such as drug resistance, auxotrophy, cytotoxic agent resistance, or surface polypeptide expression can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show other expression traits, so transformed cells can be selected. Another aspect of the present invention is to provide a Corynebacterium microorganism that expresses a fructose-4-epimerase derived from Kosmotoga olearia or a variant thereof, wherein the microorganism comprises a polynucleotide of a mutation of the present invention or a vector comprising the polynucleotide of a mutation.

[0082] The mutant polynucleotides, vectors, fructose-4-epimerase and fructose-4-epimerase variants are as described above.

[0083] As used herein, the term "a microorganism expressing Kosmotoga olearia-derived fructose-4-epimerase or a variant thereof" refers to a microorganism having a naturally weak ability to produce Kosmotoga olearia-derived fructose-4-epimerase or a variant thereof, or a microorganism obtained by imparting the ability to produce Kosmotoga olearia-derived fructose-4-epimerase or a variant thereof to a parent strain that does not have the ability to produce fructose-4-epimerase or a variant thereof. For the purpose of achieving the present invention, the microorganism is specifically a microorganism expressing fructose-4-epimerase or a variant thereof, comprising a mutated polynucleotide or a vector comprising a mutated polynucleotide, and the mutated polynucleotide may include the following mutations: i) a polynucleotide encoding leucine (L) is selected from CTC, CTG, CTT and TTG; ii) a polynucleotide encoding isoleucine (I) is selected from ATC and ATT; iii) a polynucleotide encoding threonine (T) is selected from ACT, ACG and ACC; iv) a polynucleotide encoding arginine (R) is selected from CGT, CGC and CGG; v) a polynucleotide encoding glycine (G) is selected from GGC, GGT and GGA; vi) a polynucleotide encoding valine (V) is selected from GTA, GTC, GTG and GTT; vii) a polynucleotide encoding serine (S) is selected from TCA, TCT, TCC and AGC; viii) a polynucleotide encoding alanine (A) is selected from GCG, GCT, GCC and GCA; and ix) a polynucleotide encoding glutamine (Q) is selected from CAG and CAA, but is not limited thereto.

[0084] The microorganism may be a recombinant microorganism constructed by introducing a vector containing a mutated polynucleotide into a host cell. The method for transforming the vector may include any method for introducing a polynucleotide into a cell, and can be accomplished using suitable standard techniques known in the art. For example, the method may include, but is not limited to, electroporation, calcium phosphate coprecipitation, retroviral infection, microinjection, DEAE-dextran, cationic liposomes, and heat shock.

[0085] As used herein, the term "transformation" refers to the introduction of a vector comprising a polynucleotide encoding a target protein into a host cell, so that the protein encoded by the polynucleotide can be expressed in the host cell.

[0086] The transforming gene can include a form of inserting into the host cell chromosome and a form located outside the chromosome, as long as it can be expressed in the host cell. Gene includes DNA and RNA as polynucleotides, and any gene can be used without restriction, as long as it can be introduced into the host cell and expressed in the host cell. For example, the gene can be introduced into the host cell in the form of an expression cassette, which is a polynucleotide construct comprising all elements required for self-expression. The expression cassette can generally include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal that can be operably connected to the gene. The expression cassette can adopt a recombinant vector form that can self-replicate. The gene can be introduced into the host cell in its own form or in the form of a polynucleotide construct, and can be operably connected to the sequence required for expression in the host cell.

[0087] As used herein, the term "thereby expressing a polynucleotide or polypeptide / expressing a polynucleotide or polypeptide" refers to a state in which a target polynucleotide or polypeptide is introduced into a microorganism or modified so as to be expressed in the microorganism, and when the polynucleotide or polypeptide is a protein present in the microorganism, a state in which the activity of the polynucleotide or polypeptide is enhanced compared to the inherent activity or activity before modification.

[0088] In particular, "introduction of a polypeptide (or protein)" refers to an increase in activity compared to the inherent activity or activity of the target polypeptide before modification, or the microorganism displays the activity of a specific polypeptide that the microorganism did not originally possess. For example, a polynucleotide encoding a specific polypeptide is introduced into the chromosome of a microorganism, or a vector containing a polynucleotide encoding a specific polypeptide is introduced into a microorganism and displays the activity of the specific polypeptide. "Activity enhancement" refers to an increase in activity compared to the inherent activity or activity of a specific polypeptide possessed by a microorganism before modification. "Intrinsic activity" refers to the original specific polypeptide activity of the parent strain before transformation when the characteristics of a microorganism are changed by genetic mutations caused by natural or human factors.

[0089] In particular, the activity enhancement of the present invention can be

[0090] 1) Increased copy number of a gene encoding a protein or its enzyme variant;

[0091] 2) introducing mutations into the expression control sequence of a gene encoding a protein or an enzyme variant thereof;

[0092] 3) replacing the expression control sequence of the gene encoding the protein or its enzyme variant with a sequence showing strong activity;

[0093] 4) replacing the gene encoding the native protein on the chromosome with a mutant polynucleotide;

[0094] 5) introducing additional mutations into the gene encoding the protein or its enzyme variant to increase the activity of the protein or its enzyme variant;

[0095] 6) introducing a protein or an enzyme variant thereof into a microorganism; or

[0096] 7) A combination of two or more selected from 1) to 6) above, but not limited thereto.

[0097] In the above, the increase in the copy number of the gene is not particularly limited, and can be carried out in the form of a gene operably connected vector, or can be carried out by inserting the gene into the host cell chromosome. In particular, the vector (operably connected to the polynucleotide of the mutation of the present invention) can replicate and function independently of the host and can be introduced into the host cell. Alternatively, the vector (operably connected to the polynucleotide of the mutation) can insert the polynucleotide of the mutation into the host cell chromosome and can be introduced into the host cell chromosome. Inserting the polynucleotide of the mutation into the chromosome can be achieved by any method known in the art, for example, by homologous recombination.

[0098] Secondly, the expression control sequence is modified to increase the expression of the mutated polynucleotide, but is not specifically limited thereto. Sequence mutations can be induced by deletion, insertion, or non-conservative or conservative substitution of nucleic acid sequences, or a combination thereof, to further enhance the activity of the expression control sequence, or by replacing the expression control sequence with a nucleic acid sequence that exhibits stronger activity. Expression control sequences are not specifically limited thereto and may include promoters, operator sequences, sequences encoding ribosome binding sites, sequences that control transcription and translation termination, etc.

[0099] A strong promoter may be connected to the upper part of the mutated polynucleotide expression unit instead of the original promoter, but the promoter is not limited thereto. Examples of known strong promoters include cj1-cj7 promoters (Korean Patent No. 10-0620092), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (Korean Patent No. 10-1783170), O2 promoter (Korean Patent No. 10-1632642), tkt promoter and yccA promoter, but the promoter is not limited thereto.

[0100] The modified mutated polynucleotide sequence on the chromosome is not particularly limited thereto, but can be carried out by inducing mutations in the expression control sequence by deletion, insertion, or non-conservative or conservative substitution of nucleic acid sequences, or a combination thereof, to further improve the activity of the mutated polynucleotide, or by replacing the mutated polynucleotide sequence with a mutated polynucleotide sequence to further enhance the activity.

[0101] Based on the expression, activity or concentration of the polypeptide in the wild-type or unmodified microbial strain, such introduction of the target polypeptide (or protein) and enhancement of activity may be to increase the expression, activity or concentration of the corresponding polypeptide, but is not limited thereto.

[0102] As used herein, the term "unmodified microorganism" does not exclude strains including mutations that may occur naturally in microorganisms, but refers to the natural strain itself, or a microorganism that does not contain a mutated polynucleotide and a gene encoding a target polypeptide, or a microorganism that has not been transformed with a vector containing a mutated polynucleotide and a gene encoding a target polypeptide.

[0103] For the purpose of the present invention, the target polypeptide may be fructose-4-epimerase or a variant thereof, but is not limited thereto.

[0104] Recombinant microorganism can comprise prokaryotic microorganism and eukaryotic microorganism, as long as it is the polynucleotide that comprises sudden change of the present invention or the carrier that comprises the polynucleotide of sudden change and therefore can produce the microorganism of fructose-4-epimerase or its variant.Recombinant microorganism can comprise the microorganism strain that belongs to for example Escherichia (Escherichia) genus, Erwinia (Erwinia) genus, Serratia (Serratia) genus, Providencia (Providencia) genus, Corynebacterium (Corynebacterium) genus and Brevibacterium (Brevibacterium) genus, particularly Corynebacterium genus. For example, the Corynebacterium genus can be Corynebacterium glutamicum, Corynebacterium ammoniagenes, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens, more particularly Corynebacterium glutamicum, but the recombinant microorganism is not limited thereto.

[0105] For the purposes of the present invention, a recombinant microorganism may be a microorganism that produces a greater amount of fructose-4-epimerase or a variant thereof than a wild-type or unmodified microbial strain.

[0106] The microorganism of the present invention may include all microorganisms capable of expressing the fructose-4-epimerase or a variant thereof of the present invention by various known methods other than introducing a nucleic acid or a vector.

[0107] Another aspect of the present invention is to provide a method for producing fructose-4-epimerase or a variant thereof, the method comprising culturing a microorganism of the genus Corynebacterium in a culture medium, the microorganism comprising the mutated polynucleotide of the present invention or a vector comprising the mutated polynucleotide.

[0108] The mutant polynucleotide, vector, fructose-4-epimerase, fructose-4-epimerase variant and microorganism are as described above.

[0109] For the purpose of the present invention, the microorganism belonging to the genus Corynebacterium is a microorganism that can express fructose-4-epimerase derived from Kosmotoga olearia or a variant thereof, but is not limited thereto.

[0110] As used herein, the term "cultivation" refers to growing microorganisms under appropriately controlled environmental conditions. The cultivation process of the present invention can be implemented using appropriate culture media and culture conditions known in the art. Such cultivation processes can be readily adapted and used by those skilled in the art based on the selected strain. In particular, the cultivation can be batch, continuous, or fed-batch, but is not limited thereto.

[0111] As used herein, the term "culture medium" refers to a material containing the nutrients required for the mixed culture of microorganisms as the main components, and provides nutrients and growth factors including water necessary for survival and development. In particular, as a culture medium and other culture conditions for culturing the microorganisms of the present invention, any culture medium can be used without any particular limitation, as long as it is a culture medium conventionally used for culturing microorganisms. The microorganisms of the present invention can be cultured in a conventional culture medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin, etc., under aerobic conditions, while controlling temperature, pH, etc.

[0112] In the present invention, the carbon source may include carbohydrates such as glucose, fructose, sucrose and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid and citric acid; and amino acids such as glutamic acid, methionine and lysine. Natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, waste sugar cane and corn steep liquor can be used. In particular, carbohydrates such as glucose and sterile pretreated molasses (i.e., molasses converted into reducing sugars) can be used. Appropriate amounts of other carbon sources can be used variously without limitation. These carbon sources can be used alone or in combination of two or more species, but the carbon source is not limited thereto.

[0113] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used; and organic nitrogen sources such as amino acids such as glutamic acid, methionine and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products and defatted soybean cake or its decomposition products can be used alone or in combination of two or more types, but the nitrogen source is not limited thereto.

[0114] The phosphorus source may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or their corresponding sodium salts. As inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. may be used. In addition to these, the inorganic compound may include amino acids, vitamins, and / or appropriate precursors. These components or precursors can be added to the culture medium in batches or continuously. However, the culture medium is not limited thereto.

[0115] In the present invention, during the microbial culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture medium in a suitable manner to adjust the culture medium pH. During the culture, defoaming agents such as fatty acid polyethylene glycol esters can be used to suppress bubble formation. Oxygen or oxygen-containing gas can be injected into the culture medium to maintain the aerobic state of the culture medium, or nitrogen, hydrogen or carbon dioxide gas can be injected or gas may not be injected to maintain anaerobic and microaerobic conditions, but the conditions are not limited thereto.

[0116] The culture medium temperature may be 20-50° C., particularly 30-37° C., but not limited thereto. Cultivation can be continued until the desired amount of useful substances is obtained, and the culture period may be particularly 10-100 hours, but not limited thereto.

[0117] The fructose-4-epimerase or fructose-4-epimerase variant produced by culture may be maintained in the cells with or without being added to the culture medium.

[0118] The production method may include the step of recovering the fructose-4-epimerase or its variant from the culture medium or the microorganism.

[0119] The fructose-4-epimerase or variant thereof produced in the culturing step of the present invention can be recovered by collecting the desired enzyme from the culture broth using a suitable method known in the art, depending on the culturing method. For example, centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC can be used. The desired enzyme can be recovered from the culture medium or microorganism using any suitable method known in the art.

[0120] The recovery step may include a purification process, which may be performed using suitable methods known in the art. Thus, the recovered enzyme may be in a purified form or in the form of an enzyme-containing broth fermented by a microorganism (Introduction to Biotechnology and Genetic Engineering, AJ Nair, 2008).

[0121] Another aspect of the present invention provides a composition for producing tagatose, comprising a microorganism of the genus Corynebacterium comprising the mutated polynucleotide of the present invention or a vector comprising the mutated polynucleotide; or a culture of the microorganism.

[0122] Another aspect of the present invention provides use of a microorganism of the genus Corynebacterium for producing tagatose, wherein the microorganism comprises the mutated polynucleotide of the present invention or a vector comprising the mutated polynucleotide.

[0123] The mutant polynucleotide, vector, fructose-4-epimerase, fructose-4-epimerase variant and microorganism are as described above.

[0124] For the purpose of the present invention, the microorganism belonging to the genus Corynebacterium is a microorganism that can express fructose-4-epimerase derived from Kosmotoga olearia or a variant thereof, but is not limited thereto.

[0125] The tagatose-producing composition may further include fructose, but is not limited thereto.

[0126] The tagatose-producing composition of the present invention may further comprise any suitable excipient commonly used in tagatose-producing compositions, such as, but not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0127] The composition for producing tagatose in the present invention may further comprise a metal ion or a metal salt. In one embodiment, the metal of the metal ion or metal salt may be a metal containing a divalent cation. In particular, the metal of the present invention may be nickel (Ni), iron (Fe), cobalt (Co), magnesium (Mg) or manganese (Mn). More particularly, the metal salt may be MgSO 4 , FeSO 4 , NiSO 4 , NiCl 2 , MgCl 2 , CoSO 4 , MnCl 2 or MnSO 4 .

[0128] Another aspect of the present invention provides a method for producing tagatose, comprising contacting a Corynebacterium microorganism; or a culture of the microorganism, wherein the microorganism comprises the mutated polynucleotide of the present invention or a vector comprising the mutated polynucleotide, with fructose.

[0129] The mutant polynucleotide, vector, fructose-4-epimerase, fructose-4-epimerase variant and microorganism are as described above.

[0130] For the purpose of the present invention, the microorganism belonging to the genus Corynebacterium is a microorganism that can express fructose-4-epimerase derived from Kosmotoga olearia or a variant thereof, but is not limited thereto.

[0131] The step of contacting the Corynebacterium microorganism or the culture of the microorganism with fructose may be a step of contacting the Corynebacterium microorganism or the culture of the microorganism with fructose to convert the fructose into tagatose, but is not limited thereto.

[0132] The fructose-4-epimerase variant can be used as a fructose-4-epimerase to produce tagatose from fructose, but is not limited thereto.

[0133] For example, the contacting of the present invention can be carried out at pH 5.0-pH 9.0 and at 30-80°C and / or for 0.5-48 hours. In particular, the contacting of the present invention can be carried out at pH 6.0-pH 9.0 or pH 7.0-pH 9.0. The contacting of the present invention can be carried out at a temperature of 35°C-80°C, 40°C-80°C, 45°C-80°C, 50°C-80°C, 55°C-80°C, 60°C-80°C, 30°C-70°C, 35°C-70°C, 40°C-70°C, 45°C-70°C, 50°C-70°C, 55°C-70°C, 60°C-70°C, 30°C-65°C, 35°C-65°C, 40°C-65°C, 45°C-65°C, 50°C-65°C, 55°C-65°C, 30°C-60°C, 35°C-60°C, 40°C-60°C, 45°C-60°C, 50°C-60°C or 55°C-60°C. The contacting of the present invention may be carried out for 0.5 hours to 36 hours, 0.5 hours to 24 hours, 0.5 hours to 12 hours, 0.5 hours to 6 hours, 1 hour to 48 hours, 1 hour to 36 hours, 1 hour to 24 hours, 1 hour to 12 hours, 1 hour to 6 hours, 3 hours to 48 hours, 3 hours to 36 hours, 3 hours to 24 hours, 3 hours to 12 hours, 3 hours to 6 hours, 6 hours to 48 hours, 6 hours to 36 hours, 6 hours to 24 hours, 6 hours to 12 hours, 12 hours to 48 hours, 12 hours to 36 hours, 12 hours to 24 hours, 18 hours to 48 hours, 18 hours to 36 hours, or 18 hours to 30 hours.

[0134] The contacting of the present invention can be carried out in the presence of metal ions or metal salts. The metal ions or metal salts that can be used are the same as those in the above aspect.

[0135] The preparation method of the present invention may further include a step of separating and / or purifying the prepared tagatose. Separation and / or purification may be carried out by conventional methods in the field of the present invention. As non-limiting examples, dialysis, precipitation, adsorption, electrophoresis, ion exchange chromatography, and fractional crystallization may be used. Purification may be carried out by only one method or by two or more methods.

[0136] The preparation method of the present invention may further include a decolorization and / or desalting step before or after the separation and / or purification step. By decolorization and / or desalting, tagatose of excellent quality can be obtained.

[0137] As another example, the preparation method of the present invention may further include a step of crystallizing tagatose after the step of converting fructose into tagatose, the separation and / or purification step, or the decolorization and / or desalination step of the present invention. Crystallization can be carried out by a conventional crystallization method. For example, crystallization can be carried out by cooling crystallization.

[0138] The preparation method of the present invention may further include a step of concentrating tagatose before the crystallization step. Concentration can increase crystallization efficiency.

[0139] As another example, the preparation method of the present invention may further include the step of contacting unreacted fructose with the enzyme of the present invention, a microorganism expressing the enzyme, or a microbial culture after the separation and / or purification step of the present invention; reusing the mother liquor obtained from the separation and / or purification step after the crystallization step of the present invention; or a combination thereof.

[0140] Example

[0141] Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these Examples and Experimental Examples are for the purpose of illustrating the present invention, and the scope of the present invention is not limited to these Examples and Experimental Examples.

[0142] Example 1: Verification of the expression level of Kosmotoga olearia-derived fructose-4-epimerase expressed from a mutant polynucleotide

[0143] To increase the gene expression efficiency of the fructose-4-epimerase derived from Kosmotoga olearia, known in Korean Patent Publication No. 10-2018-0111678A, in a coryneform bacterium strain (Corynebacterium glutamicum), a mutant polynucleotide fructose-4-epimerase gene (SEQ ID NO: 3) was constructed. A 6X His-tag sequence was added for isolation during fructose-4-epimerase gene construction.

[0144] As a plasmid, pHCP plasmid is used for high copy number, in which the 21st position of orfA2 (parB) is mutated to adenine (A) (Korean Patent Publication No. 10-2018-0092110A). In the present invention, an attempt was made to obtain a plasmid with a higher copy number than the existing pHCP. To this end, a 98bp sequence was randomly changed and screened, which is the ctRNA part of pHCP. Therefore, a polynucleotide in which the 22nd nucleotide of the ctRNA part was replaced with C (cytosine) and a polynucleotide in which the 79th nucleotide was replaced with C (cytosine) were finally identified. The high copy number plasmid containing this is called pHCP7 vector (SEQ ID NO: 8), and the vector was used to construct a plasmid containing the fructose-4-epimerase gene sequence.

[0145] Each sudden change fructose-4-epimerase gene inserts the pHCP7 carrier, is transformed into coryneform bacteria strain (Corynebacterium glutamicum ATCC13032).The coryneform bacteria strain of conversion is 30 ℃ in 10mL BHI substratum (comprising 25 μ g / mL kantlex (Km)), and 200rpm stirs and cultivates 24 hours down.Afterwards, substratum places the fresh BHI substratum of 50mL (comprising 25 μ g / mL Km) and 30 ℃, and 200rpm stirs and cultivates 24 hours down with 1 / 100 volume ratio.

[0146] The cultured strain was recovered at an OD 600nm of 4, suspended in 300 μL of PBS, and sonicated in an ice bucket (50% pulse and 20% amplitude for a total of 7 minutes). The supernatant obtained by centrifugation was mixed with 5x sample buffer and heated at 100°C for 5 minutes. The mixture was loaded onto a 12% SDS-PAGE, and proteins were identified using Coomassie Brilliant Blue staining and destaining. To analyze protein expression levels, the expression level of fructose-4-epimerase in total protein was confirmed using a gel analyzer program.

[0147] Results, such as Figure 1 As shown, it was confirmed that the expression level of fructose-4-epimerase expressed by the mutant polynucleotide was increased by about 6% or more compared to the expression level of fructose-4-epimerase wild-type (before mutation) polynucleotide (WT).

[0148] Example 2: Verification of tagatose conversion efficiency of fructose-4-epimerase expressed from mutant polynucleotides

[0149] 45 ml of the culture broth of the Corynebacterium strain cultured in Example 1 was centrifuged at 3,500 rpm and 4°C for 10 minutes, followed by washing with 10 mL of Tris-HCl (pH 8.0) buffer. Centrifugation and weighing were then performed under the same conditions as above. An amount equivalent to 20% by weight of the recovered strain in the total reaction volume was inoculated into a 2 mL tube and preheated at 60°C for 30 minutes. The CoSO₄ or NiSO₄ to be added to the tagatose conversion reaction was also preheated. The strain was mixed with 50 mM tris-HCl (pH 8.0) and 3 mM CoSO₄ or NiSO₄ containing the substrate (30% fructose) and reacted at 60°C for 2 hours. After the reaction was completed, the sample was centrifuged at 13,000 rpm and 4°C for 10 minutes to recover the supernatant, which was then analyzed by HPLC. The HPLC analysis used an Aminex HPX-87H column using 5 mM H₂SO₄ as solvent to analyze the amount of tagatose produced.

[0150] Results, such as Figure 2 As shown, it was confirmed that the conversion rate of fructose-4-epimerase expressed from the mutant polynucleotide was about 22%, which was as much as 7% higher than that of WT (#7_KO (KNF4E)).

[0151] Example 3: Verification of expression levels of fructose-4-epimerase variants expressed from mutated polynucleotides

[0152] Experiments were conducted to confirm whether the expression level of the fructose-4-epimerase derived from Kosmotoga olearia of Example 1 was increased even if some amino acid sequence mutations were observed. The gene sequences of two enzyme variants (N97Y, T124W, N367V mutations: SEQ ID NO: 4, T124W mutation: SEQ ID NO: 6) obtained by mutating amino acids in the fructose-4-epimerase gene (SEQ ID NO: 3) were constructed, respectively, which are mutated polynucleotides. The strain containing the enzyme variant (SEQ ID NO: 4) is named Corynebacterium glutamicum CF01-0014 and was deposited on October 18, 2019, at the Korean Collection of Microorganisms, a trustee institution under the Budapest Treaty, with accession number KCCM12610P.

[0153] Each mutant fructose-4-epimerase gene was inserted into the pHCP7 vector and introduced into a coryneform bacterium strain. The expression levels of the two enzyme variants in the strain were confirmed by Western blotting according to the method of Example 1.

[0154] Results, such as Figure 3As shown, it was confirmed that the expression level of the fructose-4-epimerase N97Y, T124W, and N367V variants (lane 4) expressed from the mutant polynucleotides increased by about 8% compared to the WT (lane 2), and the expression level of the T124W variant (lane 5) increased by about 12% or more compared to the fructose-4-epimerase wild-type (before mutation) polynucleotide (WT), confirming that the expression level was similar to that of the fructose-4-epimerase (lane 3) expressed from the mutant polynucleotides.

[0155] Example 4: Verification of tagatose conversion efficiency of fructose-4-epimerase variants expressed from mutated polynucleotides

[0156] The tagatose conversion rates of the two enzyme variants in Example 3 were confirmed by the method of Example 2.

[0157] Results, such as Figure 4 As shown in Figure 3, the tagatose conversion rate of the N97Y, T124W, and N367V variants (3aa mutations) was approximately 32%, a 19% increase compared to the WT. The tagatose conversion rate of the T124W variant was also as high as 27.8%.

[0158] Comparative Example 1: Verification of the expression level and tagatose conversion rate of the new Thermotoga apollogenes-derived fructose-4-epimerase with the same nucleotide mutation

[0159] Mutated polynucleotide variants 2 and 1 (SEQ ID NOs: 12 and 13) were constructed in which the same nucleotide mutations as those in Example 1 were applied to the nucleic acid sequence (SEQ ID NO: 9) encoding a variant (SEQ ID NO: 10) of fructose-4-epimerase (hexuronic acid C4-epimerase, SEQ ID NO: 9) derived from the new Thermotoga apollogenes known in Korean Patent Publication No. 10-2017-0015250A, inserted into a pHCP vector, and introduced into a coryneform bacterium strain, and then the expression level and tagatose conversion rate were confirmed by the methods of Examples 1 and 2.

[0160] Results, such as Figure 5 As shown, the expression levels of the fructose-4-epimerase variants (Variant 1 / TN_Variant_1, Variant 2 / TN_Variant_2, CJ) expressed from the mutated polynucleotides were lower than that of the WT, and the tagatose conversion rates of the fructose-4-epimerase variants were also lower than that of the WT ( Figure 6 ).

[0161] In particular, in Korean Patent Publication No. 10-2017-0015250A, a fructose-4-epimerase variant showed higher activity than the wild-type (pre-mutation) fructose-4-epimerase, but it was confirmed that when the polynucleotide of the enzyme variant was mutated, the activity was lower than that of the wild-type (pre-mutation) fructose-4-epimerase, and thus the expression level was significantly reduced.

[0162] Therefore, it was confirmed that the nucleotide mutations of the present invention were effective on the Kosmotoga olearia-derived enzyme.

[0163] Based on the above description, it will be understood by those skilled in the art that the present invention can be implemented in different specific forms without changing its technical spirit or basic characteristics. Therefore, it should be understood that the above embodiments are not restrictive, but illustrative in all aspects. The scope of the present invention is defined by the appended claims, rather than the preceding description thereof, and all changes and modifications that fall within the scope of the claims or equivalents of these scopes are intended to be covered by the claims.

[0164] Sequence Listing <110> CJ CheilJedang Co., Ltd. Korea Advanced Institute of Science and Technology <120> Composition for preparing tagatose and method for preparing tagatose using the same <130> OPA21006 <150> KR 10-2020-0007490 <151> 2020-01-20 <160> 13 <170> KoPatentIn 3.0 <210> 1 <211> 1308 <212> DNA <213> Unknown <220> <223> Kosmotoga olearia fructose-4-epimerase Wild type_ polynucleotide <400> 1 atgaaaaaac atcctcttca ggacattgtt tcattgcaaa aacagggaat acccaaaggg 60 gttttctctg tatgtagtgc caatagattt gttattgaaa cactctgga attgcgaag 120 atgaaaggga caacggttct tatagaggcc acctgcaatc aggtaacca gttcggtggc 180 tacaccggta tgactcctgc tgatttcaga gaaatggtttt tttctcgc tgaggatatt 240 ggactccca aaaataaat catccttggt ggcgaccatc tggcccaa tccctggaag 300 gtcagccgt cagatcaggc tatgcgtaac gccattgaaa tgattcgaga atacgctaaa 360 gctgggttta ccaagcttca tctggatgcc agcatgcgtc ttgcagacga tccgggaac 420 gaaaacgagc cgctgaaccc ggaagttata gcggaaagaa cagctctctct ctgtcttgaa 480 gccgagagggg cttttaaaga atccgccggt tctctccggc ctgtttacgt tattggtacg 540 gatgttccgc caccgggtgg agcgcaaac gaagtaaat cgattcatgt aaccagtgtt 600 caggatttg agcgtaccgt tgagttgacc aaaaggcat tttcgacca tggtttgtat 660 gaagcctggg gaagggtgat tgcggttgtt gtgcaaccgg gagtagaatt cgggaatgaa 720 catatattcg atatgatag aaatcgagcg agagaactta ctgaggcgat aaaaagcat 780 ccaaatatag ttttgagg tcactcgaca gattatcaa cggcaaagc attgaaagaa 840 atggtagaag acggtgtagc catactcaag gttggggccag ctctacatt tgcgctcaga 900 gaggcttttt ttgcgttgag cagcattgaa aaagagttat tttatgat acccggggctt 960 tgttcaact tgttgaagt tgtcgagaga gcgatgcttg acaatccaaa acattgggaa 1020 aaatattacc agggagaga gagaaaat agattaagccc gtaaatacag ctttctcgat 1080 cgcttgaggt attactggaa tctcctgag gttagacag cggtgaataa gctgataacc 1140 aaccttgaaa caaagaat cccgttaacg cttataagcc agttcatgcc gatgcagtac 1200 CAAAAAATCA gaacggttt gctaagaag gatccataa gccttaataa agatcgaatt 1260 acccttgttc ttgatgacta ctatttcgca actcaccctg aatgttga 1308 <210> 2 <211> 435 <212> PRT <213> Unknown <220> <223> Kosmotoga olearia fructose-4-epimerase_amino acid <400> 2 Met Lys Lys Pro Leu Gln Asp Ile Val Ser Leu Gln Lys Gln Gly 1 5 10 15 Ile Pro Lys Gly Val Phe Ser Val Cys Ser Ala Asn Arg Phe Val Ile 20 25 30 Glu Thr Thr Leu Glu Tyr Ala Lys Met Lys Gly Thr Thr Val Leu Ile 35 40 45 Glu Ala Thr Cys Asn Gln Val Asn Gln Phe Gly Gly Tyr Thr Gly Met 50 55 60 Thr Pro Ala Asp Phe Arg Glu Met Val Phe Ser Ile Ala Glu Asp Ile 65 70 75 80 Gly Leu Pro Lys Asn Lys Ile Ile Leu Gly Gly Asp His Leu Gly Pro 85 90 95 Asn Pro Trp Lys Gly Gln Pro Ser Asp Gln Ala Met Arg Asn Ala Ile 100 105 110 Glu Met Ile Arg Glu Tyr Ala Lys Ala Gly Phe Thr Lys Leu His Leu 115 120 125 Asp Ala Ser Met Arg Leu Ala Asp Asp Pro Gly Asn Glu Asn Glu Pro 130 135 140 Leu Asn Pro Glu Val Ile Ala Glu Arg Thr Ala Leu Leu Cys Leu Glu 145 150 155 160 Ala Glu Arg Ala Phe Lys Glu Ser Ala Gly Ser Leu Arg Pro Val Tyr 165 170 175 Val Ile Gly Thr Asp Val Pro Pro Pro Gly Gly Ala Gln Asn Glu Gly 180 185 190 Lys Ser Ile His Val Thr Ser Val Gln Asp Phe Glu Arg Thr Val Glu 195 200 205 Leu Thr Lys Lys Ala Phe Phe Asp His Gly Leu Tyr Glu Ala Trp Gly 210 215 220 Arg Val Ile Ala Val Val Val Gln Pro Gly Val Glu Phe Gly Asn Glu 225 230 235 240 His Ile Phe Glu Tyr Asp Arg Asn Arg Ala Arg Glu Leu Thr Glu Ala 245 250 255 Ile Lys Lys His Pro Asn Ile Val Phe Glu Gly His Ser Thr Asp Tyr 260 265 270 Gln Thr Ala Lys Ala Leu Lys Glu Met Val Glu Asp Gly Val Ala Ile 275 280 285 Leu Lys Val Gly Pro Ala Leu Thr Phe Ala Leu Arg Glu Ala Phe Phe 290 295 300 Ala Leu Ser Ser Ile Glu Lys Glu Leu Phe Tyr Asp Thr Pro Gly Leu 305 310 315 320 Cys Ser Asn Phe Val Glu Val Val Glu Arg Ala Met Leu Asp Asn Pro 325 330 335 Lys His Trp Glu Lys Tyr Gln Gly Glu Glu Arg Glu Asn Arg Leu 340 345 350 Ala Arg Lys Tyr Ser Phe Leu Asp Arg Leu Arg Tyr Tyr Trp Asn Leu 355 360 365 Pro Glu Val Arg Thr Ala Asn Lys Leu Ile Thr Asn Leu Glu Thr 370 375 380 Lys Glu Ile Pro Leu Thr Leu Ile Ser Gln Phe Met Pro Met Gln Tyr 385 390 395 400 Gln Lys Ile Arg Asn Gly Leu Leu Arg Lys Asp Pro Ile Ser Leu Ile 405 410 415 Lys Asp Arg Ile Thr Leu Val Leu Asp Asp Tyr Tyr Phe Ala Thr His 420 425 430 Pro Glu Cys 435 <210> 3 <211> 1305 <212> DNA <213> Artificial Sequence <220> <223> Kosmotoga olearia fructose-4-epimerase Variant polynucleotide_ polynucleotide <400> 3 atgaaaaagc atccattgca ggacatcgta tcactgcaga agcaggtat cccaagggc 60 gtcttctcag tctgttccgc taaccgtttc gtcatcgaga ccactctgga atacgccaag 120 atgaagggca ctacggttct tattgaagca acctgtaacc aggtgaacca gtttggcggc 180 tacaccggta tgactcctgc cgacttccgc gagatggtct tctccattgc tgaagacatt 240 ggccttccta agaacaaaat cattctcggc ggcgaccacc tcggtcccaa tccgtggaag 300 ggtcagcctt ccgaccaggc tatgcggaac gctattgaaa tgattcgcga gtacgccaaa 360 gcaggcttta ccaaactcca cttggacgct tcaatgcggc tggctgacga tcctggaaac 420 gagaacgagc ccttgaaccc agaagtcatt gcagagcgga ctgctctctt gtgcctggaa 480 gccgaacgtg cctttaaaga atccgcaggt tctctccgcc ctgtttacgt cattggcacg 540 gacgttccgc caccaggagg tgctcagaat gagggaaaga gcattcacgt tacctccgtg 600 caggacttcg aacgtaccgt tgagcttacc aagaaagcat tctttgacca tggtctctat 660 gaggcctggg gccgtgtgat cgccgttgtt gtgcagcccg gcgttgaatt cggtaacgag 720 cacatttttg aatatgatcg gaaccgtgca cgcgaattga ccgaagctat caaaaaacac 780 cctaacattg tttcgaagg ccatagcacg gactaccaga cggccaaagc attgaaggaa 840 atggttgaag acggagtagc aattttgaag gtaggccccg cactgacctt tgcgcttcgg 900 gaagcattt ttgcgctttc ttccatcgag aaggaacttt tctacgatac ccctggactt 960 tgtagcaact tcgttgaagt tgtagagcgg gctatgctgg acaaccctaa gcactgggaa 1020 aagtactacc agggagagga acggggagaat cggcttgcac gcaaatacag ctttcttgac 1080 cgcctccgtt actattggaa tctgcccgag gtacgtactg ccgtgaataa gttgatcacc 1140 aatttggaaa cgaaagaaat cccattgacg ctcattagcc agttcatgcc gatgcagtat 1200 caaaagattc gcaatggtct gctgcggaaa gatcccatct ctctgatcaa ggaccggatc 1260 accttggtcc tggacgatta ctattttgca acccatcctg aatgt 1305 <210> 4 <211> 1305 <212> DNA <213> Artificial Sequence <220> <223> Kosmotoga olearia fructose-4-epimerase Variant polynucleotide_WVY_ polynucleotide <400> 4 atgaaaaagc atccattgca ggacatcgta tcactgcaga agcagggtat ccccaagggc 60 gtcttctcag tctgttccgc taaccgtttc gtcatcgaga ccactctgga atacgccaag 120 atgaagggca ctacggttct tattgaagca acctgtaacc aggtgaacca gtttggcggc 180 tacaccggta tgactcctgc cgacttccgc gagatggtct tctccattgc tgaagacatt 240 ggccttccta agaacaaaat cattctcggc ggcgaccacc tcggtcccta cccgtggaag 300 ggtcagcctt ccgaccaggc tatgcggaac gctattgaaa tgattcgcga gtacgccaaa 360 gcaggcttt ggaaactcca cttggacgct tcaatgcggc tggctgacga tcctggaaac 420 gagaacgagc ccttgaaccc agaagtcatt gcagagcgga ctgctctctt gtgcctggaa 480 gccgaacgtg cctttaaaga atccgcaggt tctctccgcc ctgtttacgt cattggcacg 540 gacgttccgc caccaggagg tgctcagaat gagggaaaga gcattcacgt tacctccgtg 600 caggacttcg aacgtaccgt tgagcttacc aagaaagcat tctttgacca tggtctctat 660 gaggcctggg gccgtgtgat cgccgttgtt gtgcagcccg gcgttgaatt cggtaacgag 720 cacatttttg aatatgatcg gaaccgtgca cgcgaattga ccgaagctat caaaaaacac 780 cctaacattg tttcgaagg ccatagcacg gactaccaga cggccaaagc attgaaggaa 840 atggttgaag acggagtagc aattttgaag gtaggccccg cactgacctt tgcgcttcgg 900 gaagcattt ttgcgctttc ttccatcgag aaggaacttt tctacgatac ccctggactt 960 tgtagcaact tcgttgaagt tgtagagcgg gctatgctgg acaaccctaa gcactgggaa 1020 aagtactacc agggagagga acggggagaat cggcttgcac gcaaatacag ctttcttgac 1080 cgcctccgtt actattgggt tctgcccgag gtacgtactg ccgtgaataa gttgatcacc 1140 aatttggaaa cgaaagaaat cccattgacg ctcattagcc agttcatgcc gatgcagtat 1200 caaaagattc gcaatggtct gctgcggaaa gatcccatct ctctgatcaa ggaccggatc 1260 accttggtcc tggacgatta ctattttgca acccatcctg aatgt 1305 <210> 5 <211> 435 <212> PRT <213> Artificial Sequence <220> <223> Kosmotoga olearia fructose-4-epimerase Variant_WVY_amino acid <400> 5 Met Lys Lys His Pro Leu Gln Asp Ile Val Ser Leu Gln Lys Gln Gly 1 5 10 15 Ile Pro Lys Gly Val Phe Ser Val Cys Ser Ala Asn Arg Phe Val Ile 20 25 30 Glu Thr Thr Leu Glu Tyr Ala Lys Met Lys Gly Thr Thr Val Leu Ile 35 40 45 Glu Ala Thr Cys Asn Gln Val Asn Gln Phe Gly Gly Tyr Thr Gly Met 50 55 60 Thr Pro Ala Asp Phe Arg Glu Met Val Phe Ser Ile Ala Glu Asp Ile 65 70 75 80 Gly Leu Pro Lys Asn Lys Ile Ile Leu Gly Gly Asp His Leu Gly Pro 85 90 95 Tyr Pro Trp Lys Gly Gln Pro Ser Asp Gln Ala Met Arg Asn Ala Ile 100 105 110 Glu Met Ile Arg Glu Tyr Ala Lys Ala Gly Phe Trp Lys Leu His Leu 115 120 125 Asp Ala Ser Met Arg Leu Ala Asp Asp Pro Gly Asn Glu Asn Glu Pro 130 135 140 Leu Asn Pro Glu Val Ile Ala Glu Arg Thr Ala Leu Leu Cys Leu Glu 145 150 155 160 Ala Glu Arg Ala Phe Lys Glu Ser Ala Gly Ser Leu Arg Pro Val Tyr 165 170 175 Val Ile Gly Thr Asp Val Pro Pro Pro Gly Gly Ala Gln Asn Glu Gly 180 185 190 Lys Ser Ile His Val Thr Ser Val Gln Asp Phe Glu Arg Thr Val Glu 195 200 205 Leu Thr Lys Lys Ala Phe Phe Asp His Gly Leu Tyr Glu Ala Trp Gly 210 215 220 Arg Val Ile Ala Val Val Val Gln Pro Gly Val Glu Phe Gly Asn Glu 225 230 235 240 His Ile Phe Glu Tyr Asp Arg Asn Arg Ala Arg Glu Leu Thr Glu Ala 245 250 255 Ile Lys Lys His Pro Asn Ile Val Phe Glu Gly His Ser Thr Asp Tyr 260 265 270 Gln Thr Ala Lys Ala Leu Lys Glu Met Val Glu Asp Gly Val Ala Ile 275 280 285 Leu Lys Val Gly Pro Ala Leu Thr Phe Ala Leu Arg Glu Ala Phe Phe 290 295 300 Ala Leu Ser Ser Ile Glu Lys Glu Leu Phe Tyr Asp Thr Pro Gly Leu 305 310 315 320 How To Make Asn Phe Val Glu Val Val Glu Arg Ala Met Leu Asp Asn Pro 325 330 335 Lys His Trp Glu Lys Tyr Gln Gly Glu Glu Arg Glu Asn Arg Leu 340 345 350 Ala Arg Lys Tyr Ser Phe Leu Asp Arg Leu Arg Tyr Tyr Trp Val Leu 355 360 365 Pro Glu Val Arg Thr Ala Asn Lys Leu Ile Thr Asn Leu Glu Thr 370 375 380 Lys Glu Ile Pro Leu Thr Leu Ile Ser Gln Phe Met Pro Met Gln Tyr 385 390 395 400 Gln Lys Ile Arg Asn Gly Leu Leu Arg Lys Asp Pro Ile Ser Leu Ile 405 410 415 Lys Asp Arg Ile Thr Leu Val Leu Asp Asp Tyr Tyr Phe Ala Thr His 420 425 430 Pro Glu Cys 435 <210> 6 <211> 1308 <212> DNA <213> Artificial Sequence <220> <223> Kosmotoga olearia fructose-4-epimerase Variant polynucleotide_W_ polynucleotide <400> 6 atgaaaaagc atccattgca ggacatcgta tcactgcaga agcagggtat ccccaagggc 60 gtcttctcag tctgttccgc taaccgtttc gtcatcgaga ccactctgga atacgccaag 120 atgaagggca ctacggttct tattgaagca acctgtaacc aggtgaacca gtttggcggc 180 tacaccggta tgactcctgc cgacttccgc gagatggtct tctccattgc tgaagacatt 240 ggccttccta agaacaaaat cattctcggc ggcgaccacc tcggtcccaa tccgtggaag 300 ggtcagcctt ccgaccaggc tatgcggaac gctattgaaa tgattcgcga gtacgccaaa 360 gcaggcttt ggaaactcca cttggacgct tcaatgcggc tggctgacga tcctggaaac 420 gagaacgagc ccttgaaccc agaagtcatt gcagagcgga ctgctctctt gtgcctggaa 480 gccgaacgtg cctttaaaga atccgcaggt tctctccgcc ctgtttacgt cattggcacg 540 gacgttccgc caccaggagg tgctcagaat gagggaaaga gcattcacgt tacctccgtg 600 caggacttcg aacgtaccgt tgagcttacc aagaaagcat tctttgacca tggtctctat 660 gaggcctggg gccgtgtgat cgccgttgtt gtgcagcccg gcgttgaatt cggtaacgag 720 cacatttttg aatatgatcg gaaccgtgca cgcgaattga ccgaagctat caaaaaacac 780 cctaacattg ttttcgaagg ccatagcacg gactaccaga cggccaaagc attgaaggaa 840 atggttgaag acggagtagc aattttgaag gtaggccccg cactgacctt tgcgcttcgg 900 gaagcatttt ttgcgctttc ttccatcgag aaagacttt tctacgatac ccctggactt 960 tgtagcaact tcgttgaagt tgtagagcgg gctatgctgg aaaccctaa gcactgggaa 1020 aagtactacc agggagga acgggagaat cggcttgcac gcaaatacag ctttcttgac 1080 cgcctccgtt actattggaa tctgcccgag gtacgtactg ccgtgaataa gttgatcacc 1140 aatttggaaa cgaaagaaat cccattgacg ctcattagcc agttcatgcc gatgcagtat 1200 caaaagattc gcaatggtct gctgggaaa gatcccatct ctctgatcaa ggaccggatc 1260 accttggtcc tggacgatta ctatttttgca acccatcctg aatgttga 1308 <210> 7 <211> 435 <212> PRT <213> Artificial Sequence <220> <223> Kosmotoga olearia fructose-4-epimerase Variant_W_amino acid <400> 7 Met Lys Lys His Pro Leu Gln Asp Ile Val Ser Leu Gln Lys Gln Gly 1 5 10 15 Ile Pro Lys Gly Val Phe Ser Val Cys Ser Ala Asn Arg Phe Val Ile 20 25 30 Glu Thr Thr Leu Glu Tyr Ala Lys Met Lys Gly Thr Thr Val Leu Ile 35 40 45 Glu Ala Thr Cys Asn Gln Val Asn Gln Phe Gly Gly Tyr Thr Gly Met 50 55 60 Thr Pro Ala Asp Phe Arg Glu Met Val Phe Ser Ile Ala Glu Asp Ile 65 70 75 80 Gly Leu Pro Lys Asn Lys Ile Ile Leu Gly Gly Asp His Leu Gly Pro 85 90 95 Asn Pro Trp Lys Gly Gln Pro Ser Asp Gln Ala Met Arg Asn Ala Ile 100 105 110 Glu Met Ile Arg Glu Tyr Ala Lys Ala Gly Phe Trp Lys Leu His Leu 115 120 125 Asp Ala Ser Met Arg Leu Ala Asp Asp Pro Gly Asn Glu Asn Glu Pro 130 135 140 Leu Asn Pro Glu Val Ile Ala Glu Arg Thr Ala Leu Leu Cys Leu Glu 145 150 155 160 Ala Glu Arg Ala Phe Lys Glu Ser Ala Gly Ser Leu Arg Pro Val Tyr 165 170 175 Val Ile Gly Thr Asp Val Pro Pro Pro Gly Gly Ala Gln Asn Glu Gly 180 185 190 Lys Ser Ile His Val Thr Ser Val Gln Asp Phe Glu Arg Thr Val Glu 195 200 205 Leu Thr Lys Lys Ala Phe Phe Asp His Gly Leu Tyr Glu Ala Trp Gly 210 215 220 Arg Val Ile Ala Val Val Val Gln Pro Gly Val Glu Phe Gly Asn Glu 225 230 235 240 His Ile Phe Glu Tyr Asp Arg Asn Arg Ala Arg Glu Leu Thr Glu Ala 245 250 255 Ile Lys Lys His Pro Asn Ile Val Phe Glu Gly His Ser Thr Asp Tyr 260 265 270 Gln Thr Ala Lys Ala Leu Lys Glu Met Val Glu Asp Gly Val Ala Ile 275 280 285 Leu Lys Val Gly Pro Ala Leu Thr Phe Ala Leu Arg Glu Ala Phe Phe 290 295 300 Ala Leu Ser Ser Ile Glu Lys Glu Leu Phe Tyr Asp Thr Pro Gly Leu 305 310 315 320 Cys Ser Asn Phe Val Glu Val Val Glu Arg Ala Met Leu Asp Asn Pro 325 330 335 Lys His Trp Glu Lys Tyr Tyr Gln Gly Glu Glu Arg Glu Asn Arg Leu 340 345 350 Ala Arg Lys Tyr Ser Phe Leu Asp Arg Leu Arg Tyr Tyr Trp Asn Leu 355 360 365 Pro Glu Val Arg Thr Ala Val Asn Lys Leu Ile Thr Asn Leu Glu Thr 370 375 380 Lys Glu Ile Pro Leu Thr Leu Ile Ser Gln Phe Met Pro Met Gln Tyr 385 390 395 400 Gln Lys Ile Arg Asn Gly Leu Leu Arg Lys Asp Pro Ile Ser Leu Ile 405 410 415 Lys Asp Arg Ile Thr Leu Val Leu Asp Asp Tyr Tyr Phe Ala Thr His 420 425 430 Pro Glu Cys 435 <210> 8 <211> 98 <212> DNA <213> Artificial Sequence <220> <223> pHCP7 <400> 8 gctgcacgaa tacctgaaaa acgttgaacg cccgtgagc ggtactcac agggcgtcgg 60 ctaaccccca gtccaaacct gggagaaagc gctcaaaa 98 <210> 9 <211> 481 <212> PRT <213> Unknown <220> <223> Thermotoga neapolitana fructose-4-epimerase <400> 9 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Arg Glu Asn His Glu Asn With Glu Val With Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Ser Val Arg Glu 115 120 125 Asn Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Leu Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Thr Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Ser Glu Arg Glu Lys Asn Glu Met Phe Glu Glu Ile Leu Lys 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Thr Val Leu 210 215 220 Gly Glu Arg Leu Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe His Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly <210> 10 <211> 481 <212> PRT <213> Artificial Sequence <220> <223> Thermotoga neapolitana fructose-4-epimerase Variant <400> 10 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Asn Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Leu Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Thr Ile Asp Pro Gln Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Ser Glu Arg Glu Lys Asn Glu Met Phe Glu Glu Ile Leu Lys 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Thr Val Leu 210 215 220 Gly Glu Arg Leu Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Met Thr Val Asp Glu Asp 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Met Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe His Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Val Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385,390,395,400 Lys Ser Thr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465,470,475,480 Gly <210> 11 <211> 1447 <212> DNA <213> Artificial Sequence <220> <223> Thermotoga neapolitana fructose-4-epimerase_ polynucleotide <400> 11 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tcttttctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggacgtgag ggagaacgag agaacgggaa ggacctggag agacgtgctg 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 acaatcgatc ctcaggatca tgtgaggaat ctttcaaaac tcagtgaaag agaaaagaac 600 gagatgttcg aggaaatact gaaaaaagag cgaatcgaca ggatctatct tgggaaaaaa 660 tacaccgtcc tcggtgaaag actggagttc gacgagaaaa atttgaggga tgctgctctg 720 gtgtactatg atgcgatcgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaat zgagttgac gaagatgaaa ctcccacgag tcctctcttc 840 cacattttcg tgtggaaga actcagacga agaggtgtg agttcaccaa tcttgccctg 900 agattcatcg gcgaatga aaagggaata gattacaag gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaatcgca aggatgttcg aaggataca atatcactc 1020 cactctggaa gcgacaatt tccgtgtat cctgctttg cttccgcgac aggaggcctt 1080 ttccacgtga agacagccgg aacgagttat cttgaggcgg tgaggtcat atccatggtc 1140 aacccggagc tcttcgttga gatctacagg tgtgctcg atcacttga ggaagacaga 1200 aagtccacac acatactgc ggatctgtcg aaagttccgg aagtagagaa agtgaagat 1260 gaagagatcttc caggtcttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaagatgc atctttgaa gaacggctgt ttagacgct tgaacaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggctaa 1447 <210> 12 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> variant 2 <400> 12 atggtcctga aagtcttcaa ggaccacttt ggacgaggat acgaagttta cgaaaagtct 60 taccgcgaaa aggactctct ttctttcttc ctgaccaagg gagaggaagg caaaatcctc 120 gttgtggccg gtgaaaaagc acctgagggc ctgtcgtttt tcaagaaaca gcgagtggag 180 ggagtctcgt tctttttctg tgagcgcaat catgagaacc tcgaagtact gcgtaaatac 240 tttccagacc ttaagccagt gcgcgcaggt cttcgtgcct cttttggaac cggcgaccgc 300 cttggcatca ccaccccggc acacgttcgc gcacttaagg actcaggtct ctttcccatc 360 tttgctcagc aggacgtccg cgagaacgag cgcaccggac gcacctggcg agacgttctt 420 gacgatgcta cctggggtgt gttccaggag ggctactccg agggcttcgg tgcagacgct 480 gatcacgtta agcgcccgga ggacctcgta tcggctgcac gtgaaggatt caccatgttc 540 actatcgatc ctcaggacca cgttcgcaat ctctcaaaac tgtccgaacg cgaaaagaac 600 gagatgttcg aggaaattct taaaaaggag cgcatcgatc gtatctactt gggaaaaaaa 660 tataccgtcc ttggagaacg cctggagttc gacgagaaaa atctgcgcga cgctgcactt 720 gtctactatg acgccatcgc acacgttgac atgatgtacc aaatcttgaa agatgaaact 780 ccggacttcg acttcgaaat gaccgtcgat gaagatgaaa ctcccacttc ccctctgttc 840 cacatcttcg tcgtggaaga actgcgccgc cgtggcgttg agttcacgaa tctggcactc 900 cgcttcatcg gcgaaatgga aaaaggcatc gactacaaag gtgacctggc ccagttcgag 960 cgcgaaatca aaatgcacgc tgaaatcgca cgcatgttcg aaggatacaa aatttcactc 1020 cactctggaa gcgacaaatt ttccgtctac cctgcatttg cttccgccac gggaggactc 1080 ttccacgtga aaaccgcagg tacgtcctac ctggaggcag tcaaagtcat ctccatggtg 1140 aacccggagc tgttcgttga gatctatcgc tgcgcactgg accattttga ggaagaccgt 1200 aagtccactc acatctctgc agatctgtcg aaggtgccgg aagtcgagaa ggtcaaagac 1260 gaagaccttc caggtctttt tgaagacatc aacgttcgcc agttgatcca cgtcacctac 1320 ggctctgtcc tgaaagatgc ttctttgaag gaacgcctct ttaaaaccct cgaacaaaat 1380 gaggaactct tctacgagac cgttgctaaa cacatcaagc gccacgttga tctcttgaaa 1440 in 1446 <210> 13 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> variant 1 <400> 13 atggtgctga aggtgttcaa ggatcacttc ggccgcggct acgaagtgta cgaaaagtcc 60 taccgcgaaa aggactccct gtccttcttc ctgaccaagg gcgaagaagg caagatcctg 120 gtggtggccg gcgaaaagc cccagaaggc ctgtccttct ttaagaagca gcgcgtggaa 180 ggcgtgtcct tcttcttg cgaacgcaac cacgaaaacc tggaagtgct gcgcaagtac 240 ttcccagatc tgaagccagt gcgcgccggc ctgcgcgcat cttcggtac tggcgaccgc 300 ctgggcatca ccaccccagc tcacgtgcgc gccctgaagg actccggcct gttcccaatc 360 ttcgcccagc aggacgtgcg cgaaaacgaa cgcaccggcc gcacctggcg cgacgttttg 420 gacgacgcaa cctggggcgt gttccaggaa ggctactccg aaggcttcgg cgcagatgca 480 gatcacgtga agcgcccaga agatctggtg tccgcagcac gcgaaggctt caccatgttc 540 accatcgacc cacaggatca cgtgcgcaac ctgtccaagc tgtccgaacg cgaaaagaac 600 gaaatgttcg aagaaatcct gaaaggaa cgcatcgacc gcatctacct gggcaagaag 660 tacaccgtgc tgggcgaacg cctggaattc gatgaaaaga acctgcgcga tgccgcactg 720 gtgtactacg atgccatcgc ccacgtggac atgatgtacc agatcctgaa ggatgaaacc 780 ccagacttcg acttcgaaat gaccgtggat gaagacgaaa ccccaacctc cccactgttc 840 cacatcttcg tggtggaaga actgcgccgc cgcggcgtgg aattcaccaa cctggcactg 900 cgcttcatcg gcgaaatgga aaagggcatc gactacaagg gcgacctggc acagttcgaa 960 cgcgaaatca agatgcacgc agaaatcgca cgcatgttcg aaggctacaa gatctccctg 1020 cactccggct ccgataagtt ctccgtgtac ccagcattcg catccgcaac cggcggcctg 1080 ttccacgtga agaccgcagg cacctcctac ctggaagccg tgaaggtcat ttccatggtg 1140 aacccagaac tgttcgtgga aatctaccgga tgcgcactgg atcacttgga aagaatcgc 1200 aagtccaccc acatctccgc cgatctgtcc aaggtgccag aagtggaaaa ggtgaaggat 1260 gaagacctgc caggcctgtt cgaagacatc aacgtgcgcc agctgatcca cgtgacctac 1320 1380 gaaactgt tctacgaaac cgtggccaag cacatcaagc gccacgtgga cctgctgaag 1440 ggctaa 1446

Claims

1. A mutant polynucleotide encoding a fructose-4-epimerase or a fructose-4-epimerase variant, wherein the mutant polynucleotide consists of any one nucleic acid sequence selected from the group consisting of: SEQ ID NO:4 and SEQ ID NO:

6. A vector comprising the mutated polynucleotide according to claim 1 .

3. A Corynebacterium microorganism expressing a fructose-4-epimerase or a fructose-4-epimerase variant derived from Kosmotoga olearia, wherein the microorganism comprises the mutated polynucleotide according to claim 1 or a vector comprising the mutated polynucleotide.

4. A method for producing fructose-4-epimerase or a fructose-4-epimerase variant, the method comprising culturing a microorganism of the genus Corynebacterium in a culture medium, the microorganism comprising the mutated polynucleotide according to claim 1 or a vector comprising the mutated polynucleotide.

5. A composition for producing tagatose, comprising a microorganism of the genus Corynebacterium, the microorganism comprising the mutated polynucleotide of claim 1 or a vector comprising the mutated polynucleotide; or a culture of the microorganism. The tagatose-producing composition according to claim 5 , wherein the composition further comprises fructose.

7. A method for producing tagatose, comprising contacting a microorganism of the genus Corynebacterium or a culture of the microorganism with fructose, the microorganism comprising the mutated polynucleotide according to claim 1 or a vector comprising the mutated polynucleotide.

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