KR102114865B1 - Novel psicose-6-phosphate phosphatase, composition for producing psicose including the phosphatase, and method for producing psicose using the phosphatase - Google Patents

Abstract

The present application relates to a novel D-psicose-6-phosphate dephosphorylation enzyme, a composition for producing D-psicose containing the enzyme, and a method for preparing D-psicose using the enzyme.

Description

Novel Psycho-6-Phosphate Dephosphorylase, Psycho Production Composition Containing the Enzyme, Method for Producing Psycho Using the Enzyme {NOVEL PSICOSE-6-PHOSPHATE PHOSPHATASE, COMPOSITION FOR PRODUCING PSICOSE INCLUDING THE PHOSPHATASE , AND METHOD FOR PRODUCING PSICOSE USING THE PHOSPHATASE}

The present application relates to a novel D-psicose-6-phosphate dephosphorylase, a composition for producing D-psicose containing the enzyme, and a method for preparing D-psicose using the enzyme.

Psycho, a type of rare sugar, is a monosaccharide rarely found in nature. It is present in small amounts in figs, grapes, and jackfruits. Psychos have a sweetness level of about 70% of sugar, but can be used as a low-calorie sweetener because calories are only 5% of sugar. In addition, the effect of reducing body fat and regulating blood sugar is recognized.

Initially, Psycos was produced by a chemical isomerization reaction under conditions of a catalyst or an organic solvent, but the production efficiency was very low. In order to replace this, a biological method has been developed in which fructose is epimerized using epimerization enzyme to convert psychos, and to date, enzymes having such activity in various microorganism species have been reported.

Psychos-3-epimerase (EC 5.1.3.30) and tagatose-3-epimerase (D-) Tagatose-3-epimerase (EC 5.1.3.31) can be used to produce D-psicose by 3-epimerization (3-epimerization).

Alternatively, Chan et al. (2008. Biochem. 47: 9608-9617) for fructose-6-phosphate and D-psicose-6-phosphate D-ribulose-5-phosphate-3-epimerase and E. coli from Streptococcus pyogenes capable of carrying out 3-epimerization reaction Derived D-psicose-6-phosphate-3-epimerase was reported, and these enzymes were used to generate D-psicose-6-phosphate from fructose-6-phosphate. After that, psychos can be prepared through a dephosphorylation reaction. Fructose 6-phosphate, which can be used for enzymatic reactions, can be produced by various enzyme combination reactions from raw materials such as starch, maltodextrin, sucrose, and glucose.

The single enzyme reaction using the enzyme is a reversible reaction, and a certain level of reaction equilibrium exists between the fructose as a substrate and the product Psychos, and the reaction efficiency is reported to be about 20 to 35% of the substrate. Due to the reaction equilibrium of the enzyme, an additional purification process is required to separate the fructose (substrate) remaining in the enzyme reaction solution in order to prepare a high-purity psychos. Therefore, a high-efficiency and economical method for manufacturing psychos suitable for industrialization is required.

Under these backgrounds, the applicant has developed a method for producing a high efficiency of psychos, and as a result of diligent research, a novel psychos-6-phosphate dephosphorylation enzyme was discovered and the enzymes were compared to other sugars. The present application was completed by confirming that phosphoric acid was highly selectively dephosphorylated to efficiently produce psychos.

One object of the present application is derived from a strain of the genus Meioothermus (Meiothermus), characterized in that the selective dephosphorylation of psychos-6-phosphate, psychos-6-phosphate dephosphorylation enzyme (psicose-6-phosphate) phosphatase).

Another object of the present application is to provide a nucleic acid encoding the D-psicose-6-phosphate dephosphatease described herein, a vector comprising the nucleic acid and / or a transformant expressing the vector.

Another object of the present application is to provide a composition for producing psychos comprising the dephosphorylase, a transformant expressing the same, or a culture of the transformant.

Another object of the present application is to provide a method for producing psychos from fructose using a psychos-6-phosphate dephosphorylation enzyme derived from a strain of the genus Meiothermus.

Hereinafter, the contents of the present application will be described in detail as follows. On the other hand, the description and embodiment of one aspect disclosed in the present application may be applied to the description and embodiment of another aspect for common matters. Also, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, the scope of the present application is not considered to be limited by the specific descriptions described below.

In order to achieve one object of the present application, the present application provides, as an aspect, a psychos-6-phosphate dephosphorylation enzyme derived from a strain of the genus Meiothermus.

The Psychos-6-phosphate dephosphorylase of the present application may include Meiothermus silvanus, Meiothermus taiwanensis, Meiothermus timidus, Meiothermus cerbereus , Meiothermus chliarophilus, Meiothermus ruber, Meiothermus rufus, Meiothermus terrae, Meiothermus granatius, Meiothermus granaticius, Meiothermus granaticius It is selected from the group consisting of Meiothermus hypogaeus, Meiothermus luteus, Meiothermus rosaceus, Meiothermus roseus, Meiothermus cateniformans. It is characterized by being an enzyme derived from any one microorganism.

Psychos-6-phosphate dephosphorylase of the present application undergoes a dephosphorylation reaction more selectively (specifically) to Psychos-6-phosphate, glucose-1-phosphate, glucose- It may be non-specific for 6-phosphate (D-glucose-6-phosphate) or fructose-6-phosphate.

Psychos-6-phosphate dephosphorylase of the present application can be suitably expressed under the conditions of pH 5.0 to 9.0, specifically pH 6.0 to 9.0, and more specifically pH 6.5 to 8.5. In addition, the Psychos-6-phosphate dephosphorylase of the present application can be appropriately expressed in its activity at conditions ranging from 40 ° C to 80 ° C, specifically, from 40 ° C to 60 ° C or from 45 ° C to 55 ° C. have.

The molecular weight of Psychos-6-phosphate dephosphorylase in the present application may be about 20 kDa to about 28 kDa, specifically about 22 kDa to about 26 kDa, and more specifically about 24 kDa.

Psychos-6-phosphate dephosphorylase of the present application transforms the enzyme itself or a DNA expressing it (eg, the nucleotide sequence of SEQ ID NOs: 5 to 8) in the strain and cultures it to obtain a culture. And, by crushing the culture, it may be purified through a column or the like. Examples of the strain for transformation include Escherichia coli, Corynebacterum glutamicum, Aspergillus oryzae, or Bacillus subtilis.

According to the exemplary embodiment of the present application, the psychos-6-phosphate dephosphorylase of the present application is composed of any one of amino acid sequences of SEQ ID NOs: 1 to 4, or 70%, 75%, 80%, 85% of each of them , 90%, 95%, 97%, 99% or 100%, or a protein consisting of or comprising a sequence having homology or identity within a range defined by any two of the above values. Can be. For example, if the amino acid sequence having the homology or identity and consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 4 or showing the corresponding efficacy with the protein containing the sequence, some sequences are deleted, modified, substituted Or, even if it has an added amino acid sequence, it is obvious that it is included within the scope of the present application.

In addition, if the protein is made of the amino acid sequence of SEQ ID NO: 1 to 4 of the present application or has a corresponding activity with the Psycho-6-phosphate dephosphorylation enzyme containing the sequence, the amino acid sequence of any one of SEQ ID NO: 1 to 4 Does not exclude a sequence that does not have any effect on protein mutations or mutations that can occur naturally, or synonymous mutations thereof. And, the protein comprising the amino acid sequence of SEQ ID NO: 1 to 4 is also within the scope of the present application.

This application provides, as another aspect, a nucleic acid encoding the Psychos-6-phosphate dephosphorylation enzyme of the present application.

The term "nucleic acid" in the present application has the meaning of comprehensively including a DNA or RNA molecule, and a nucleotide which is a basic structural unit in a nucleic acid may include not only natural nucleotides, but also sugar or base site modified analogs (see Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

Specifically, the nucleic acid encoding the Psychos-6-phosphate dephosphorylation enzyme of the present application may be made of any one of the nucleotide sequences of SEQ ID NOs: 5 to 8 or include the above sequence, and more specifically, the sequence of the present application It may be a nucleotide sequence having at least 70%, 80%, 90%, 95%, 97%, or 99% homology or identity with the nucleotide sequence of any one of numbers 5 to 8. Conucleotide degeneracy (codon degeneracy) consisting of any one of the amino acid sequence of SEQ ID NO: 1 to 4 or a polynucleotide that can be translated into a protein comprising the sequence or a protein having homology or identity therewith also of the present application It is obvious that it can be included in the scope.

Or consists of any one of the amino acid sequence of SEQ ID NO: 1-4 of the present application is an enzyme comprising the sequence, Mayo sseomeoseu Silva Nurse (Meiothermus silvanus), Mayo sseomeoseu Taiwan cis norbornene (Meiothermus taiwanensis), It is characterized by an enzyme derived from any one microorganism selected from the group consisting of Meiothermus timidus or Meiothermus cerbereus .

In the present application, the term 'homology' or 'identity' refers to the degree of correlation with two given amino acid sequences or nucleotide sequences and may be expressed as a percentage.

The terms homology and identity can often be used interchangeably.

The sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, and default gap penalties established by the program used can be used together. Substantially, homologous or identical sequences are generally at least about 50%, 60%, 70%, 80% of the entire or full-length sequence in medium or high stringent conditions. Or it can hybridize to 90% or more. Hybridization also contemplates polynucleotides containing degenerate codons instead of codons in the polynucleotide.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity, see, eg, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: Using the default parameters as in 2444, it can be determined using known computer algorithms such as the "FASTA" program. Or, as performed in the Needleman program of 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), It can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453). (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215] : 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA /.] (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.

The homology, similarity or identity of a polynucleotide or polypeptide is, for example, Smith and Waterman, Adv. Appl. As known in Math (1981) 2: 482, for example, Needleman et al. (1970), J Mol Biol. 48: 443 can be determined by comparing sequence information using a GAP computer program. In summary, the GAP program defines the total number of symbols in the shorter of the two sequences, divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). The default parameters for the GAP program are (1) a binary comparison matrix (contains values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: Weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or gap open penalty 10, gap extension penalty 0.5); And (3) no penalty for the end gap. Thus, as used herein, the term “homology” or “identity” refers to relevance between sequences.

In another aspect of the present application, the vector comprising the nucleic acid encoding the psychos-6-phosphate dephosphorylase described herein, or the nucleic acid encoding the psychos-6-phosphate dephosphatase of the present application or the present application Provided is a transformant comprising a vector comprising a nucleic acid encoding Psychos-6-phosphate dephosphorylase.

The term "vector" in this application refers to any medium for cloning and / or transfer of a base to an organism, such as a host cell. The vector may be a replication unit (replicon) capable of binding to other DNA fragments to obtain replication of the combined fragments. Herein, a “replica unit” is any genetic unit (eg, plasmid, phage, cosmid, chromosome, virus) that functions as an autologous unit of DNA replication in vivo, ie, is capable of replicating by self-regulation. Says The term "vector" includes viral and non-viral mediators for introducing a base into an organism, such as a host cell, in vitro, ex vivo or in vivo. The term "vector" can also include minispherical DNA. Specifically, the vector containing the nucleic acid encoding the Psychos-6-phosphate dephosphorylase of the present application is pET24a (+)-Mcer-A6Pase, pET24a (+)-Mtai-A6Pase, pET24a (+)-Msil-A6Pase Or pET24a (+)-Mtim-A6Pase.

The term "transformation" in the present application means that a vector containing a nucleic acid encoding a target protein is introduced into a host cell so that the protein encoding the nucleic acid can be expressed in the host cell. Recombinant nucleic acids can include both, as long as they can be expressed in the host cell, whether inserted into the host cell's chromosome or located outside the chromosome. In addition, the nucleic acid includes DNA and RNA encoding a target protein. The nucleic acid may be introduced into a host cell and expressed, so long as it is introduced in any form. For example, the nucleic acid may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all elements necessary for self-expression. The expression cassette may include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, which are operably linked to the nucleic acid. The expression cassette may be in the form of an expression vector capable of replicating itself. In addition, the nucleic acid may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

In addition, the term "operably linked" in the above means that the promoter sequence and the gene sequence to initiate and mediate transcription of the nucleic acid encoding the target protein of the present application are functionally linked.

The method of transforming with the vector of the present application includes any method of introducing a nucleic acid into a cell, and can be performed by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, retroviral infection, microinjection, polyethylene glycol (PEG), DEAE-dex Tran method, cationic liposome method, and lithium acetate-DMSO method, but are not limited thereto.

As the host cell, it is preferable to use a host having a high DNA introduction efficiency and a high expression efficiency of the introduced DNA, for example, a microorganism of the genus Corynecaceae, a microorganism of the genus Escherichia or a genus of Serratia, and specifically E. coli , but is not limited thereto.

The transformants of the present application are E. coli BL21 (DE3) / Mcer-A6Pase, E. coli BL21 (DE3) / Mtai-A6Pase, E. coli BL21 (DE3) / Msil-A6Pase or E. coli BL21 (DE3) / Mtim-A6Pase.

As another example of the present application, there is provided a composition for producing psychos comprising the psychos-6-phosphate dephosphorylation enzyme, a microorganism expressing the same, or a culture of the microorganism.

The composition for the production of psychos may include an enzyme and / or a substrate [(i) starch, maltodextrin, sucrose, or a combination thereof involved in the psychos production pathway; (ii) phosphate; (iii) fructose-6-phosphate-3-epimerase; (iv) glucose-6-phosphate-isomerase; (v) phosphoglucomutase or glucose phosphorylase; And / or (vi) α-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase, sucrose phosphorylase, α-amylase, pullulanase, isoamylase, glucoamylase or sucrase ]; Microorganisms expressing enzymes involved in the psychos production pathway; Or it may additionally include a culture of microorganisms expressing the enzymes involved in the psychos production pathway. However, this is exemplary, and if it is possible to produce psychos using the psychos-6-phosphate dephosphorylation enzyme of the present application, the enzymes included in the composition for producing psychos of the present application and the substrate used for the production of psychos are limited Does not work.

Starch / maltodextrin phosphorylase (EC 2.4.1.1) and α-glucan phosphorylase of the present application phosphorylate phosphate to glucose to transfer glucose from starch or maltodextrin to glucose-1- Any protein that has a phosphoric acid producing activity may be included. Specifically, the protein having the activity of producing glucose-1-phosphate from the starch or maltodextrin may be a protein consisting of the amino acid sequence of SEQ ID NO: 17. In addition, the protein having the activity of producing glucose-1-phosphate from the starch or maltodextrin can be encoded by the base sequence of SEQ ID NO: 18.

Sucrose phosphorylase (EC 2.4.1.7) of the present application may include any protein as long as it is a protein having the activity of phosphorylating phosphate to glucose to produce glucose-1-phosphate from sucrose.

 The starch liquor saccharifying enzymes of the present application are α-amylase (a-3.2), pullulanase (EC 3.2.1.41), glucoamylase (EC-3.2.1.3) and isoamylase (isoamylase). Can include any protein as long as it has the activity of converting starch or maltodextrin to glucose.

Sucrase (EC 3.2.1.26) of the present application may include any protein as long as it has a protein that converts sucrose into glucose.

The phosphoglucomutase (EC 5.4.2.2) of the present application may include any protein as long as it has the activity of converting glucose-1-phosphate to glucose-6-phosphate. Specifically, the protein having the activity of converting glucose-1-phosphate to glucose-6-phosphate may be a protein consisting of the amino acid sequence of SEQ ID NO: 19. In addition, the protein having the activity of converting the glucose-1-phosphate to glucose-6-phosphate can be encoded by the base sequence of SEQ ID NO: 20.

Glucose phosphatase (glucokinase) may include any protein as long as it has the activity of transferring phosphoric acid to glucose and converting it to glucose-6-phosphate. Specifically, the glucose phosphatase may be a polyphosphate-dependent glucose phosphatase and more specifically, a protein consisting of the amino acid sequence of SEQ ID NO: 21 or 22. Glucose-6-phosphate-isomerase of the present application may include any protein as long as it has the activity of converting glucose-6-phosphate to fructose-6-phosphate. Specifically, the glucose-6-phosphate-isomerase may be a protein consisting of the amino acid sequence of SEQ ID NO: 23. In addition, the glucose-6-phosphate-isomerase can be encoded by the base sequence of SEQ ID NO: 24.

The fructose-6-phosphate-3-epimerase of the present application may include any protein as long as it has the activity of converting fructose-6-phosphate to psychos-6-phosphate. Specifically, the fructose-6-phosphate-3-epimerase of the present application may be a protein consisting of the amino acid sequence of SEQ ID NO: 25. In addition, the fructose-6-phosphate-3-epimerase of the present application can be encoded by the nucleotide sequence of SEQ ID NO: 26.

The composition for producing psychos of the present application may further include a protein (eg, 4-a-glucanotransferase, etc.) having the activity of converting glucose into starch, maltodextrin, or sucrose. The protein having the activity of converting the glucose into starch, maltodextrin, or sucrose may be an enzyme consisting of the amino acid sequence of SEQ ID NO: 27, and may be specifically encoded by the nucleotide sequence of SEQ ID NO: 28. The composition for producing psychos herein may further include any suitable excipients commonly used in the composition for producing psychos. Such excipients may include, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizers, and isotonic agents, but are not limited thereto.

The composition for producing psychos herein may further include a metal ion or a metal salt. In one embodiment, the metal may be at least one metal selected from the group consisting of Ni, Mg, Co, Mn, Fe and Zn. More specifically, the composition for producing psychos of the present application may further include a metal salt, and more specifically, the metal salt may be NiSO ₄ , MgSO ₄ , MgCl ₂ , NiCl ₂ , CoSO ₄ , CoCl ₂ , MnCl ₂ , It may be one or more selected from the group consisting of FeSO ₄ and ZnSO ₄ .

In another aspect of the present application, Psychos -6-phosphate is contacted with Psychos -6-phosphate dephosphorylation enzyme derived from a strain of the genus Meiothermus, a microorganism expressing the same, or a culture of the microorganism. It relates to a method for producing a psychos comprising the step of converting the psychos-6-phosphoric acid into a psychos.

In the above-described aspect, matters relating to psychos-6-phosphate dephosphorylation enzyme can be applied in the same manner in the psychos production method of the present embodiment.

Specifically, the Mousse mate authoring spp derived psicose-6-phosphate is dephosphorylated enzyme Mayo sseomeoseu Silva Nurse (Meiothermus silvanus), Mayo sseomeoseu Taiwan N-Sys (Meiothermus taiwanensis), Meiothermus timidus , Meiothermus cerbereus , Meiothermus chliarophilus , Meiothermus ruber , Meiothermus ruber ( Meiothermus rufus ) , Meiothermus terrae , Meiothermus granaticius , Meiothermus hypogaeus , Meiothermus luteus , Meiothermus rusus , Meiothermus rosace ) , Meiothermus roseus, and It may be an enzyme derived from any one microorganism selected from the group consisting of Meiothermus cateniformans .

Psychos-6-phosphate dephosphorylation enzyme in the preparation method of the present application may be an enzyme consisting of or comprising an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 described herein.

The method of the present application is fructose-6-phosphate (fructose-6-phosphate) fructose-6-phosphate-3-epimerase, prior to the step of converting psychos-6-phosphate to psychos, the fructose-6- Converting the fructose-6-phosphate to D-psicose-6-phosphate by contacting a culture of a microorganism expressing a phosphate-3-epimerase or a microorganism expressing the fructose-6-phosphate-3-epimerase. It may further include a step.

The method of the present application is glucose-6-phosphate-isomerase, glucose-6 to glucose-6-phosphate before the step of converting the fructose-6-phosphate to psychos-6-phosphate -Contacting a culture of a microorganism expressing phosphate-isomerase or a microorganism expressing the glucose-6-phosphate-isomerase, further comprising converting the glucose-6-phosphate to fructose-6-phosphate Can be.

In the method of the present application, before the step of converting the glucose-6-phosphate to fructose-6-phosphate, the phosphoglucomutase and the phosphoglucomutase are expressed in glucose-1-phosphate. It may further include the step of converting the glucose-1-phosphate to glucose-6-phosphate by contacting a microorganism or a culture of the microorganism expressing the phosphoglucomutase.

In the method of the present application, before the step of converting the glucose-6-phosphate to fructose-6-phosphate, the glucose phosphorylase in glucose, the microorganism expressing the glucose phosphorylase or the microorganism expressing the glucose phosphorylase Contacting the culture, and phosphate, may further include the step of converting the glucose to glucose-6-phosphate.

The method of the present application is α-glucan phosphorylase, starch phosphorylase, maltodextrin in starch, maltodextrin, sucrose, or a combination thereof before the step of converting the glucose-1-phosphate to glucose-6-phosphate. Phosphorylase or sucrose phosphorylase; Microorganisms expressing the phosphorylase; Alternatively, the method may further include converting the starch, maltodextrin, sucrose, or a combination thereof to glucose-1-phosphate by contacting a culture of a microorganism expressing the phosphorylase and phosphate.

The method of the present application includes α-amylase, pullulanase, glucoamylase, sucraase or isoamylase in starch, maltodextrin, sucrose or a combination thereof before the step of converting the glucose into glucose-6-phosphate; Microorganisms expressing the α-amylase, pullulanase, glucoamylase, sucrase or isoamylase; Or further comprising the step of converting the starch, maltodextrin, sucrose, or a combination thereof to glucose by contacting a culture of a microorganism expressing the α-amylase, pullulanase, glucoamylase, sucrase or isoamylase. can do.

In another aspect of the present application, starch, maltodextrin, sucrose, or a combination thereof, and phosphate (a) inositol-mono-phosphatase; Fructose-6-phosphate-3-epimerase; Glucose-6-phosphate-isomerase; Phosphoglucomutase or glucose phosphorylase; And α-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase, sucrose phosphorylase, α-amylase, pullulanase, isoamylase, glucoamylase or sucrase; Or (b) contacting a microorganism expressing the enzyme of item (a) or a culture of the microorganism, to provide a method for manufacturing psychos.

 In the preparation of Psychos according to the manufacturing method of the present application, contact of the enzyme with each substrate may be performed under conditions of pH 5.0 to 9.0, specifically pH 6.0 to 9.0, and more specifically pH 6.5 to 8.5. In addition, the contact of the present application is 30 ℃ to 80 ℃, 35 ℃ to 80 ℃, 40 ℃ to 80 ℃, 50 ℃ to 80 ℃, 55 ℃ to 80 ℃, 60 ℃ to 80 ℃, 30 ℃ to 70 ℃, 35 ℃ to 70 ℃, 40 ℃ to 70 ℃, 45 ℃ to 70 ℃, 50 ℃ to 70 ℃, 55 ℃ to 70 ℃, 60 ℃ to 70 ℃, 30 ℃ to 65 ℃, 35 ℃ to 65 ℃, 40 ℃ to 65 ℃, 45 ℃ to 65 ℃, 50 ℃ to 65 ℃, 55 ℃ to 65 ℃, 30 ℃ to 60 ℃, 35 ℃ to 60 ℃, 40 ℃ to 60 ℃, 45 ℃ to 60 ℃, 40 ℃ to 55 ℃ Or it may be carried out at a temperature of 45 ℃ to 55 ℃. In addition, the contact of the present application is 0.1 to 36 hours, 0.1 to 24 hours, 0.1 to 12 hours, 0.1 to 6 hours, 0.2 to 36 hours, 0.2 to 24 hours, 0.2 For hours to 12 hours, 0.2 hours to 6 hours, 0.3 hours to 36 hours, 0.3 hours to 24 hours, 0.3 hours to 12 hours, 0.3 hours to 6 hours, 0.4 hours to 36 hours, 0.4 hours For 24 hours, 0.4 hours to 12 hours, 0.4 hours to 6 hours, 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 For 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 For 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 For 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.

The contact conditions can be applied, for example, in the manufacture of Psychos by Psychos-6-phosphate dephosphorylation enzyme. The contacting can be performed in the presence of a buffer solution, for example, sodium phosphate or Tris-HCl buffer solution can be used. More specifically, the contact with the psychos-6-phosphate dephosphorylation enzyme can be carried out in pH 6 to 9 conditions, specifically in Tris-HCl buffer solution.

The method of the present application may further include the step of purifying the psychos. The tablet of the present application is not particularly limited, and a method commonly used in the technical field of the present application may be used. Non-limiting examples include chromatography, fractional crystallization, and ion purification. Only one purification method may be performed, or two or more methods may be performed together. For example, a psychos-generated reactant may be purified through chromatography, and separation of sugars by chromatography may be performed using a difference in a weak binding force between a sugar to be separated and a metal ion attached to an ionic resin. have.

In addition, the present application may further include performing decolorization, desalting, or both before or after the purification step of the present application. By performing the decolorization and / or desalination, a more purified psychos reactant can be obtained without impurities.

The novel D-psicose-6-phosphate dephosphorylation enzyme of the present application has heat resistance, so that a path for converting D-psicose-6-phosphate to D-psicose can be industrially performed, and is an economical raw material such as glucose or starch (eg, maltose). Dextrin) is used to enable the progress of the psychos synthesis pathway, and it is possible to significantly increase the conversion to psychos by allowing the production of psychos by the irreversible reaction pathway, the psychos-6-phosphate dephosphorylation reaction.

In addition, the method for manufacturing D-psicose of the present application includes a high-dose D-psicose reaction product according to an increase in D-psicose conversion rate because D-psicose 6-phosphate dephosphorylation enzyme having high substrate specificity for D-psicose-6 is used. As the separation and purification process can be simplified or eliminated, the manufacturing method is simple and economical.

Figure 1 shows the expression and purification of Psycho-6-phosphate dephosphorylation recombinase (SM: protein size marker, Mcer: pET24a (+)-Mcer-A6Pase, Mtai: pET24a (+)-Mtai-A6Pase, Msil: pET24a ( +)-Msil-A6Pase, Mtim: pET24a (+)-Mtim-A6Pase) is the result of protein electrophoresis (SDS-PAGE) analysis.
FIG. 2 shows recombinant enzymes (Mcer: Mcer-A6Pase, Mtai: Mtai-A6Pase, Msil: Msil-A6Pase, Mtim: Mtim-A6Pase, Kpn: Klebsiella pneumonia derived from Picos-6-phosphate dephosphorylation reaction) haloacid dehalogenase-like hydrolase) is a comparison result of dephosphorylase activity (%) against Psychos 6-phosphate.
Figure 3 dephosphorylation enzyme activity of the purified recombinant enzymes (Mcer: Mcer-A6Pase, Mtai: Mtai-A6Pase, Msil: Msil-A6Pase, Mtim: Mtim-A6Pase) at various pH conditions for Psycho 6-phosphate (%).

Hereinafter, the present application will be described in more detail through examples and experimental examples. However, these examples and experimental examples are for illustrative purposes only, and the scope of the present application is not limited to these examples and experimental examples.

Example

Example 1: Preparation of a transforming strain producing heat-resistant Psychos-6-phosphate dephosphorylation enzyme

Genes expected to have dephosphorylation enzyme activity to produce psychos by dephosphorylating psychos -6-phosphate from microorganisms of the genus Meiothermus were selected, and recombinant expression vectors and transformants including the genes were prepared.

Specifically, the make-authoring mousse in Mayo sseomeoseu theta Beretta mouse (Meiothermus cerbereus) Microbe registered in Genbank DSM11376, Mayo sseomeoseu Taiwan N-Sys (Meiothermus taiwanensis) DSM14542, Mayo sseomeoseu Silva Nurse (Meiothermus silvanus) DSM9946 and Mayo sseomeoseu thymidine Ranges (Meiothermus timidus ) The gene sequence of DSM17022 is selected, and a gene expected to be a psychos-6-phosphate dephosphorylation enzyme is selected, and the amino acid sequence of the gene (SEQ ID NO: 1, 2, 3, 4) and nucleotide sequence (SEQ ID NO: 5, 6, 7, 8) Based on the information, forward primers (SEQ ID NOs: 9, 10, 11, 12) and reverse primers (SEQ ID NOs: 13, 14, 15, 16) were designed and synthesized.

PCR reaction (genomic DNA of M. cerbereus DSM11376, M taiwanensis DSM14542, M. silvanus DSM9946, and M. timidus DSM17022 using the synthesized primer as a template (1 minute at 94 ° C, 30 seconds at 58 ° C, Genes were amplified by conducting one cycle at 72 ° C for one cycle (32 cycles). The amplified gene was purified using a PCR purification kit (purification kit, Quiagen), and inserted into the pET24a (+) (novagen, USA) vector using restriction enzymes Nde I and xho I to synthesize a recombinant expression vector. Recombinant expression vectors named pET24a (+)-Mcer-A6Pase, pET24a (+)-Mtai-A6Pase, pET24a (+)-Msil-A6Pase and pET24a (+)-Mtim-A6Pase were synthesized.

The recombinant vector was transformed into E. coli BL21 (DE3) by heat shock transformation (Sambrook and Russell: Molecular cloning, 2001), and then stored frozen in 50% glycerol. The transformed strains were E. coli BL21 (DE3) / Mcer-A6Pase, E. coli BL21 (DE3) / Msil-A6Pase, E. coli BL21 (DE3) / Mtai-A6Pase and E. coli BL21 (DE3) / Mtim -A6Pase, respectively, and deposited on November 22, 2017 at the Korean Culture Center of Microorganisms (KCCM), an international depositary organization under the Budapest Treaty, and received accession numbers KCCM12171P, KCCM12172P, KCCM12173P, and KCCM12174P, respectively. .

Example 2: Preparation of enzymes and substrates required for Psychos manufacturing pathway

Example 2-1: Enzyme Preparation Required for Psycho Production Path

Thermo-accelerated microorganism derived α-glucan phosphorylase, α-glucan phosphorylase, phosphoglucomutase, and glucose-6-phosphate-isomerase required for the Psychos manufacturing route of the present application Genes predicted by the enzymes to provide -6-phosphate-isomerase) and fructose-6-phosphate-3-epimerase (ct1 respectively in the order of description of the enzymes) , ct2, tn1 and fp3e).

Forward based on the base sequence of the selected gene [SEQ ID NOs: 18, 20, 24 and 26, respectively, in the order of description of the enzymes] and amino acid sequence [SEQ ID NOs: 17, 19, 23, and 25, respectively, in the order of description of the enzymes] Forward primers SEQ ID NOs: 29, 31, 33 and 35 and reverse primers SEQ ID NOs: 30, 32, 34 and 36 were devised and synthesized, and using them, Thermomoto Neapolitana or Thermomoto Mari The chromosomal DNA of Tima was used as a template and polymerized by PCR (PCR), denatured for 30 seconds at 95 ° C, annealed for 30 seconds at 55 ° C, and polymerized for 2 minutes at 68 ° C. Genes of each enzyme were amplified using conditions repeated once. Each amplified enzyme gene was inserted into the plasmid vector pET21a (Novagen) for E. coli expression using restriction enzymes Nde and Xho, respectively, and recombinant expression vectors named pET21a-CJ_ct1, pET21a-CJ_ct2, pET21a-CJ_tn1 and pET21a-CJ_fp3e, respectively. Was produced. E. coli BL21 (DE3) / pET21a-CJ_ct1 (KCCM11990P) was prepared by transforming the recombinant expression vector into an E. coli BL21 (DE3) strain by a conventional transformation method (Sambrook et al. 1989). ), E. coli BL21 (DE3) / pET21a-CJ_ct2 (KCCM11991P), E. coli BL21 (DE3) / pET21a-CJ_tn1 (KCCM11992P) and E. coli BL21 (DE3) / pET21a-CJ_fp3e (KCCM11848P). The strains were deposited with the Korean Culture Center of Microorganisms (KCCM) on March 20, 2017 under the Budapest Treaty, and were assigned accession numbers of KCCM11990P, KCCM11991P, and KCCM11992P, respectively, as of June 23, 2016. It has been deposited with the Korean Culture Center of Microorganisms (KCCM) and has been assigned a trust number KCCMP11848P.

Example 2-2: Preparation of substrate required for Psychos manufacturing route

From the starch, maltodextrin, sucrose, glucose-1-phosphate, glucose, glucose-6-phosphate, fructose-6-phosphate, it is possible to obtain psychos-6-phosphate, the substrate of the enzyme of the present application. It is known to those skilled in the art that conversion is possible between the liver. Accordingly, it was confirmed that psychos-6-phosphate was produced based on various substrates.

Specifically, the process of converting sucrose to glucose by treating enzymes such as sucrase is already known. As a result, Thermomotoga neapolitana heat-resistant α-glucan phosphorylase (SEQ ID NO: 17) ) To confirm the production of glucose-1-phosphate from starch and maltodextrin.

The produced glucose was treated with heat-resistant glucose phosphatase (SEQ ID NO: 21) derived from Deinococcus geothermalis , heat-resistant glucose phosphatase (SEQ ID NO: 22) derived from Anaerolinea thermophila , and glucose It was confirmed that the conversion of thiophosphate into glucose-6-phosphoric acid was performed by treating the phosphatase with heat-resistant phosphoglucomutase (SEQ ID NO: 19) derived from Neapolitana ( Thermotoga neapolitana ).

The prepared glucose-6-phosphate was treated with heat-resistant glucose-6-phosphate-isomerase (SEQ ID NO: 23) derived from Thermotoga maritima to confirm that fructose-6-phosphate was produced. Ethermoto was treated with heat-resistant fructose-6-phosphate-3-epimerase (SEQ ID NO: 25) derived from Neapolitana ( Thermotoga neapolitana ) to produce Psychos-6-phosphate, the substrate of the enzyme of the present invention. Confirmed.

Through this, it was confirmed that the enzyme can be converted to psychos-6-phosphoric acid by treating enzymes on various substrates, and the step of obtaining psychos-6-phosphoric acid from various substances was obtained using the enzymes of the present invention. Obviously, it can be carried out before the conversion of 6-phosphoric acid to psychos.

Example 3: Production and purification of heat-resistant Psychos-6-phosphate dephosphorylation enzyme

E. coli BL21 (DE3) / Mcer-A6Pase, E. coli BL21 (DE3) / Mtai-A6Pase, E. coli BL21 (DE3) / Msil-A6Pase and E. coli BL21 (DE3) prepared in Example 1 above In order to produce D-psicose-6-phosphate dephosphorylation enzyme from / Mtim-A6Pase, 4 types of E. coli BL21 (DE3) were inoculated into 5 ml medium (Difco) of LB-kanamycin and measured at 600 nm. The mixture was shaken at 37 ° C and 200 rpm until one absorbance reached 1.5. Subsequently, the shaking culture was inoculated into 500 ml LB-kanamycin medium, and when 0.5 nm IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added when absorbance at 600 nm was 0.7, 37 ° C. and 150 rpm. Incubated for 16 hours under conditions.

The cultured culture medium was centrifuged at 8000 rpm for 20 minutes to recover only the cells, washed twice with 0.85% (w / v) NaCl, and then dissolved buffer (lysis buffer, 50 mM Tris-HCl, pH 7.0, 300). mM NaCl) and then crushed at 4 ° C. for 20 minutes using a sonicator. The supernatant was recovered by centrifuging the crushing solution at 13,000 rpm for 4 to 20 minutes, and then applied to a Ni-NTA column (Ni-NTA Superflow, Qiagen) equilibrated with the lysis buffer in advance, and 250 mM imidazole (imidazole). ) Containing buffer solution (50 mM Tris-HCl, 300 mM NaCl, pH 7.0) was sequentially flowed to obtain purified Psychos-6-phosphate dephosphorylation enzyme. The four types of Mcer-A6Pase, Mtai-A6Pase, Msil-A6Pase, and Mtim-A6Pase confirmed that the size of the monomer was about 24 kDa (FIG. 1).

Example 4: Confirmation of the activity of heat-resistant Psychos-6-phosphate dephosphorylation enzyme

Are four dephosphorylation enzymes Mcer-A6Pase, Mtai-A6Pase, Msil-A6Pase, and Mtim-A6Pase produce dephosphorylated psychos using psychos-6-phosphate as a substrate, and are specific for psychos-6-phosphates Was confirmed.

Since it is difficult to purchase the Psycos 6-phosphate reagent, the enzyme reaction of A6Pase without purification is performed after producing Psycos 6-phosphate from fructose 6-phosphate using fructose 6-phosphate epimerization enzyme. It was used as a substrate. The reaction conditions for fructose 6-phosphate epimerase were fructose 6-phosphate epimerase in 50 mM Tris-HCl buffer (pH 7.0) containing 1% D-fructose 6-phosphate and 0.1 mM ZnSO ₄ Was added and reacted at 50 ° C for 18 hours. After the reaction, the solution contained fructose 6-phosphate and Psychos 6-phosphate at about 0.7 wt% and 0.3 wt%, respectively. 50% by weight of fructose 6-phosphate epimerase reaction solution (a mixture of fructose 6-phosphate and D-psicose 6), 50% by weight of 50 mM Tris-HCl buffer solution (pH 7.0) with 0.1 mM MgCl ₂ To each Psycho 6-phosphate deglycosylating enzyme was added and reacted at 50 ° C for 30 minutes.

To evaluate the activity of the enzyme on glucose 1-phosphate, each of the psychos 6-phosphate prepared above in 50 mM Tris-HCl buffer solution (pH 7.0) containing 0.5% by weight glucose 1-phosphate, 0.1 mM MgCl ₂ Deglycosylating enzyme was added and reacted at 50 ° C for 30 minutes.

Thereafter, the reaction was stopped by heating at 100 ° C. for 5 minutes, and then Psycho generation was confirmed through HPLC analysis. For the HPLC analysis, a Refractive Index Detector (Agilent 1260 RID) of HPLC (Agilent, USA) equipped with an Aminex HPX-87C column (BIO-RAD) was used, and a mobile phase solvent was water, temperature was 80 ° C., flow rate Was performed at 0.6 ml / min. Enzyme activity (%) on the phosphorylated substrate was calculated as 'amount of dephosphorylation product / amount of phosphorylated substrate X 100'. As a control, haloacid dehalogenase-like hydrolase (Kpn HAD) from Klebsiella pneumonia , which dephosphorylates Psychos 6-phosphate, was used.

As a result, the dephosphorylation titer of psychos 6-phosphoric acid was confirmed for the four dephosphorylation enzymes Mcer-A6Pase, Mtai-A6Pase, Msil-A6Pase, and Mtim-A6Pase, and the previously known psychos-6-phosphate dephosphorylation enzyme Even when compared with Kpn HAD, it was confirmed that it exhibits a higher dephosphorylation reactivity to Psychos-6-phosphate (FIG. 2). In addition, it was confirmed that the enzyme of the present invention has little selectivity and specificity for D-psicose-6-phosphate compared to other sugars because there is little dephosphorylation activity for glucose-1-phosphate.

Example 5: Dephosphorylation enzyme activity according to pH

The activity was compared at various pH ranges (pH 6-9) to determine the effect of the reaction solution on the activity of four dephosphorylation enzymes (Mcer-A6Pase, Mtai-A6Pase, Msil-A6Pase, and Mtim-A6Pase). The activity was measured in the same manner as in Example 3 except for the buffer solution used for the enzyme reaction. Sodium phosphate buffer solution at pH 6 and 7, and Tris-HCl buffer solution at pH 7, 8 and 9 were used, and the concentration of the buffer solution was 50 mM.

As a result, the four kinds of dephosphorylation enzymes (Mcer-A6Pase, Mtai-A6Pase, Msil-A6Pase, and Mtim-A6Pase) were compared to sodium phosphate buffer solution, compared to Tris-HCl buffer solution to dephosphorylation activity of psychos-6-phosphate. This was high. In addition, while Mcer-A6Pase and Mtai-A6Pase showed the highest activity at pH 7, Msil-A6Pase and Mtim-A6Pase had the highest titer at pH 8 (FIG. 3).

From the above description, those skilled in the art to which the present application pertains will appreciate that the present application may be implemented in other specific forms without changing the technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present application should be construed as including all changes or modifications derived from the meaning and scope of the following claims rather than the detailed description and equivalent concepts thereof, which are included in the scope of the present application.

Korea Microbial Conservation Center (Overseas) KCCM12171P 20171122 Korea Microbial Conservation Center (Overseas) KCCM12172P 20171122 Korea Microbial Conservation Center (Overseas) KCCM12173P 20171122 Korea Microbial Conservation Center (Overseas) KCCM12174P 20171122 Korea Microbial Conservation Center (Overseas) KCCM11990P 20170320 Korea Microbial Conservation Center (Overseas) KCCM11991P 20170320 Korea Microbial Conservation Center (Overseas) KCCM11992P 20170320 Korea Microbial Conservation Center (Overseas) KCCM11848P 20160623

<110> CJ CHEILJEDANG CORPORATION <120> NOVEL PSICOSE-6-PHOSPHATE PHOSPHATASE, COMPOSITION FOR PRODUCING          PSICOSE INCLUDING THE PHOSPHATASE, AND METHOD FOR PRODUCING          PSICOSE USING THE PHOSPHATASE <130> KPA180998-KR-P1 <150> KR 10-2017-0167873 <151> 2017-12-08 <160> 36 <170> KoPatentIn 3.0 <210> 1 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Amio acid sequence of psicose 6-phosphatase from Meiothermus          cerbereus <400> 1 Met Lys Leu Lys Ala Leu Leu Phe Asp Leu Asp Gly Thr Leu Ala Asp   1 5 10 15 Thr Asp Arg Leu His Glu Gln Ala Trp Leu Glu Gly Leu Ser Arg Tyr              20 25 30 Gly Leu Gln Gly Asp His Thr Phe Tyr Gln Thr Gln Ile Ser Gly Gly          35 40 45 Leu Asn Pro Glu Ile Val Gln Arg Leu Leu Pro Gln Leu Ser Gln Ala      50 55 60 Glu Ala Glu Ala Phe Leu Glu Gln Lys Glu Ala Arg Phe Arg Glu Leu  65 70 75 80 Ala Ser Glu Val Gln Pro Leu Pro Gly Leu Gly Thr Leu Trp Asp Trp                  85 90 95 Ala Gln Glu Gln Asn Leu Arg Arg Ala Leu Val Ser Asn Ala Pro Arg             100 105 110 Glu Asn Ala Gln Tyr Leu Leu Lys Arg Leu Gly Leu Val Phe Asp His         115 120 125 Ile Val Leu Ser Glu Glu Leu Pro Ala Gly Lys Pro Asp Pro Leu Pro     130 135 140 Tyr Arg Thr Ala Leu Gln Ala Leu Asn Ile Gly Pro Ser Glu Ala Leu 145 150 155 160 Ala Phe Glu Asp Ser Pro Ser Gly Val Arg Ser Ala Val Gly Ala Gly                 165 170 175 Ile Arg Thr Val Ala Leu Thr Thr Gly His Pro Ala His Ala Leu Glu             180 185 190 Gln Ala Gly Ala Phe Leu Cys Ile Pro Asn Phe Ala Asp Pro Arg Leu         195 200 205 Trp Ala Tyr Leu His Lys Met Gly     210 215 <210> 2 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of psicose 6-phosphatase from Meiothermus          taiwanensis <400> 2 Met Lys Leu Arg Ala Leu Leu Phe Asp Leu Asp Gly Thr Leu Ala Asp   1 5 10 15 Thr Asp Arg Leu His Glu Gln Ala Trp Leu Glu Gly Leu Ser Arg Tyr              20 25 30 Gly Ile Gln Gly Asp His Arg Phe Tyr Gln Ala Gln Ile Ser Gly Gly          35 40 45 Leu Asn Pro Glu Ile Val Ala Arg Leu Leu Pro Gln Leu Ser Pro Asp      50 55 60 Glu Gln Val Ala Phe Ile Glu Gln Lys Glu Ala Arg Phe Arg Glu Leu  65 70 75 80 Ala Ser Glu Val Gln Pro Leu Pro Gly Leu Arg Val Leu Trp Asp Trp                  85 90 95 Ala Gln Ser Gln Gly Leu Arg Arg Ala Leu Val Ser Asn Ala Pro Arg             100 105 110 Glu Asn Ala His Tyr Leu Leu Glu Arg Leu Gly Leu Met Phe Asp Ala         115 120 125 Ile Val Leu Ser Glu Asp Leu Pro Ala Gly Lys Pro Asp Pro Leu Pro     130 135 140 Tyr Arg Thr Ala Leu Gln His Leu Asn Ile Gly Pro Gln Glu Ala Leu 145 150 155 160 Ala Phe Glu Asp Ser Pro Ser Gly Val Arg Ser Ala Val Gly Ala Gly                 165 170 175 Leu Arg Thr Val Ala Leu Thr Thr Gly His Pro Pro His Ala Leu Glu             180 185 190 Gln Ala Gly Ala Phe Leu Cys Ile Pro Asp Phe Thr Asp Pro Arg Leu         195 200 205 Trp Asp Trp Leu Gln Lys Met Gly     210 215 <210> 3 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of psicose 6-phosphatase from Meiothermus          silvanus <400> 3 Met Arg Ala Leu Leu Phe Asp Leu Asp Gly Thr Leu Ala Asn Thr Asp   1 5 10 15 Arg Leu His Glu Gln Ala Trp Leu Glu Thr Leu Arg Phe Tyr Gly Ile              20 25 30 Glu Gly Asp His His Phe Tyr Gln Thr Gln Ile Ser Gly Gly Leu Asn          35 40 45 Pro Glu Ile Val Arg Arg Leu Leu Pro Gln Leu Ser Glu Ala Glu Gly      50 55 60 Glu Ala Phe Ile Ala Arg Lys Glu Arg Arg Phe Arg Glu Leu Ala Gln  65 70 75 80 Asp Leu Arg Ala Leu Pro Gly Leu Asp Ala Leu Leu Ala Trp Ala Arg                  85 90 95 Arg Lys Lys Leu Leu Thr Gly Leu Val Thr Asn Ala Pro His Glu Asn             100 105 110 Ala Arg His Val Thr Gln Ala Leu Gly Leu Ser Phe Asp Val Val Val         115 120 125 Leu Ala Glu Glu Leu Ala Ala Gly Lys Pro Asp Pro Leu Pro Tyr Arg     130 135 140 Val Ala Leu Glu Arg Leu Asp Leu Gly Ala Gln Glu Ala Leu Ala Phe 145 150 155 160 Glu Asp Ser Pro Ala Gly Val Lys Ala Ala Val Gly Ala Gly Ile Pro                 165 170 175 Thr Ile Gly Leu Thr Thr Gly His Pro Pro Glu Ala Leu Lys Ala Ala             180 185 190 Gly Ala Phe Leu Leu Ile Ala Asp Phe Thr Asp Pro Gln Leu Trp Lys         195 200 205 Tyr Leu Glu Arg Ser     210 <210> 4 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of psicose 6-phosphatase from Meiothermus          timidus <400> 4 Met Arg Ala Leu Leu Phe Asp Leu Asp Gly Thr Leu Ala Asp Thr Asp   1 5 10 15 Arg Leu His Glu Gln Ala Trp Leu Glu Val Leu Leu Pro Tyr Gly Ile              20 25 30 Arg Gly Asp His Ala Phe Tyr Gln Gln His Ile Ser Gly His Leu Asn          35 40 45 Pro Glu Ile Val Ser Arg Leu Leu Pro His Leu Pro Pro Leu Glu Arg      50 55 60 Thr Ala Leu Ile Glu Val Lys Glu Arg Arg Phe Arg Glu Leu Ala Gln  65 70 75 80 Gly Leu Lys Ala Leu Pro Gly Leu Glu Gly Leu Trp Arg Trp Ala Arg                  85 90 95 Glu Arg Gly Leu Thr Leu Ala Leu Val Thr Asn Ala Pro Arg Pro Asn             100 105 110 Ala Glu His Val Leu Gln Ala Leu Gly Ala Glu Phe Asp Leu Val Val         115 120 125 Leu Ala Glu Glu Leu Ala Ala Gly Lys Pro Asp Pro Leu Pro Tyr Arg     130 135 140 Thr Ala Leu Gly Arg Leu Gly Leu Asp Pro Ala Glu Ala Leu Ala Phe 145 150 155 160 Glu Asp Ser Pro Ser Gly Val Arg Ser Ala Val Gly Ala Gly Ile Arg                 165 170 175 Thr Ile Gly Leu Thr Thr Gly His Asp Pro Arg Gly Leu Leu Glu Ala             180 185 190 Gly Ala Phe Leu Leu Ile Asp Asp Phe Ser Asp Gly Arg Leu Trp Glu         195 200 205 Tyr Leu Glu Gly Glu Ser Asp Ser     210 215 <210> 5 <211> 651 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequences of psicose 6-phosphatase from Meiothermus          cerbereus <400> 5 atgaagctta aagccctgct cttcgacctc gatggcaccc tggccgacac cgaccgcctg 60 cacgagcaag cctggctcga gggcctctcc cgctacggcc tgcagggcga ccacaccttt 120 taccagaccc agattagcgg gggcctgaac cccgagattg tgcagcgcct gctgccccaa 180 ctttcccagg ctgaggctga ggcttttctc gagcaaaaag aggcccgctt ccgcgaactg 240 gcctccgagg tgcagcccct accgggcttg gggacgttgt gggactgggc ccaggagcaa 300 aacctgcgcc gggccctggt gagcaacgcc cccagggaga acgcacagta cctgctaaag 360 cggctggggt tggtgttcga ccacatcgtg ctatccgaag agctgcccgc gggcaagccc 420 gacccgctgc cctaccgcac cgccctgcaa gcactcaaca tcggccccag cgaggccctg 480 gccttcgaag actccccctc cggggtgcgc tcggcggtgg gggccggtat ccgcaccgtg 540 gccctgacca ccggccatcc cgcccatgcg ctggaacaag ccggggcgtt cctttgcatc 600 cccaactttg ccgacccgcg cctgtgggcg tatctgcaca aaatgggcta g 651 <210> 6 <211> 651 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequences of psicose 6-phosphatase from Meiothermus          taiwanensis <400> 6 atgaagctga gggccctgct cttcgacctt gacggcaccc tggccgacac cgaccgcctg 60 cacgagcagg cctggctcga gggcctgtcc cgctacggca tccagggcga ccaccgcttc 120 taccaggccc agatcagcgg gggcctcaac cccgagattg tagcgcgcct gctgccccag 180 ctttcccccg acgagcaggt ggcctttatc gagcaaaaag aggcccgctt ccgcgagctg 240 gcatccgaag tgcaacccct accgggcttg agggtgctgt gggactgggc ccaaagccag 300 ggtttgcgcc gggctctggt aagcaacgcc cccagggaga acgcccacta cctgctagag 360 cggctggggc taatgttcga cgccatcgtc ctgtccgaag acctgcccgc cggcaagccc 420 gacccgctgc cctaccgcac ggccctgcag cacctcaaca tcggccccca ggaggccctg 480 gcctttgagg actccccctc cggggtgcgc tcggcggtgg gggccggcct ccgcaccgtg 540 gccctcacca ccggccaccc accccatgcc ctggaacagg ccggggcctt tctctgcatc 600 cccgacttta ccgacccgcg cctgtgggac tggctgcaaa agatgggcta g 651 <210> 7 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequences of psicose 6-phosphatase from Meiothermus          silvanus <400> 7 atgcgcgccc tactgtttga cctcgacgga accctagcca acaccgaccg gctacacgaa 60 caagcctggc tcgagactct gcgcttttat ggcatcgagg gggaccatca cttctatcag 120 acccagatca gcgggggcct caaccccgag atcgtgcggc ggctcctgcc ccagctttcc 180 gaggctgagg gagaggcttt tatcgcccgc aaagagcgcc gcttccgcga gctggcccaa 240 gacttacggg cattgccggg gctcgacgcg ttgctcgcgt gggcccggcg gaaaaaactc 300 ctgaccggcc tggtcaccaa cgccccccac gagaacgccc ggcacgtgac ccaagccctg 360 ggtctgagct tcgacgtagt cgtgctggcc gaggagttag cagcgggcaa gcccgacccg 420 ctcccctacc gggtggcgct cgagcggctc gatttggggg cacaagaggc cctggccttc 480 gaggattccc ctgcaggggt gaaggccgcc gtgggcgcgg gtatccccac catcggcctc 540 accaccggac acccccccga agccctgaag gccgccggag cctttctcct catcgccgac 600 tttacggatc cgcaactgtg gaagtatctc gagcggagtt ag 642 <210> 8 <211> 651 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequences of psicose 6-phosphatase from Meiothermus          timidus <400> 8 atgcgcgcct tgctgtttga tctcgatggc accctagccg acaccgaccg cctgcacgag 60 caggcttggc tcgaggtcct gctcccctac ggcatccggg gggatcatgc cttctaccag 120 cagcacatca gcggccacct caaccccgag atcgtatccc ggttgctgcc tcaccttccc 180 ccgctcgagc gcaccgcgtt gatcgaggtc aaggagcggc gtttccgcga gctggctcag 240 ggcctgaagg ccctgccggg gttggaaggg ttgtggcgct gggccaggga gcggggcctg 300 acgctggcct tggtgaccaa cgcccctcgc cctaacgccg agcacgtgct ccaggccctg 360 ggcgcggaat tcgacctggt ggtgctggcc gaggaactgg ccgccggaaa gcccgatccc 420 ctgccctacc gcactgccct ggggcggctg gggctagacc ccgccgaggc cctggccttc 480 gaggattccc cctcgggggt gcgctcggcg gtgggagcgg ggattcgcac catcggcctg 540 accaccggtc acgacccccg gggcctgctc gaggctgggg ccttcttgct gatcgacgat 600 ttcagcgacg ggcggctgtg ggaatacctg gaaggggaat ccgatagctg a 651 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of Mcer-A6Pase <400> 9 catatgaagc ttaaagccct gct 23 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of Mtai-A6Pase <400> 10 catatgaagc tgagggccct gc 22 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of Msil-A6Pase <400> 11 catatgcgcg ccctactgtt t 21 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of Mtim-A6Pase <400> 12 catatgcgcg ccttgctgtt tg 22 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of Mcer-A6Pase <400> 13 ctcgaggccc attttgtgca gatac 25 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of Mtai-A6Pase <400> 14 ctcgaggccc atcttttgca gccag 25 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of Msil-A6Pase <400> 15 ctcgagactc cgctcgagat actt 24 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of Mtim-A6Pase <400> 16 ctcgaggcta tcggattccc cttcc 25 <210> 17 <211> 823 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequences of CT1 <400> 17 Met Leu Lys Lys Leu Pro Glu Asn Leu Glu His Leu Glu Glu Leu Ala   1 5 10 15 Tyr Asn Leu Trp Trp Ser Trp Ser Arg Pro Ala Gln Arg Leu Trp Arg              20 25 30 Lys Ile Asp Pro Glu Gly Trp Glu Glu His Arg Asn Pro Val Lys Ile          35 40 45 Leu Lys Glu Val Ser Asp Glu Arg Leu Glu Glu Leu Ser Lys Asp Asp      50 55 60 Asp Phe Ile Ser Leu Tyr Glu Leu Thr Ile Glu Arg Phe Lys Asp Tyr  65 70 75 80 Met Glu Lys Glu Asp Thr Trp Phe Asn Val Asn Tyr Pro Glu Trp Asp                  85 90 95 Glu Lys Ile Val Tyr Met Cys Met Glu Tyr Gly Leu Thr Lys Ala Leu             100 105 110 Pro Ile Tyr Ser Gly Gly Leu Gly Ile Leu Ala Gly Asp His Leu Lys         115 120 125 Ser Ala Ser Asp Leu Gly Leu Pro Leu Ile Ala Ile Gly Leu Leu Tyr     130 135 140 Lys His Gly Tyr Phe Thr Gln Gln Ile Asp Arg Asp Gly Lys Gln Ile 145 150 155 160 Glu Ile Phe Pro Asp Tyr Asn Pro Glu Asp Leu Pro Met Lys Pro Leu                 165 170 175 Lys Asp Glu Lys Gly Asn Gln Val Ile Val Glu Val Pro Leu Asp Ser             180 185 190 Thr Val Val Lys Ala Arg Val Phe Glu Val Lys Val Gly Arg Val Ser         195 200 205 Leu Tyr Leu Leu Asp Pro Asp Ile Glu Glu Asn Glu Glu Arg Tyr Arg     210 215 220 Lys Ile Cys Asn Tyr Leu Tyr Asn Pro Glu Pro Asp Val Arg Val Ser 225 230 235 240 Gln Glu Ile Leu Leu Gly Ile Gly Gly Met Lys Leu Leu Arg Ala Leu                 245 250 255 Asn Leu Lys Pro Gly Val Ile His Leu Asn Glu Gly His Pro Ala Phe             260 265 270 Ser Ser Leu Glu Arg Ile Lys Asn Tyr Met Glu Glu Gly Tyr Ser Phe         275 280 285 Thr Glu Ala Leu Glu Ile Val Arg Gln Thr Ser Val Phe Thr Thr His     290 295 300 Thr Pro Val Pro Ala Gly His Asp Arg Phe Pro Phe Asp Leu Val Glu 305 310 315 320 Lys Lys Leu Ser Lys Phe Phe Glu Gly Phe Glu Lys Arg Asn Leu Leu                 325 330 335 Met Asp Leu Gly Lys Asp Glu Thr Gly Ser Phe Asn Met Thr Tyr Leu             340 345 350 Ala Leu Arg Thr Ser Ser Phe Ile Asn Gly Val Ser Lys Leu His Ala         355 360 365 Glu Val Ser Arg Arg Met Phe Lys Asn Val Trp Gln Gly Val Pro Val     370 375 380 Glu Glu Ile Pro Ile Glu Gly Ile Thr Asn Gly Val His Met Gly Thr 385 390 395 400 Trp Ile Asn Arg Glu Met Arg Lys Leu Tyr Asp Arg Tyr Leu Gly Arg                 405 410 415 Val Trp Arg Asp His Thr Asp Leu Glu Gly Ile Trp Tyr Gly Val Asp             420 425 430 Arg Ile Pro Asp Glu Glu Leu Trp Gln Ala His Leu Arg Ala Lys Lys         435 440 445 Arg Phe Ile Glu Tyr Ile Lys Glu Ser Val Arg Arg Arg Asn Glu Arg     450 455 460 Leu Gly Ile Asp Glu Asp Val Pro Asn Ile Asp Glu Asn Ser Leu Ile 465 470 475 480 Ile Gly Phe Ala Arg Arg Phe Ala Thr Tyr Lys Arg Ala Val Leu Leu                 485 490 495 Leu Ser Asp Leu Glu Arg Leu Lys Lys Ile Leu Asn Asp Pro Glu Arg             500 505 510 Pro Val Tyr Val Val Tyr Ala Gly Lys Ala His Pro Arg Asp Asp Ala         515 520 525 Gly Lys Glu Phe Leu Lys Arg Ile Tyr Glu Val Ser Gln Met Pro Glu     530 535 540 Phe Lys Asn Arg Ile Ile Val Leu Glu Asn Tyr Asp Ile Gly Met Ala 545 550 555 560 Arg Leu Met Val Ser Gly Val Asp Val Trp Leu Asn Asn Pro Arg Arg                 565 570 575 Pro Met Glu Ala Ser Gly Thr Ser Gly Met Lys Ala Ala Ala Asn Gly             580 585 590 Val Leu Asn Ala Ser Val Tyr Asp Gly Trp Trp Val Glu Gly Tyr Asn         595 600 605 Gly Arg Asn Gly Trp Val Ile Gly Asp Glu Ser Val Leu Pro Glu Thr     610 615 620 Glu Val Asp Asp Pro Arg Asp Ala Glu Ala Leu Tyr Asp Leu Leu Glu 625 630 635 640 Asn Glu Ile Ile Pro Thr Tyr Tyr Glu Asn Lys Glu Lys Trp Ile Phe                 645 650 655 Met Met Lys Glu Ser Ile Lys Ser Val Ala Pro Arg Phe Ser Thr Thr             660 665 670 Arg Met Leu Lys Glu Tyr Thr Glu Lys Phe Tyr Ile Lys Gly Leu Val         675 680 685 Asn Lys Glu Trp Leu Glu Arg Lys Glu Asn Ala Glu Arg Phe Gly Ala     690 695 700 Trp Lys Glu Arg Ile Leu Arg Asn Trp Ser Ser Val Ser Ile Glu Arg 705 710 715 720 Ile Val Leu Glu Asp Thr Arg Ser Val Glu Val Thr Val Lys Leu Gly                 725 730 735 Asp Leu Ser Pro Asp Asp Val Leu Val Glu Leu Leu Ile Gly Arg Gly             740 745 750 Glu Ser Met Glu Asp Leu Glu Ile Trp Lys Val Ile Gln Ile Arg Lys         755 760 765 His Arg Arg Glu Gly Asp Leu Phe Ile Tyr Ser Tyr Val Asn Gly Ala     770 775 780 Leu Gly His Leu Gly Ser Pro Gly Trp Phe Tyr Ala Val Arg Val Leu 785 790 795 800 Pro Tyr His Pro Lys Leu Pro Thr Arg Phe Leu Pro Glu Ile Pro Val                 805 810 815 Val Trp Lys Lys Val Leu Gly             820 <210> 18 <211> 2472 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequences of CT1 <400> 18 atgctgaaga aactcccgga gaatctggag catctggaag aactcgccta caacctctgg 60 tggagctggt ctaggcccgc tcagagactc tggagaaaga tagatccgga aggctgggag 120 gaacacagaa accccgttaa aatactgaaa gaagtttctg atgaaaggct cgaagaactt 180 tcaaaagatg atgatttcat atccctctac gaactcacca ttgaaaggtt caaggattac 240 atggagaaag aagacacctg gttcaacgtg aactaccccg aatgggacga gaagatcgtc 300 tacatgtgta tggagtacgg tttgaccaaa gcccttccga tctactccgg tggtcttgga 360 atcctcgcgg gagaccatct caaatccgca agcgatcttg gacttcctct catagcgatc 420 ggacttctct acaaacatgg atatttcacc cagcagatcg acagagatgg aaaacagata 480 gagattttcc ctgattacaa cccagaggac ttacccatga agcccctgaa ggatgaaaag 540 ggaaaccagg tgatcgtgga ggttcctctc gacagtaccg tggtgaaggc acgtgttttt 600 gaagtgaagg taggaagggt gagtctgtac ctgctcgatc cggacatcga ggaaaacgag 660 gaacgataca gaaagatctg caactacctt tacaacccgg aacccgatgt gagggtctcc 720 caggagatac tcctcggaat tgggggaatg aagcttctca gggctctgaa cctgaaacca 780 ggagtcatcc atctgaacga aggacatccg gcgttctctt ccctcgaaag gataaagaac 840 tacatggaag aaggatattc cttcacagag gcccttgaga tcgtgagaca gacgagtgtg 900 tttacaaccc acacacccgt tcccgctgga cacgacagat ttccctttga cctcgtggaa 960 aagaaacttt cgaaattctt cgaaggattc gaaaagagaa atcttctcat ggatcttggg 1020 aaagatgaaa caggcagttt caacatgacg tatcttgccc tgagaacgtc ctctttcata 1080 aacggcgtga gcaaactgca tgcggaagtt tccagaagga tgttcaaaaa cgtgtggcag 1140 ggtgttcccg tggaggaaat accgatcgaa gggataacga acggcgttca catgggaacc 1200 tggatcaacc gtgagatgag aaaactgtac gacagatatc tcggaagggt atggagagat 1260 cacaccgacc ttgagggtat ctggtacggt gttgacagga ttccagatga agaactctgg 1320 caggctcacc tgagggcaaa gaagagattc atcgagtaca taaaagaatc ggtaagaaga 1380 agaaacgaga gactgggaat cgacgaagat gtgccgaaca tcgatgaaaa ttcgctcatc 1440 ataggttttg caagaaggtt tgccacttac aagagggcag ttctcctgct cagcgatctg 1500 gagagactca agaagatcct caacgatcca gaaagacccg tttacgtggt ctatgcgggg 1560 aaggcccatc caagggacga tgcggggaag gaatttttga aacgcatcta cgaagtctcg 1620 cagatgcctg agttcaaaaa caggatcatc gtactggaaa actacgacat tggaatggca 1680 cggctcatgg tgtcgggagt ggatgtgtgg ctgaacaacc cgagaagacc catggaagca 1740 agtggaacaa gcggaatgaa ggcagcagcc aacggagttc ttaacgcgag tgtttacgat 1800 ggatggtggg ttgaagggta caacggcaga aacggctggg tcataggcga tgaaagcgtt 1860 cttccagaga cggaagtgga cgatcccagg gacgcagaag cactctacga tctcctcgaa 1920 aacgaaatca tcccaaccta ctacgaaaac aaagaaaagt ggatcttcat gatgaaagag 1980 agcataaaga gtgttgctcc aagattcagc accaccagaa tgctcaaaga atacacggag 2040 aagttctaca taaagggact tgtgaacaaa gaatggcttg aaagaaaaga aaacgccgaa 2100 aggtttggtg catggaagga aaggatcctc agaaactgga gcagcgtttc catagaaaga 2160 atcgtccttg aggacacaag gagtgttgag gtgacggtga aactgggaga cctttcacct 2220 gatgatgtac tggttgaact tttgattgga agaggagaaa gcatggaaga tctggagatc 2280 tggaaggtga tacagataag aaagcacaga agggaagggg atctgttcat ctacagttat 2340 gtcaacggtg ccctcggtca tcttggctct ccgggatggt tctacgcggt gagggtgcta 2400 ccttatcatc cgaaacttcc caccagattc ttgccggaga tacctgtggt gtggaaaaag 2460 gttctcgggt ga 2472 <210> 19 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequences of CT2 <400> 19 Met Ile Gly Tyr Gln Ile Tyr Val Arg Ser Phe Arg Asp Gly Asn Phe   1 5 10 15 Asp Gly Val Gly Asp Phe Lys Gly Leu Lys Gly Ala Ile Ser Tyr Leu              20 25 30 Lys Glu Leu Gly Val Asp Phe Val Trp Leu Met Pro Val Phe Ser Ser          35 40 45 Ile Ser Phe His Gly Tyr Asp Val Val Asp Phe Tyr Ser Phe Lys Ala      50 55 60 Glu Tyr Gly Asp Glu Lys Asp Phe Arg Glu Met Ile Glu Ala Phe His  65 70 75 80 Asp Asn Gly Ile Lys Val Val Leu Asp Leu Pro Ile His His Thr Gly                  85 90 95 Phe Leu His Val Trp Phe Gln Lys Ala Leu Lys Gly Asp Pro His Tyr             100 105 110 Arg Asp Tyr Tyr Val Trp Ala Ser Glu Lys Thr Asp Leu Asp Glu Arg         115 120 125 Arg Glu Trp Asp Asn Glu Arg Ile Trp His Pro Leu Glu Asp Gly Arg     130 135 140 Phe Tyr Arg Gly Leu Phe Gly Pro Leu Ser Pro Asp Leu Asn Tyr Asp 145 150 155 160 Asn Pro Gln Val Phe Glu Glu Met Lys Lys Val Val Tyr His Leu Leu                 165 170 175 Glu Met Gly Val Asp Gly Phe Arg Phe Asp Ala Ala Lys His Met Arg             180 185 190 Asp Thr Leu Glu Gln Asn Val Arg Phe Trp Arg Tyr Phe Leu Ser Asp         195 200 205 Ile Glu Gly Ile Phe Leu Ala Glu Ile Trp Ala Glu Ser Lys Val Val     210 215 220 Asp Glu His Gly Arg Ile Phe Gly Tyr Met Leu Asn Phe Asp Thr Ser 225 230 235 240 His Cys Ile Lys Glu Ala Val Trp Lys Glu Asn Phe Lys Val Leu Ile                 245 250 255 Glu Ser Ile Glu Arg Ala Leu Val Gly Lys Asp Tyr Leu Pro Val Asn             260 265 270 Phe Thr Ser Asn His Asp Met Ser Arg Leu Ala Ser Phe Glu Gly Gly         275 280 285 Leu Ser Glu Glu Lys Val Lys Leu Ser Leu Ser Ile Leu Phe Thr Leu     290 295 300 Pro Gly Val Pro Leu Ile Phe Tyr Gly Asp Glu Leu Gly Met Lys Gly 305 310 315 320 Ile Tyr Arg Lys Pro Asn Thr Glu Val Val Leu Asp Pro Phe Pro Trp                 325 330 335 Ser Glu Asn Met Cys Val Glu Gly Gln Thr Phe Trp Lys Trp Pro Ala             340 345 350 Tyr Asn Asp Pro Phe Ser Gly Val Ser Val Glu Tyr Gln Arg Arg Asn         355 360 365 Arg Asp Ser Ile Leu Ser His Thr Met Arg Trp Ala Gly Phe Arg Gly     370 375 380 Glu Asn His Trp Leu Asp Arg Ala Asn Ile Glu Phe Leu Cys Lys Glu 385 390 395 400 Glu Lys Leu Leu Val Tyr Arg Leu Val Asp Glu Gly Arg Ser Leu Lys                 405 410 415 Val Ile His Asn Leu Ser Asn Gly Glu Met Val Phe Glu Gly Val Arg             420 425 430 Val Gln Pro Tyr Ser Thr Glu Val Val         435 440 <210> 20 <211> 1326 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequences of CT2 <400> 20 atgataggct accagatcta cgtgagatca ttcagggatg gaaacttcga tggtgtgggg 60 gatttcaaag gattgaaagg tgcgatttcc tacctgaaag aactgggtgt tgattttgtc 120 tggctcatgc ccgtcttttc ctccatttcc ttccacgggt atgacgtggt ggatttttat 180 tctttcaaag ccgagtacgg agacgagaaa gactttagag agatgatcga ggcgttccac 240 gacaacggta taaaagtcgt tctcgatctt cccatccatc atactggttt cctccatgtg 300 tggtttcaga aagccctgaa aggagatcca cactacaggg attattacgt atgggcgagt 360 gaaaaaacgg atctggacga aagaagagag tgggacaacg aaaggatctg gcatcctctg 420 gaggacggaa ggttctacag aggacttttc ggtcccctct cacccgatct gaactacgat 480 aacccgcagg tttttgaaga gatgaagaag gtggtttatc accttcttga aatgggagtg 540 gacggattca gattcgacgc agcaaagcac atgagagata ctctggaaca gaacgttcgc 600 ttttggaggt atttcctctc cgatattgag ggaatattcc ttgcggaaat ctgggcagaa 660 tccaaagttg tggatgaaca cggcaggata ttcggctaca tgctaaattt cgatacctca 720 cactgtatta aggaagcggt gtggaaggaa aacttcaaag tgttgatcga gtcgatcgaa 780 agggccctgg ttggaaaaga ttatctgccg gtgaacttca catcgaacca tgatatgtca 840 aggcttgcga gtttcgaagg agggttgagt gaagagaagg tgaaactctc actttccatt 900 ctgttcacgc ttcccggggt tcctctcata ttctacggag acgaactggg aatgaaagga 960 atctatcgaa aaccgaacac ggaagtcgtg ctggatccgt tcccctggag cgaaaacatg 1020 tgtgttgaag gccagacatt ttggaaatgg cccgcgtata acgatccatt ctccggtgtt 1080 tctgttgagt atcagaggag aaatcgtgat tcgattctct cacacacgat gaggtgggca 1140 ggattcagag gggaaaatca ctggctggac agggcaaaca tcgaatttct gtgcaaagaa 1200 gaaaaactgc tcgtgtacag actggtcgat gaagggcgtt ctctgaaagt gatacacaac 1260 ctgtcgaatg gtgaaatggt gtttgaggga gtgcgcgtac aaccctacag cacggaggtg 1320 gtttga 1326 <210> 21 <211> 270 <212> PRT <213> Artificial Sequence <220> <223> Polyphospate-dependent glucokinase derived from Deinococcus          geothermalis <400> 21 Met Leu Ala Ala Ser Asp Ser Ser Gln His Gly Gly Lys Ala Val Thr   1 5 10 15 Leu Ser Pro Met Ser Val Ile Leu Gly Ile Asp Ile Gly Gly Ser Gly              20 25 30 Ile Lys Gly Ala Pro Val Asp Thr Ala Thr Gly Lys Leu Val Ala Glu          35 40 45 Arg His Arg Ile Pro Thr Pro Glu Gly Ala His Pro Asp Ala Val Lys      50 55 60 Asp Val Val Val Glu Leu Val Arg His Phe Gly His Ala Gly Pro Val  65 70 75 80 Gly Ile Thr Phe Pro Gly Ile Val Gln His Gly His Thr Leu Ser Ala                  85 90 95 Ala Asn Val Asp Lys Ala Trp Ile Gly Leu Asp Ala Asp Thr Leu Phe             100 105 110 Thr Glu Ala Thr Gly Arg Asp Val Thr Val Ile Asn Asp Ala Asp Ala         115 120 125 Ala Gly Leu Ala Glu Ala Arg Phe Gly Ala Gly Ala Gly Val Pro Gly     130 135 140 Glu Val Leu Leu Leu Thr Phe Gly Thr Gly Ile Gly Ser Ala Leu Ile 145 150 155 160 Tyr Asn Gly Val Leu Val Pro Asn Thr Glu Phe Gly His Leu Tyr Leu                 165 170 175 Lys Gly Asp Lys His Ala Glu Thr Trp Ala Ser Asp Arg Ala Arg Glu             180 185 190 Gln Gly Asp Leu Asn Trp Lys Gln Trp Ala Lys Arg Val Ser Arg Tyr         195 200 205 Leu Gln Tyr Leu Glu Gly Leu Phe Ser Pro Asp Leu Phe Ile Ile Gly     210 215 220 Gly Gly Val Ser Lys Lys Ala Asp Lys Trp Gln Pro His Val Ala Thr 225 230 235 240 Thr Arg Thr Arg Leu Val Pro Ala Ala Leu Gln Asn Glu Ala Gly Ile                 245 250 255 Val Gly Ala Ala Met Val Ala Ala Gln Arg Ser Gln Gly Asp             260 265 270 <210> 22 <211> 253 <212> PRT <213> Artificial Sequence <220> <223> Polyphospate-dependent glucokinase derived from Anaerolinea          thermophila <400> 22 Met Gly Arg Gln Gly Met Glu Ile Leu Gly Ile Asp Ile Gly Gly Ser   1 5 10 15 Gly Ile Lys Gly Ala Pro Val Asp Val Glu Thr Gly Gln Leu Thr Ala              20 25 30 Glu Arg Tyr Arg Leu Pro Thr Pro Glu Asn Ala Leu Pro Glu Glu Val          35 40 45 Ala Leu Val Val Ala Gln Ile Val Glu His Phe Gln Trp Lys Gly Arg      50 55 60 Val Gly Ala Gly Phe Pro Ala Ala Ile Lys His Gly Val Ala Gln Thr  65 70 75 80 Ala Ala Asn Ile His Pro Thr Trp Ile Gly Leu His Ala Gly Asn Leu                  85 90 95 Phe Ser Glu Lys Cys Gly Cys Pro Val Ser Val Leu Asn Asp Ala Asp             100 105 110 Ala Ala Gly Leu Ala Glu Met Ile Phe Gly Ala Gly Lys Gly Gln Lys         115 120 125 Gly Val Val Leu Met Ile Thr Ile Gly Thr Gly Ile Gly Thr Ala Leu     130 135 140 Phe Thr Asp Gly Ile Leu Val Pro Asn Thr Glu Leu Gly His Ile Glu 145 150 155 160 Ile Arg Gly Lys Asp Ala Glu Gln Arg Ser Ser Glu Ala Ala Arg Gln                 165 170 175 Arg Lys Asp Trp Thr Trp Gln Gln Trp Ala Lys Arg Leu Asn Glu His             180 185 190 Leu Glu Arg Leu Glu Ala Leu Phe Trp Pro Asp Leu Phe Ile Leu Gly         195 200 205 Gly Gly Ala Val Lys Asn His Glu Lys Phe Phe Pro Tyr Leu Lys Leu     210 215 220 Arg Thr Pro Phe Val Ala Ala Lys Leu Gly Asn Leu Ala Gly Ile Val 225 230 235 240 Gly Ala Ala Trp Tyr Ala His Thr Gln Glu Thr Gln Ala                 245 250 <210> 23 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequences of TN1 <400> 23 Met Lys Lys Met Ala Leu Lys Phe Asp Phe Ser Asn Leu Phe Glu Pro   1 5 10 15 Asn Ile Ser Gly Gly Leu Arg Glu Glu Asp Leu Glu Ser Thr Lys Glu              20 25 30 Lys Val Ile Glu Ala Ile Lys Asn Phe Thr Glu Asn Thr Pro Asp Phe          35 40 45 Ala Arg Leu Asp Arg Lys Trp Ile Asp Ser Val Lys Glu Leu Glu Glu      50 55 60 Trp Val Val Asn Phe Asp Thr Val Val Val Leu Gly Ile Gly Gly Ser  65 70 75 80 Gly Leu Gly Asn Leu Ala Leu His Tyr Ser Leu Arg Pro Leu Asn Trp                  85 90 95 Asn Glu Met Ser Arg Glu Glu Arg Asn Gly Tyr Ala Arg Val Phe Val             100 105 110 Val Asp Asn Val Asp Pro Asp Leu Met Ala Ser Val Leu Asp Arg Ile         115 120 125 Asp Leu Lys Thr Thr Leu Phe Asn Val Ile Ser Lys Ser Gly Ser Thr     130 135 140 Ala Glu Val Met Ala Asn Tyr Ser Ile Ala Arg Gly Ile Leu Glu Ala 145 150 155 160 Asn Gly Leu Asp Pro Lys Glu His Ile Leu Ile Thr Thr Asp Pro Glu                 165 170 175 Lys Gly Phe Leu Arg Lys Val Val Lys Glu Glu Gly Phe Arg Ser Leu             180 185 190 Glu Val Pro Pro Gly Val Gly Gly Arg Phe Ser Val Leu Thr Pro Val         195 200 205 Gly Leu Phe Ser Ala Met Ala Glu Gly Ile Asp Ile Glu Glu Leu His     210 215 220 Asp Gly Ala Arg Asp Ala Phe Glu Arg Cys Lys Lys Glu Asp Leu Phe 225 230 235 240 Glu Asn Pro Ala Ala Met Ile Ala Leu Thr His Tyr Leu Tyr Leu Lys                 245 250 255 Arg Gly Lys Ser Ile Ser Val Met Met Ala Tyr Ser Asn Arg Met Thr             260 265 270 Tyr Leu Val Asp Trp Tyr Arg Gln Leu Trp Ala Glu Ser Leu Gly Lys         275 280 285 Arg Tyr Asn Leu Lys Gly Glu Glu Val Phe Thr Gly Gln Thr Pro Val     290 295 300 Lys Ala Ile Gly Ala Thr Asp Gln His Ser Gln Ile Gln Leu Tyr Asn 305 310 315 320 Glu Gly Pro Asn Asp Lys Val Ile Thr Phe Leu Arg Leu Glu Asn Phe                 325 330 335 Asp Arg Glu Ile Ile Ile Pro Asp Thr Gly Arg Glu Glu Leu Lys Tyr             340 345 350 Leu Ala Arg Lys Arg Leu Ser Glu Leu Leu Leu Ala Glu Gln Thr Gly         355 360 365 Thr Glu Glu Ala Leu Arg Lys Asn Asp Arg Pro Asn Met Lys Val Ile     370 375 380 Phe Asp Arg Leu Thr Ser Tyr Asn Val Gly Gln Phe Phe Ala Tyr Tyr 385 390 395 400 Glu Ala Ala Thr Ala Phe Met Gly Tyr Leu Leu Glu Ile Asn Pro Phe                 405 410 415 Asp Gln Pro Gly Val Glu Leu Gly Lys Lys Ile Thr Phe Ala Leu Met             420 425 430 Gly Arg Glu Gly Tyr Glu Tyr Glu Ile Lys Asp Arg Thr Lys Lys Val         435 440 445 Ile Ile Glu     450 <210> 24 <211> 1356 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequences of TN1 <400> 24 atgaaaaaga tggctttgaa atttgatttt tcaaatcttt ttgaaccgaa catctccggt 60 ggactgagag aggaagatct ggaaagcaca aaagaaaagg tgatagaggc gataaagaat 120 ttcactgaga acacaccgga ttttgccaga ctggacagaa aatggatcga ttcggtgaag 180 gaactcgagg agtgggtggt gaacttcgac acggtggtcg ttctgggaat tgggggatcc 240 ggtcttggaa accttgccct tcattattcg ttgagaccac tgaactggaa cgagatgtcg 300 agagaggaaa gaaacggtta tgcgagagtc ttcgtggtgg acaacgtaga tcccgatctc 360 atggcctccg tccttgatag gatagatctg aagacaacgc tgttcaacgt gatctcaaaa 420 tctggatcca cggctgaggt tatggcgaat tactcgatcg caaggggaat cctggaggct 480 aatggtctgg acccgaaaga acacatcctc atcacaacag atccagagaa gggctttttg 540 agaaaagtag tgaaagaaga gggcttcaga agtcttgagg tccctcccgg cgttggagga 600 aggttcagcg tgctgacgcc cgttggcctc ttctctgcca tggcggaggg tatcgacata 660 gaagaactcc acgacggtgc ccgggatgcg ttcgagagat gcaagaagga agacctgttc 720 gaaaatccag cggcgatgat cgccctcaca cactatctct atctgaagag aggaaagagc 780 atctccgtca tgatggccta ctccaacagg atgacctacc tcgtggactg gtacagacag 840 ctgtgggcag aaagtctggg aaagagatac aacctgaaag gagaggaggt cttcacgggt 900 cagaccccgg tgaaggcaat aggagccacc gatcagcact ctcagataca gctttacaac 960 gagggcccaa acgacaaagt gataacgttt ttgcggttgg aaaacttcga tagagagatc 1020 ataataccgg acaccggaag agaagagctc aaataccttg caagaaaaag actctctgaa 1080 cttctccttg cagaacagac aggaacagag gaagccctaa ggaaaaacga cagaccgaac 1140 atgaaggtga tcttcgacag actcacctct tacaatgtgg gccagttctt cgcttattat 1200 gaagccgcaa ctgctttcat ggggtatctc ctcgagatca acccgtttga tcagccgggt 1260 gtggaacttg gaaagaagat cacgtttgcc ctcatgggaa gggaaggtta cgaatacgaa 1320 ataaaagatc gcaccaagaa ggtgatcata gaatga 1356 <210> 25 <211> 221 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequences of FP3E <400> 25 Met Met Val Lys Ile Ala Ala Ser Ile Leu Ala Cys Asp Leu Ala Arg   1 5 10 15 Leu Ala Asp Glu Val Lys Arg Val Glu Glu His Ile Asp Met Val His              20 25 30 Phe Asp Val Met Asp Gly His Phe Val Pro Asn Ile Ser Phe Gly Leu          35 40 45 Pro Val Leu Lys Ala Leu Arg Lys Glu Thr Ser Leu Pro Ile Ser Val      50 55 60 His Leu Met Ile Thr Asn Pro Glu Asp Tyr Val Asp Arg Phe Val Glu  65 70 75 80 Glu Gly Ala Asp Met Val Ala Val His Tyr Glu Thr Thr Pro His Leu                  85 90 95 His Arg Ile Val His Arg Ile Lys Asp Leu Gly Ala Lys Ala Phe Val             100 105 110 Ala Leu Asn Pro His Thr Pro Val Phe Leu Leu Ser Glu Ile Ile Thr         115 120 125 Asp Val Asp Gly Val Leu Val Met Ser Val Asn Pro Gly Phe Ser Gly     130 135 140 Gln Arg Phe Ile Ala Arg Ser Leu Glu Lys Ile Arg Ser Leu Lys Lys 145 150 155 160 Met Ile Arg Asp Leu Gly Leu Glu Thr Glu Ile Met Val Asp Gly Gly                 165 170 175 Val Asn Glu Glu Asn Ala Ser Ile Leu Ile Lys Asn Gly Ala Thr Ile             180 185 190 Leu Val Met Gly Tyr Gly Ile Phe Lys Asn Glu Asn Tyr Val Glu Leu         195 200 205 Val Arg Ser Ile Lys Gln Glu Arg Gly Glu Ser Ala Gly     210 215 220 <210> 26 <211> 666 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequences of FP3E <400> 26 atgatggtaa agatcgccgc ttcaatcctt gcgtgtgatc ttgcaagact cgccgatgag 60 gtaaaaaggg tggaagaaca catagacatg gttcacttcg atgtcatgga tggacacttc 120 gttccgaaca tctcgttcgg attgcccgtt ctcaaagccc tgagaaaaga aaccagcctt 180 cctataagtg ttcatctgat gatcacaaat ccagaggact atgtggaccg tttcgtggaa 240 gagggagcgg acatggtggc ggtccactac gagacaacgc cgcaccttca caggatagtg 300 cacaggataa aggatctcgg ggcgaaggcg ttcgtcgccc tcaacccaca cacaccggtt 360 tttctcctgt ctgagatcat aacggatgtg gatggcgtac tcgtgatgag tgtgaacccg 420 ggcttttctg gtcagagatt cattgcaagg agtctggaaa aaataaggag tctgaagaag 480 atgataaggg atctgggact cgaaacggag atcatggtcg atggtggtgt caacgaagaa 540 aacgcttcta tcttaataaa gaacggtgcg acgatccttg taatggggta cggtatcttc 600 aaaaacgaaa actatgtgga actggtgaga tccatcaagc aggaaagagg ggaatctgct 660 ggctga 666 <210> 27 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequences of TN2 <400> 27 Met Ile Gly Tyr Gln Ile Tyr Val Arg Ser Phe Arg Asp Gly Asn Phe   1 5 10 15 Asp Gly Val Gly Asp Phe Lys Gly Leu Lys Gly Ala Ile Ser Tyr Leu              20 25 30 Lys Glu Leu Gly Val Asp Phe Val Trp Leu Met Pro Val Phe Ser Ser          35 40 45 Ile Ser Phe His Gly Tyr Asp Val Val Asp Phe Tyr Ser Phe Lys Ala      50 55 60 Glu Tyr Gly Asp Glu Lys Asp Phe Arg Glu Met Ile Glu Ala Phe His  65 70 75 80 Asp Asn Gly Ile Lys Val Val Leu Asp Leu Pro Ile His His Thr Gly                  85 90 95 Phe Leu His Val Trp Phe Gln Lys Ala Leu Lys Gly Asp Pro His Tyr             100 105 110 Arg Asp Tyr Tyr Val Trp Ala Ser Glu Lys Thr Asp Leu Asp Glu Arg         115 120 125 Arg Glu Trp Asp Asn Glu Arg Ile Trp His Pro Leu Glu Asp Gly Arg     130 135 140 Phe Tyr Arg Gly Leu Phe Gly Pro Leu Ser Pro Asp Leu Asn Tyr Asp 145 150 155 160 Asn Pro Gln Val Phe Glu Glu Met Lys Lys Val Val Tyr His Leu Leu                 165 170 175 Glu Met Gly Val Asp Gly Phe Arg Phe Asp Ala Ala Lys His Met Arg             180 185 190 Asp Thr Leu Glu Gln Asn Val Arg Phe Trp Arg Tyr Phe Leu Ser Asp         195 200 205 Ile Glu Gly Ile Phe Leu Ala Glu Ile Trp Ala Glu Ser Lys Val Val     210 215 220 Asp Glu His Gly Arg Ile Phe Gly Tyr Met Leu Asn Phe Asp Thr Ser 225 230 235 240 His Cys Ile Lys Glu Ala Val Trp Lys Glu Asn Phe Lys Val Leu Ile                 245 250 255 Glu Ser Ile Glu Arg Ala Leu Val Gly Lys Asp Tyr Leu Pro Val Asn             260 265 270 Phe Thr Ser Asn His Asp Met Ser Arg Leu Ala Ser Phe Glu Gly Gly         275 280 285 Leu Ser Glu Glu Lys Val Lys Leu Ser Leu Ser Ile Leu Phe Thr Leu     290 295 300 Pro Gly Val Pro Leu Ile Phe Tyr Gly Asp Glu Leu Gly Met Lys Gly 305 310 315 320 Ile Tyr Arg Lys Pro Asn Thr Glu Val Val Leu Asp Pro Phe Pro Trp                 325 330 335 Ser Glu Asn Met Cys Val Glu Gly Gln Thr Phe Trp Lys Trp Pro Ala             340 345 350 Tyr Asn Asp Pro Phe Ser Gly Val Ser Val Glu Tyr Gln Arg Arg Asn         355 360 365 Arg Asp Ser Ile Leu Ser His Thr Met Arg Trp Ala Gly Phe Arg Gly     370 375 380 Glu Asn His Trp Leu Asp Arg Ala Asn Ile Glu Phe Leu Cys Lys Glu 385 390 395 400 Glu Lys Leu Leu Val Tyr Arg Leu Val Asp Glu Gly Arg Ser Leu Lys                 405 410 415 Val Ile His Asn Leu Ser Asn Gly Glu Met Val Phe Glu Gly Val Arg             420 425 430 Val Gln Pro Tyr Ser Thr Glu Val Val         435 440 <210> 28 <211> 1326 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequences of TN2 <400> 28 atgataggct accagatcta cgtgagatca ttcagggatg gaaacttcga tggtgtgggg 60 gatttcaaag gattgaaagg tgcgatttcc tacctgaaag aactgggtgt tgattttgtc 120 tggctcatgc ccgtcttttc ctccatttcc ttccacgggt atgacgtggt ggatttttat 180 tctttcaaag ccgagtacgg agacgagaaa gactttagag agatgatcga ggcgttccac 240 gacaacggta taaaagtcgt tctcgatctt cccatccatc atactggttt cctccatgtg 300 tggtttcaga aagccctgaa aggagatcca cactacaggg attattacgt atgggcgagt 360 gaaaaaacgg atctggacga aagaagagag tgggacaacg aaaggatctg gcatcctctg 420 gaggacggaa ggttctacag aggacttttc ggtcccctct cacccgatct gaactacgat 480 aacccgcagg tttttgaaga gatgaagaag gtggtttatc accttcttga aatgggagtg 540 gacggattca gattcgacgc agcaaagcac atgagagata ctctggaaca gaacgttcgc 600 ttttggaggt atttcctctc cgatattgag ggaatattcc ttgcggaaat ctgggcagaa 660 tccaaagttg tggatgaaca cggcaggata ttcggctaca tgctaaattt cgatacctca 720 cactgtatta aggaagcggt gtggaaggaa aacttcaaag tgttgatcga gtcgatcgaa 780 agggccctgg ttggaaaaga ttatctgccg gtgaacttca catcgaacca tgatatgtca 840 aggcttgcga gtttcgaagg agggttgagt gaagagaagg tgaaactctc actttccatt 900 ctgttcacgc ttcccggggt tcctctcata ttctacggag acgaactggg aatgaaagga 960 atctatcgaa aaccgaacac ggaagtcgtg ctggatccgt tcccctggag cgaaaacatg 1020 tgtgttgaag gccagacatt ttggaaatgg cccgcgtata acgatccatt ctccggtgtt 1080 tctgttgagt atcagaggag aaatcgtgat tcgattctct cacacacgat gaggtgggca 1140 ggattcagag gggaaaatca ctggctggac agggcaaaca tcgaatttct gtgcaaagaa 1200 gaaaaactgc tcgtgtacag actggtcgat gaagggcgtt ctctgaaagt gatacacaac 1260 ctgtcgaatg gtgaaatggt gtttgaggga gtgcgcgtac aaccctacag cacggaggtg 1320 gtttga 1326 <210> 29 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward primer DNA sequence of ct1 <400> 29 aggagaaact catatgctga agaaactccc ggag 34 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer DNA sequence ct1 <400> 30 agccccctcg agcccgagaa c 21 <210> 31 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Forward primer DNA sequence of ct2 <400> 31 aaagggcata tgatcctgtt tggaac 26 <210> 32 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer DNA sequence of ct2 <400> 32 ataccagtct cgagcagttt caggatc 27 <210> 33 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward primer DNA sequence of tn1 <400> 33 ttactgaggg catatgaaaa agatggcttt gaaa 34 <210> 34 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer DNA sequence of tn1 <400> 34 aagacgcgtc gacttctatg atcaccttct 30 <210> 35 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward primer DNA sequence of fp3e <400> 35 ggaacatatg atggtaaaga tcgccgcttc aatc 34 <210> 36 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer DNA sequence of fp3e <400> 36 catactcgag cttcccctct cctatct 27

Claims (18)

delete delete delete delete delete delete delete Psycho-6-phosphate dephosphorylation enzyme consisting of any one amino acid sequence selected from SEQ ID NOs: 1 to 4, a microorganism for expressing it, or a composition for producing psychos comprising a culture of the microorganism. The composition of claim 8, wherein the composition for producing psychos,
(a) (i) starch, maltodextrin, sucrose or combinations thereof,
(ii) phosphate,
(iii) fructose-6-phosphate-3-epimerase,
(iv) glucose-6-phosphate-isomerase,
(v) phosphoglucomutase or glucose phosphatase, and
(vi) a group consisting of α-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase, sucrose phosphorylase, α-amylase, pullulanase, isoamylase, glucoamylase or sucrase At least one selected from;
or
(b) A composition for producing a psychos, further comprising a microorganism expressing the enzyme of item (a) or a culture of the microorganism. Psycho-6-Phosphate-6-Phosphoric acid dephosphorylation enzyme consisting of any one amino acid sequence selected from SEQ ID NOs: 1-4, is contacted with a microbial or microbial culture expressing the Psicos-6. -A method of manufacturing a psychos comprising the step of converting phosphoric acid to a psychos. The method of claim 10, wherein the contact is performed at pH 6 to 9. The fructose-6-phosphate-3-epimerase to fructose-6-phosphate, according to claim 10, wherein the method comprises: A method of manufacturing a psychos, further comprising the step of converting the fructose-6-phosphate to a psychos-6-phosphate by contacting a microorganism expressing it or a culture of the microorganism. The method of claim 12, wherein the method, prior to the step of converting the fructose-6-phosphate to Psycho-6-phosphate, glucose-6-phosphate-isomerase to glucose-6-phosphate, A method for producing psychos, further comprising the step of converting the glucose-6-phosphate to fructose-6-phosphate by contacting a microorganism expressing the same or a culture of the microorganism. The method of claim 13, wherein the method, before the step of converting the glucose-6-phosphate to fructose-6-phosphate, phosphoglucomutase in glucose-1-phosphate, a microorganism expressing it Or further comprising the step of converting the glucose-1-phosphate to glucose-6-phosphate by contacting the culture of the microorganism. The method of claim 13, wherein the method, prior to the step of converting the glucose-6-phosphate to fructose-6-phosphate, contact glucose with a glucose phosphatase, a microorganism expressing it, or a culture of the microorganism and phosphate. Let, the glucose further comprises the step of converting to glucose-6-phosphate, Psycho production method. The method of claim 14, wherein the method comprises α-glucan phosphorylase, starch phosphorylase in starch, maltodextrin, sucrose, or a combination thereof before the step of converting the glucose-1-phosphate to glucose-6-phosphate. Ases, maltodextrin phosphorylase or sucrose phosphorylase; Microorganisms expressing this; Or further comprising the step of converting the starch, maltodextrin, sucrose, or a combination thereof to glucose-1-phosphate by contacting the culture of the microorganism. 16. The method of claim 15, wherein the method comprises α-amylase, pullulanase, glucoamylase, sucrase or iso in starch, maltodextrin, sucrose, or combinations thereof, prior to the step of converting the glucose to glucose-6-phosphate. Amylase; Microorganisms expressing this; Or further comprising the step of converting the starch, maltodextrin, sucrose, or a combination thereof to glucose by contacting the culture of the microorganism, a method for manufacturing psychos. Starch, maltodextrin, sucrose or combinations thereof, and phosphates (a) inositol-mono-phosphatase; Fructose-6-phosphate-3-epimerase; Glucose-6-phosphate-isomerase; Phosphoglucomutase or glucose phosphorylase; α-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase, sucrose phosphorylase, α-amylase, pullulanase, isoamylase, glucoamylase or sucrase; And a Picos-6-phosphate dephosphorylation enzyme consisting of any one amino acid sequence selected from SEQ ID NOs: 1 to 4; Or (b) contacting a microorganism expressing the enzyme of item (a) or a culture of the microorganism, a method for manufacturing psychos.

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