WO2014193052A1 - Corynebacterium including polynucleotide for coding psicose 3-epimerase enzyme and method for producing psicose by using same - Google Patents

Abstract

The present invention relates to a corynebacterium including a polynucleotide for coding a psicose 3-epimerase enzyme, and a method for producing psicose by using the same, and more specifically, to: a corynebacterium including a polynucleotide for coding a psicose 3-epimerase enzyme having a catalytic activity for the conversion of fructose into psicose, wherein the polynucleotide can mass-produce psicose from fructose safely and efficiently by coding the amino acid sequence represented by SEQ ID NO: 1; and a method for producing psicose using the same.

Description

Corynebacterium comprising a polynucleotide encoding a Pycos 3-epimerase enzyme and a method of producing Pycos using the same

The present invention relates to a Corynebacterium comprising a polynucleotide encoding a Pycos 3-epimerase enzyme and a method of producing Pycos using the same.

D-psicose is an epimer of carbon number 3 of fructose (D-fructose), which is sweet like ordinary sugars, but is not metabolized in the human body. It is a functional sugar that can be used as a sugar substitute functional sweetener. In addition, it has the function of reducing the abdominal obesity by inhibiting the enzyme activity involved in lipid synthesis in the liver, and is a sugar currently being studied as a therapeutic agent for diabetes and atherosclerosis.

As such, psychos is gaining popularity as a sweetener, and there is an increasing need for development of a method for efficiently producing psychos in the food industry. Psycos are present in natural substances in very small amounts during molasses or glucose isomerization, so the production of conventional psychos is mainly through chemical processes. Bilik et al. Developed a technique for producing cycos from fructose using the catalytic action of molybdic acid ions. McDonald is 1,2: 4,5-di-o-isopropylidene-beta-D-fractopyranose (1,2: 4,5-di-o-isppropylidene-bata-D-fructopyranose) Psychos were produced from a three-step chemical treatment. In addition, Donner produced cyclose by heating fructose with ethanol and triethylamine. However, while the production of psychose by these chemical methods is expensive, the production efficiency is low and there is a problem in that excessive production of by-products occurs.

As a method of producing physics by biological methods, Ken Izumori et al. Used galacitol, D-tagatose or D-talitol by utilizing microbial cell reactions. It has been shown that it is possible to produce psychocos from. However, these substrates also have the disadvantage that they are relatively rare in nature and have a high cost.

As an enzyme conversion method, D-tagatose-3-epimerase of isolated microorganism Pseudomonas cichorii ST-24 is produced and purified from recombinant E. coli. There is a method of enzymatic conversion to cyclose, and Izmori et al. Produced a cyclic cyclic conversion system with a conversion rate of about 25% by immobilizing di-tagatose-3-epimerase.

According to this prior art, in order to produce cyclose from fructose, enzymes have been purified and immobilized the purified enzyme has been studied in the direction of increasing the cyclic productivity. As such, the process of purifying enzymes requires a lot of time and money.

Therefore, there is a need for a strain that can be produced with low production cost and high efficiency by eliminating the process of enzymatic purification and a method of producing a cycos using the same.

Korean Laid-Open Patent Publication No. 2006-125971 discloses a method for producing a psychos by a psychos epimerase.

<Preceding technical literature>

<Patent Documents>

Korean Laid-Open Patent No. 2006-125971

It is an object of the present invention to provide a Corynebacterium comprising a polynucleotide encoding a Psychos 3-epimerase enzyme, which has a high efficiency of Psycose production efficiency.

It is an object of the present invention to provide a method for producing a psychos which can mass-produce the psychos with a safe and high efficiency therefrom.

1. A Corynebacterium microorganism comprising a polynucleotide encoding an enzyme having an activity catalyzing the conversion of fructose to a psychose, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 1.

2. The microorganism of 1 above, wherein the microorganism is the polynucleotide introduced into a Corynebacterium glutamicum strain.

3. The microorganism according to the above 1, wherein the microorganism is the polynucleotide introduced into Corynebacterium glutamicum ATCC13032.

4. The microorganism according to any one of the preceding items, wherein the polynucleotide is introduced into the microorganism by itself or using a vector.

5. The gene of any one of 1 to 3, wherein at least one of a gene having at least 80% homology with the amino acid sequence of SEQ ID NO: 2 and a gene having at least 80% homology with the amino acid sequence of SEQ ID NO: 3 is deleted or Inactivated, microorganisms.

6. The microorganism of any one of 1 to 3, wherein at least one of ptsF having an amino acid sequence of SEQ ID NO: 2 and mtlD having an amino acid sequence of SEQ ID NO: 3 is deleted or inactivated.

7. culturing the microorganism of any one of the above 1 to 3 in a medium containing fructose; And recovering the psychoses from the culture of the microorganisms.

8. The method according to the above 7, wherein the fructose is in a concentration of 1% (w / v) to 80% (w / v).

9. The method according to the above 7, wherein the medium is nutrient medium or life-saving medium, method.

10. In the above 7, wherein the microorganism of any one of claims 1 to 3 before the step of culturing the microorganism in the medium containing no fructose encoding an enzyme having an activity that catalyzes the conversion of fructose to psychocos Expressing the polynucleotide; And recovering the microorganisms from the culture of the microorganisms.

11. The method according to the above 10, wherein the recovered microorganism is inoculated at a concentration such that the OD ₆₀₀ is 0.01 to 300 in the medium containing fructose, the method.

12. The method according to the above 11, wherein the fructose concentration of the fructose-containing medium is 1% (w / v) to 80% (w / v).

13. The method of 7 above, recovering the microorganisms from the culture of the microorganisms, further comprising the step of culturing the isolated microorganisms in a medium containing fructose again.

14. The method according to the above 13, wherein the isolated microorganism is cultured by inoculating the medium containing fructose at a concentration such that OD ₆₀₀ is from 5 to 150.

15. The method according to the above 13, wherein the fructose concentration of the medium containing fructose is 10% (w / v) to 80% (w / v).

The microorganisms of the present invention are capable of mass production of psychoses from fructose with high efficiency while being safe.

Figure 1 shows the results of measuring the amount of psychos from the Corynebacterium glutamicum transformants incorporating cyclose 3-epimerase. The gray bar is the result of 24 hours of psychos production, and the dark gray bar is the result of 48 hours of psychos production.

Figure 2 is a result of measuring the amount of Pseudopsis while recovering and cultured four times the Corynebacterium glutamicum transformed cells incorporating Pycos 3-epimerase. Panel A shows the results of the cycle production, panel B shows the concentration of the cells, and Panel C shows the results of dividing the cycles produced by the cell concentration.

The present invention includes a polynucleotide encoding an enzyme having an activity of catalyzing the conversion of fructose to a psychocos, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 1, thereby safely and efficiently purifying the fructose from fructose. The present invention relates to a Corynebacterium microorganism capable of mass production, and a method for producing a psychosis using the same.

This specification includes all the contents described in the specification and drawings of Korean Patent Application No. 10-2013-0060703.

Corynebacterium microorganism of the present invention comprises a polynucleotide encoding an enzyme having an activity that catalyzes the conversion of fructose to the psychos, the polynucleotide encodes the amino acid sequence of SEQ ID NO: 1.

Amino acid sequence of SEQ ID NO: 1 is a MKHGIYYSYW EHEWSAKFGP YIEKVAKLGF DIIEVAAHHI NEYSDAELAT IRKSAKDNGI ILTAGIGPSK TKNLSSEDAA VRAAGKAFFE RTLSNVAKLD IHTIGGALHS YWPIDYSQPV DKAGDYARGV EGINGIADFA NDLGINLCIE VLNRFENHVL NTAAEGVAFV KDVGKNNVKV MLDTFHMNIE EDSFGDAIRT AGPLLGHFHT GESNRRVPGK GRMPWHEIGL ALRDINYTGA VIMEPFVKTG GTIGSDIKVW RDLSGGADIA KMDEDARNAL AFSRFVLGG.

Corynebacterium microorganism of the present invention is not particularly limited as long as it contains the polynucleotide, for example, Corynebacterium glutamicum, preferably Corynebacterium glutamicum ATCC13032 (Corynebacterium glutamcium ATCC13032, taxid GenBank NID: NC_003450, ATCC13032) may be introduced into the polynucleotide.

Corynebacterium glutamicum ATCC13032 has been deposited with ATCC (American Type Culture Collection, Manassas, USA).

Corynebacterium glutamicum is a GRAS (Generally Recognized as Safe) strain, and in particular, Corynebacterium glutamicum ATCC13032 is an enzyme having an activity that catalyzes the conversion of fructose to psychos, when the polynucleotide comprises the polynucleotide. Can be produced with excellent efficiency, and the conversion rate of fructose to psychos is very excellent.

The enzyme may be an enzyme derived from Agrobacterium tumefaciens.

The polynucleotide encodes the amino acid sequence of SEQ ID NO: 1.

The polynucleotide may be introduced by any method known in the art. The polynucleotide may be introduced into the polynucleotide itself or by a vector, for example.

The term "vector" refers to a nucleic acid molecule capable of delivering other nucleic acids to which it is linked. From the standpoint of nucleic acid sequences that mediate the introduction of specific genes, in the present invention, a vector is interpreted to be used interchangeably with a nucleic acid construct and a cassette.

Vectors include, for example, plasmids or viral derived vectors. Plasmid refers to a circular double stranded DNA ring to which additional DNA can be linked. Vectors used in the present invention include, for example, a plasmid expression vector, a plasmid shuttle vector, a virus expression vector (e.g., a replication defective retrovirus vector, a retrovirus vector, an adenovirus vector, a herpes simplex virus vector, a poxvirus vector, a lenti Viral vectors, adeno-associated viral vectors, etc.) and viral vectors capable of performing equivalent functions thereof are included, but are not limited to these.

The polynucleotide may be operably linked with one or more elements consisting of a promoter and a transcription terminator. In addition, the polynucleotide may be operably linked with a regulatory element. For example, the regulatory element may be selected from the group consisting of chemical inducible elements and temperature sensitive elements. The chemical inducible element may be selected from the group consisting of lac operon and arabinose operon.

The microorganism of the genus Corynebacterium according to the present invention is a ptsF (EII ^Fru , fruA, NCgl1861, GI: 19553141, GI: 19553141, EC 2.7), which is a PTS transport system that transports into cells while converting endogenous di-fractose into di-fractose 1-phosphate. .1.69) may be missing or inactivated gene having a homology of 80% or more, preferably the same gene may be missing or inactivated.

Psychoses are generated from D-fructose, so depletion or inactivation of the gene can inhibit the phosphorylation of fructose, which can significantly improve the production efficiency of psychos.

The ptsF gene may be encoding the amino acid sequence of SEQ ID NO: 2.

Amino acid sequence of SEQ ID NO: 2 is MNSVNNSSLV RLDVDFGDST TDVINNLATV IFDAGRASSA DALAKDALDR EAKSGTGVPG QVAIPHCRSE AVSVPTLGFA RLSKGVDFSG PDGDANLVFL IAAPAGGGKE HLKILSKLAR SLVKKDFIKA LQEATTEQEI VDVVDAVLNP APKTTEPAAA PAAAAVAESG AASTSVTRIV AITACPTGIA HTYMAADSLT QNAEGRDDVE LVVETQGSSA VTPVDPKIIE AADAVIFATD VGVKDRERFA GKPVIESGVK RAINEPAKMI DEAIAASKNP NARKVSGSGV AASAETTGEK LGWGKRIQQA VMTGVSYMVP FVAAGGLLLA LGFAFGGYDM ANGWQAIATQ FSLTNLPGNT VDVDGVAMTF ERSGFLLYFG AVLFATGQAA MGFIVAALSG YTAYALAGRP GIAPGFVGGA ISVTIGAGFI GGLVTGILAG is LIALWIGSWK VPRVVQSLMP VVIIPLLTSV VVGLVMYLLL GRPLASIMTG LQDWLSSMSG SSAILLGIIL GLMMCFDLGG PVNKAAYLFG TAGLSTGDQA SMEIMAAIMA AGMVPPIALS IATLLRKKLF TPAEQENGKS SWLLGLAFVS EGAIPFAAAD PFRVIPAMMA GGATTGAISM ALGVGSRAPH GGIFVVWAIE PWWGWLIALA AGTIVSTIVV IALKQFWPNK AVAAEVAKQE AQQAAVNA.

In addition, the microorganism of the genus Corynebacterium according to the present invention has a homology of 80% or more with mtlD (NCgl0108, GI: 19551360, EC 1.1.1.67) encoding mannitol 2-dehydrogenase The gene may be deleted or inactivated, and preferably, the same gene may be deleted or inactivated.

Since fructose is converted to D-mannitol by mannitol 2-dihydrogenase, deletion or inactivation of the gene can suppress the reduction of fructose, thereby significantly improving the production efficiency of psychos. can do.

The mtlD gene may be encoding the amino acid sequence of SEQ ID NO: 3.

Amino acid sequence of SEQ ID NO: 3 is MNTPLQLNTE NLQEIASTSG VQIPAFNRAD VAPGIVHFGV GGFHRAHQAM YLNELMNEGK ALDWGIIGMG VMPSDVRMRD ALASQDHLYT LTTKAPDGTL DQKIIGSIID YVFAPEDPAR AVATLAQDSI RIVSLTVTEG GYNIDPATED FDHTNPRIVA DREALQAGDT STLQTFFGLI TAALISRKES GSTPFTIMSC DNIQGNGDLA KRFFLAFAHS VSSELGEWVE NNVAFPNSMV DRITPETTDG DRDDIKEIGY IDAWPVVSED FTQWVLEDAF TQGRPAYEEV GVQVVSDVEP YELMKLRLLN ASHQGLCYFG HLAGHHMVHD VMADTRFQDF LLAYMEREAT PTLKELPGVD LDAYRRQLIA RFGNAAVKDT VPRLCAESSD RIPKWLLPVV RENLAAGRDV TLSAAIVASW ARYAEGTDEQ GNPIKIVDRL SERVQENASG NRTDILSFIR DRGIFGDLVD AEPFTKAYSE TLSSLHDRGA EATIDALLTQ VTV.

The term "deleted" or "inactivated" means that the expression of the gene is reduced or not expressed. The "inactivation" can be made by methods known in the art. For example, it may be inactivated by homologuous recombination. The homologous recombination may be mediated by, for example, transposon mutagenesis or P1 transduction.

The microorganism itself can obtain advantages such as enzyme immobilization as an enzyme carrier or a container containing the enzyme. In the case of microorganisms, only microorganisms can be recovered and reused through centrifugation, but not in enzymes. In addition, the enzyme requires a secondary process of immobilization on the carrier in order to create an environment in which the activity can be maintained for a long time, but in the case of microorganisms, it is possible to reuse it by preventing the enzyme activity from rapidly degrading by sufficiently serving as a carrier. Do it.

Therefore, the present invention shows a remarkably improved effect compared to the conventional method for producing a psychos using an enzyme or the like.

The present invention also provides a method for producing a psychos.

One embodiment of the method of producing a psychosis of the present invention will be described in more detail in the steps.

First, the microorganism is cultured in a medium containing fructose.

The culture conditions of the microorganisms are not particularly limited, and conditions used for culturing the transformant of the genus Corynebacterium known in the art may be used. For example, the culture temperature may be 26 to 38 ℃.

The medium may be a nutrient medium containing yeast extract and nitrogen sources, such as 2YT medium, LB medium, TB medium.

In addition, carbon sources, including glucose, glycerol and the like; Nitrogen sources, including ammonia, urea, and the like; Essential metal ions such as sodium, potassium, calcium, magnesium, and manganese; It may be a life medium (defined medium) commonly used in the art including vitamins and the like.

The fructose concentration contained in the medium is not particularly limited, and may be included, for example, at a concentration of 1% (w / v) to 80% (w / v), preferably 1% (w / v) to 50%. (w / v).

The culture can be continuous, semi-continuous, or batch type culture.

The culturing may be carried out by further adding a substance that induces the expression of a polynucleotide encoding an enzyme having an activity of catalyzing the conversion of fructose to sicose.

The substance inducing the expression of the polynucleotide is not particularly limited and may be a substance commonly used in the art.

Thereafter, the cycos is recovered from the culture of the microorganism.

The method for recovering the psychocos is not particularly limited and may be based on methods known in the art, and examples thereof include centrifugation, filtration, crystallization, ion exchange chromatography, and the like.

Specifically. The culture may be carried out by centrifugation to separate the culture from the microorganism, and by separating the psychose from the culture by the recovery method.

The method for producing a psychos of the present invention, before the step of culturing the microorganisms in the medium containing fructose, the enzyme having an activity that catalyzes the conversion of fructose to psychos by culturing the microorganisms in a medium not containing fructose Expressing a polynucleotide encoding a nucleotide; And recovering the microorganisms from the culture of the microorganisms.

The medium not containing fructose may be the same category of medium as the medium containing fructose, except that the fructose is not included.

The method for recovering the microorganism from the culture of the microorganism is not particularly limited, and a method known in the art may be used, and examples thereof include centrifugation and filtration.

The microorganisms recovered by this method can be used at a desired concentration, for example in a concentrated state. For example, the transformant of Corynebacterium glutamicum can be inoculated and cultured in a medium containing the fructose at high concentration. Therefore, the microorganism used in the culturing in the medium containing fructose may be the recovered microorganism.

The recovered microorganism has a turbidity (measured at 600 nm absorbance, OD ₆₀₀ ) of cells in a medium containing fructose in a range of 0.01 to 300, for example, 1 to 300, 10 to 300, 20 to 300, 5 to 300, or It can be inoculated at a concentration such that 40 to 300. Thus, by using a cell containing a high concentration of the enzyme, it is possible to efficiently convert fructose to a psychos in a medium containing high concentration of fructose in the medium.

In the method, when the recovered cells are used, the fructose may be included in the medium at a concentration of, for example, 1% (w / v) to 80% (w / v). Within this range, for example, 1% (w / v) to 35% (w / v), 10% (w / v) to 80% (w / v), 20% (w / v) to 80% (w / v), 30% (w / v) to 80% (w / v), 30% (w / v) to 80% (w / v) and the like.

The method for producing a psychosis of the present invention may further include recovering the microorganism from the culture of the microorganism, and culturing the separated microorganism in a medium containing fructose again.

The isolated microorganisms can be used for the production of 2 to 10 times, preferably 3 to 10 times, more preferably 4 to 10 times. The isolated microorganism may be inoculated at a concentration such that, for example, OD ₆₀₀ is 5 to 150 in the medium. Within this range, for example, 5 to 150, 10 to 150, 20 to 150, 10 to 100, 20 to 100, 20 to 80, 20 to 40, 40 to 100, and the like.

Thus, by using a microorganism containing a high concentration of the enzyme can inhibit the growth of microorganisms in a high concentration of fructose, it is possible to efficiently convert fructose to a psychos in a medium containing a high concentration of fructose in the medium.

When the isolated Corynebacterium glutamicum cells are used, the fructose is 1% (w / v) to 80% (w / v), 1% (w / v) to 35% (w / v) , 10% (w / v) to 80% (w / v), 20% (w / v) to 80% (w / v), or 30% (w / v) to 80% (w / v) or It may be included in the medium at a concentration of 30% (w / v) to 50% (w / v).

Hereinafter, the present invention will be described in detail with reference to Examples.

Example 1 Preparation of Corynebacterium Transformants

The pCES208 (J. Microbiol. Biotechnol., 18: 639-647, 2008), an E. coli-Corinebacterium shuttle vector, was modified and used to construct a pSGT208 shuttle vector in which a terminator and a lac promoter were inserted.

Psychos 3-epimerase (Agrobacterium tumefaciens str. C58; taxid: 176299; GenBank NID: NC_003062, ATCC33970) for the production of Psychos from Corynebacterium glutamicum AGR_L_260, GI: 15890243) were introduced into the pSGT208 shuttle vector thus prepared and used.

In detail, the primers 1 and 2 were used to amplify the dpe gene from the Agrobacterium tumefaciens genome, cut it with restriction enzymes KpnI and BamHI, insert the same site of the pSGT208 shuttle vector, and then synthesize the Pycos 3-epimerase. A pS208-dpe recombinant shuttle vector was constructed.

Later, in order to increase the expression level of Psychos 3-epimerase in Corynebacterium glutamicum, the lac promoter was replaced with the trc promoter derived from pTrc99a in pS208-dpe, which was named pS208cT-dpe.

Recombinant vectors pS208-dpe, pS208cT-dpe and pSGT208 vector as a negative control thereof were introduced into the wild type Corynebacterium glutamicum ATCC 13032 and transformed into fructose. It was used for the production of the psychos. Transformation followed the method specified in the Handbook of Corynebacterium glutamicum (Lothar Eggeling et al., ISBN 0-8493-1821-1, 2005 by CRC press).

The primers used are listed in Table 1 below.

Table 1

Example 2 Production of Pycos from Fructose Using Transformants of Corynebacterium glutamicum

When high concentrations of fructose are added, it is possible to produce high concentrations of psychos if it is possible to add high concentrations of cells cultured separately to solve the problem of inhibiting cell growth.

In order to secure high concentration of cells, the Corynebacterium glutamicum transformant prepared in Example 1 was inoculated in 5 ml of LB medium (Difco) containing 20 µg / ml of kanamycin and subjected to 30 ° C. and 250 rpm conditions. After incubation with minimal media with 5 g / L glucose and 20 μg / ml kanamycin (1 g K ₂ HPO ₄ , 10 g (NH ₄ ) ₂ SO ₄ , 0.4 g MgSO ₄ 7H ₂ O, 20 mg FeSO ₄ 7H ₂ O, 20mg MnSO ₄ H ₂ O, 50mg NaCl, 2g urea, 0.1mg biotin, 0.1mg thiamine) was inoculated and cultured.

The main culture was incubated for 36 hours at 30 ° C. and 180 rpm at a volume of 50 ml in a slotted 300 ml Erlenmeyer flask to induce sufficient cell mass and sufficient expression of protein.

The obtained culture solution was centrifuged to remove the supernatant and the cells were recovered, and the cell concentration was resuspended at 65 OD ₆₀₀ in the same medium as above containing 40% (w / v) fructose as a substrate. The cells were cultured at 180 rpm.

Concentrations of fructose and psychocos were measured using high performance liquid chromatography (HPLC). HPLC was used for SCL-10A (Shimadzu, Japan), Kromasil 5NH ₂ column (4.6 mm x 250 mm), mobile phase was separated at 40 ℃ while flowing at 1.5 mL / min using 75% acetonitrile and then RI (Reflective Index) detector was used for analysis. Under the above conditions, the retention time of fructose was 5.5 minutes, and the cycle course was 4.6 minutes.

The measurement result is shown in FIG. The gray bar is the result of 24 hours of psychos production, and the dark gray bar is the result of 48 hours of psychos production.

Referring to FIG. 1, in the case of Corynebacterium glutamicum ATCC13032 in which only the pSGT208 shuttle vector was introduced as a negative control, no psychoses were detected, and thus Corynebacterium glutamicum intrinsically produces cycos from fructose. It seems to have no path.

In the experimental group using the pS208-dpe recombinant shuttle vector, approximately 7.4 g / L of Psychos were produced at 24 hours, indicating that Pycos 3-Epimerase was significantly expressed in Corynebacterium glutamicum transformants. It was confirmed that at this time, about 8.7 g / L is produced in 48 hours was confirmed that the production was almost finished in 24 hours.

In the experimental group using the pS208cT-dpe recombinant shuttle vector, the production of psychos was greatly increased to about 51.5 g / L at 24 hours and about 61.2 g / L at 48 hours. Nebacterium glutamicum shows high activity, and the conversion rate of fructose to psychos can be seen as a result indicating that it is dependent on the amount of expression.

Example 3: Continuous Production of Pycos from Fructose by Recovery after Reaction of Corynebacterium glutamicum Transformed Cells

As a result of Example 2, it can be seen that the psychos 3-epimerase showed a maximum activity at 24 hours in the Corynebacterium glutamicum transformant and then did not increase significantly.

Based on these results, in order to confirm how long the activity of converting the fructose to the psychos after 24 hours, the cells were incubated for 24 hours in the presence of fructose, and the cells were separated from the culture. Then, the process of culturing the cells for 24 hours in the presence of fructose was repeated four times.

Culture conditions and analysis methods were performed in the same manner as in Example 2.

The measurement result is shown in FIG. Panel A shows the results of the cycle production, panel B shows the concentration of the cells, and Panel C shows the results of dividing the cycles produced by the cell concentration.

Referring to FIG. 2, the cell concentration (Panel B of FIG. 2) tended to decrease as the number of reactions increased, which was caused by the centrifugation of the cell reaction solution to separate the reaction supernatant and the cell. This is because of the loss.

The conversion level of Psychos was about 50-60 g / L during 4 repetitions, which did not show a big difference (Panel A of FIG. 2), thereby resulting in cells of recombinant Corynebacterium glutamicum transformants. And it can be seen that the activity of Pseudomonas 3-epimerase expressed in cells is stable even if repeated several times. These results can be confirmed in more detail that in the panel C of FIG.

Claims (15)

1. A polynucleotide encoding an enzyme having an activity that catalyzes the conversion of fructose to psychos,

   The polynucleotide is a Corynebacterium microorganism that encodes the amino acid sequence of SEQ ID NO: 1.
2. The microorganism of claim 1, wherein the polynucleotide is introduced into a Corynebacterium glutamicum strain.
3. The microorganism of claim 1, wherein the polynucleotide is introduced into Corynebacterium glutamicum ATCC13032.
4. The microorganism of claim 1, wherein the polynucleotide is introduced into the microorganism by itself or using a vector.
5. The method according to any one of claims 1 to 3, wherein at least one of a gene having at least 80% homology with the amino acid sequence of SEQ ID NO: 2 and a gene having at least 80% homology with the amino acid sequence of SEQ ID NO: 3 is deleted or inactivated , Microorganisms.
6. The microorganism of claim 1, wherein at least one of ptsF having the amino acid sequence of SEQ ID NO: 2 and mtlD having the amino acid sequence of SEQ ID NO: 3 is deleted or inactivated.
7. Culturing the microorganism of any one of claims 1 to 3 in a medium containing fructose; And

   Recovering the psychos from the culture of the microorganisms.
8. The method of claim 7, wherein the fructose is at a concentration of 1% (w / v) to 80% (w / v).
9. The method of claim 7, wherein the medium is a nutrient medium or a life preserver.
10. The method of claim 7, wherein prior to the step of culturing the microorganism

    Culturing the microorganism of any one of claims 1 to 3 in a medium free of fructose to express a polynucleotide encoding an enzyme having an activity that catalyzes the conversion of fructose to psychos; And

    Recovering the microorganisms from the culture of the microorganisms.
11. The method of claim 10, wherein the recovered microorganism is inoculated and cultured at a concentration such that the OD ₆₀₀ is 0.01 to 300 in a medium containing fructose.
12. The method of claim 11, wherein the fructose concentration of the medium comprising fructose is between 1% (w / v) and 80% (w / v).
13. The method of claim 7, further comprising recovering the microorganisms from the culture of the microorganisms, and culturing the separated microorganisms again in a medium containing fructose.
14. The method of claim 13, wherein the isolated microorganism is inoculated and cultured at a concentration such that the OD ₆₀₀ is 5 to 150 in a medium containing fructose.
15. The method of claim 13, wherein the fructose concentration of the fructose-containing medium is 10% (w / v) to 80% (w / v).

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