Method for preparing D-chiro-inositol using microbes

Abstract

The present invention relates to a method for preparing D-chiro-inositol from myo-inositol by using a transformed host cell which expresses enzymes such as a myo-inositol transporter, inositol dehydrogenase, and inosose isomerase. According to the method of the present invention, myo-inositol can be converted into D-chiro-inositol at a high yield.

Claims

1. A method for preparing D-chiro-inositol from myo-inositol, comprising: (a) transforming a host cell with (i) a recombinant vector comprising a myo-inositol transporter coding DNA sequence operatively linked to a promoter; and (ii) a recombinant vector comprising an inositol dehydrogenase coding DNA sequence and an inosose isomerase coding DNA sequence operatively linked to a promoter; and (b) culturing the host cell of (a) in a medium comprising myo-inositol, thereby preparing D-chiro-inositol, wherein the host cell is selected from the group consisting of a mammalian cell, an insect cell, a yeast cell, and a prokaryotic cell, wherein the myo-inositol transporter has the amino acid sequence set forth in SEQ ID NO: 3, wherein the inositol dehydrogenase has the amino acid sequence set forth in SEQ ID NO: 31, and wherein the inosose isomerase has the amino acid sequence set forth in SEQ ID NO: 30.

2. The method of claim 1, wherein the host cell is a prokaryotic cell.

3. The method of claim 1, further comprising (c) separating D-chiro-inositol from a culture product of the transformed host cell after the step (b).

4. A host cell, transformed with a recombinant vector, the recombinant vector comprising: (i) a myo-inositol transporter coding DNA sequence operatively linked to a promoter; and (ii) an inositol dehydrogenase coding DNA sequence and an inosose isomerase coding DNA sequence which are operatively linked to a promoter, wherein the host cell is selected from the group consisting of a mammalian cell, an insect cell, a yeast cell, and a prokaryotic cell, wherein the myo-inositol transporter has the amino acid sequence set forth in SEQ ID NO: 3, wherein the inositol dehydrogenase has the amino acid sequence set forth in SEQ ID NO: 31, and wherein the inosose isomerase has the amino acid sequence set forth in SEQ ID NO: 30.

5. A composition for preparing D-chiro-inositol from myo-inositol comprising the host cell of claim 4.

6. A recombinant vector kit for preparing D-chiro-inositol from myo-inositol, comprising: (i) a recombinant vector comprising a myo-inositol transporter DNA coding sequence operatively linked to a promoter; and (ii) a recombinant vector comprising an inositol dehydrogenase coding DNA sequence and an inosose isomerase coding DNA sequence operatively linked to a promoter as an active component, wherein the myo-inositol transporter has the amino acid sequence set forth in SEQ ID NO: 3, wherein the inositol dehydrogenase has the amino acid sequence set forth in SEQ ID NO: 31, and wherein the inosose isomerase has the amino acid sequence set forth in SEQ ID NO: 30.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a chemical structure of myo-inositol and D-chiro-inositol.

(2) FIG. 2 shows an HPLC chromatogram on myo-inositol, D-chiro-inositol and a stereoisomer or derivatives thereof.

(3) FIG. 3 shows results of measuring a conversion ratio of D-chiro-inositol from myo-inositol and a bacterial growth rate in a transformed E. coli obtained by additionally introducing an inositol transporter of various origins to E. coli, which expresses myo-inositol dehydrogenase and inosose isomerase. Each test group for inositol transporter shows a result from culturing for 24 and 48 hours recombinant E. coli prepared by introducing a transporter expression vector along with pCOLAD-sAtiep-sAtiepf. A white rod indicates a value measured after culturing for 24 hours, while a gray rod represents a value measured after culturing for 48 hours.

(4) FIG. 4 shows a phylogenetic tree by homology analysis of myo-inositol dehydrogenase (iolG).

(5) FIG. 5 shows a phylogenetic tree by homology analysis of inosose isomerase (ioll).

(6) FIGS. 6a to 6c show a result of measuring a conversion ratio of D-chiro-inositol from myo-inositol in an inventive transformed E. coli according to culture temperatures. A test group for each symbol is as shown below; .circle-solid.: 30° C. and o: 37 in FIG. 6a. o: microbe growth, .circle-solid.: pH and .box-tangle-solidup.: amount of remaining glycerol at the temperature of 30° C. in FIG. 6b. o: microbe growth, .circle-solid.: pH and .box-tangle-solidup.: amount of remaining glycerol at the temperature of 37° C. in FIG. 6c.

(7) FIG. 7 shows a result of measuring a conversion ratio of D-chiro-inositol from myo-inositol according to induction time when culturing the transformed E. coli of the present invention. A test group for each symbol is as shown below; .square-solid.: no induction, o: induction at time 0, A: induction at OD 0.6, V: induction at OD 3 and .diamond-solid.: induction at OD 10.

(8) FIG. 8 shows a result of measuring a conversion ratio of D-chiro-inositol from myo-inositol according to induction types when culturing the transformed E. coli of the present invention. A test group for each symbol is as shown below; .square-solid.: add 1 mM IPTG at OD 0.6, o: add 0.5% lactose at time 0, A: add 0.5% lactose at OD 0.6, V: add 0.5% lactose at time OD 7, and .diamond-solid.: add 0.1% lactose at time OD 0.6.

(9) FIG. 9 shows a result of measuring a D-chiro-inositol production rate (panel A), a microbe growth (panel B) and a gradient of production speed (panel C), with regard to a recombinant strain obtained by introducing the inositol dehydrogenase and inosose isomerase genes derived from various strains and a combination thereof. An experiment was performed by introducing an inositol transporter expression recombinant plasmid vector pACYCD-StiolT1-StiolT2 together to a transformed E. coli. A test group for each symbol is as shown below; .square-solid.: pCOLAD-Cgiep-Paioll, o: pCOLAD-sAtiep-sAtiepf, A: pCOLAD-AGRL628-AGRL627, V: pCOLAD-BsiolG-Bsioill, .diamond-solid.: pCOLAD-CgiolG-CgiollC>169 and >: pCOLAD-Paidh-Paioll.

(10) FIG. 10 shows a result of checking the preparation of D-chiro-inositol from myo-inositol by performing 1 L fermentation under optimal conditions by using the transformed E. coli which is selected in the present invention.

(11) FIG. 11 shows a result of performing GC-MS analysis on a D-chiro-inositol fraction prepared by a stain of the present invention.

Best Mode

(12) Hereinafter, the present invention will be described in detail in light of the following exemplary embodiments. While those exemplary embodiments are intended to describe the present invention more clearly, it would be obvious to those skilled in the art that they are not used to limit the range of the present invention disclosed in the claims.

Exemplary Embodiment

Exemplary Embodiment 1: Cloning of Myo-Inositol Transporter Gene and Preparation of a Recombinant Vector Including the Same

(13) Each of major and minor genes of a myo-inositol transporter was cloned from Bacillus subtilis, Salmonella typhimurium or Agrobacterium tumefaciens strains, which are reported to use myo-inositol as a carbon source.

(14) In case of B. subtilis, a myo-inositol transporter gene is an iolT gene and an iolF gene. In case of S. typhimurium, a myo-inositol transporter gene is an iolT 1 gene and an iolT2 gene. In case of A. tumefaciens, a myo-inositol transporter gene is an Atu5935 gene and an Atu2525 gene. Information on the genes above is as shown in Table 1.

(15) TABLE-US-00001 TABLE 1 Genbank Accession No. Strain Gene SEQ ID NO: (GI, NID, PID) B. subtilis BsioIT SEQ ID NO: 1 BSU06230, GI: 2632936 BsioIF SEQ ID NO: 2 BSU39710, GI: 225185479 S. typhimurium StioIT1 SEQ ID NO: 3 STM4418, GI16422981 StioIT2 SEQ ID NO: 4 SMT4419, GI: 16422982 A. tumefaciens Atu5935 SEQ ID NO: 5 Atu5935, G1: 16119622 Atu2525 SEQ ID NO: 6 Atu2525, GI: 15889790

(16) 1-1. Preparation of Recombinant Vector pACYCD-BsiolT(F2) and pACYCD-BsiolT-BsiolF(F2)

(17) In case of B. subtilis strain, a major transporter iolT and a minor transporter iolF are introduced from a genome DNA of Bacillus subtilis subsp. subtilis_str. 168 (taxid:224308; GenBank NID: NC_000964, ATCC23857) into a pACYCDuet-1 expression vector, so that pACYCD-BsiolT(F2) and pACYCD-BsiolT-BsiolF(F2) is constructed. To describe in detail, iolT is amplified by using primer BsiolT-F2 and BsiolT-R from the genome DNA of B. subtilis, cut off with restriction enzymes SacI and BamHl, and inserted into the same region of vector pACYDuet-1 (Novagen), so that pACYCD-BsiolT(F2) is prepared. Also, iolF is amplified by using primer BsiolF-F and BsiolF-R, cut off with restriction enzymes Ndel and Sail, and inserted into the Ndel and Xhol regions of the pACYCD-BsiolT(F2) prepared above, so that pACYCD-BsiolT-BsiolF(F2) is prepared.

(18) 1-2. Preparation of Recombinant Vector pACYCD-StiolT1(F2) and pACYCD-StiolH-StiolT2(F2)

(19) In case of S. typhimurium strain, iolT 1, known as a major transporter, and iolT2, known as a minor transporter, are amplified from Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 ATCC700720 (taxid:99287; GenBank NID: NC_006511, ATCC700720), introduced into pACYCDuet-1 expression vector, so that pACYCD-StiolH (F2) and pACYCD-StiolT1-StiolT2(F2) is constructed. To describe in detail, iolT 1 is amplified from the genome DNA of S. typhimurium by using primer StiolT 1-F2 and StiolT 1-R, cut off with restriction enzymes Ncol and BamHl, and inserted into the same region of pACYDuet-1 vector, so that pACYCD-StiolTl (F2) is prepared. Also, iolT2 is amplified by using primer StiolT2-F and StiolT2-R, cut off with restriction enzymes Ndel and Sail, and inserted into the Ndel and Xhol regions of the pACYCD-StiolT 1 (F2) prepared above, so that pACYCD-StiolT1-StiolT2(F2) is prepared.

(20) 1-3. Preparation of Recombinant Vector pACYCD-Atu5935(F2) and pACYCD-Atu5935-Atu2525(F2)

(21) In case of A. tumefaciens, Atu5935 is amplified from the genome DNA of Agrobacterium tumefaciens str. C58 (taxid:176299; GenBank NID: NC_003062, ATCC33970) by using primer Atu5935-F2 and Atu5935-R, cut off with restriction enzymes Ncol and BamHl, and inserted into the same region of pACYCDuet-1 vector, so that pACYCD-Atu5935(F2) is prepared. Also, Atu2525 is amplified by using primer Atu2525-F and Atu2525-R, cut off with restriction enzymes Ndel and Sail, and inserted into the Ndel and Xhol regions of the pACYCD-Atu2525(F2) prepared above, so that pACYCD-Atu5935-Atu2525(F2) is prepared.

(22) 1-4. Preparation of Recombinant Vector pCOLAD-sAtiep-sAtiepf

(23) To evaluate the activity of the cloned myo-inositol transporter, an inositol dehydrogenase gene (iolG) and an inosose isomerase gene (ioll), which convert myo-inositol into D-chiro-inositol, are cloned from A. tumefaciens and introduced into vector pCOLADuet-1 (Novagen), so that a recombinant plasmid vector pCOLAD-sAtiep-sAtiepf is prepared. To describe in more detail, DNA synthesis [GenScript Inc., 860 Centennial Ave., Piscataway, N.J. 08854, USA] is performed based on amino acid sequences of BD171257_CDS1 and CDS2 of Agrobacterium sp. AB10121 (W002/055715A), so that a synthesized DNA molecule sAtiep (SQ ID No. 50) and a synthesized DNA molecule sAtiepf (SQ ID No. 51) is prepared respectively. The synthesized DNA molecule sAtiep, as a template, is PCR-amplified by using primer sAtiep-TF and sAtiep-TR, cut off with restriction enzymes BspHl and SacI, and inserted into the same restriction enzyme region of pCOLADuet-1, so that pCOLAD-sAtiep is prepared. Then, the synthesized DNA molecule sAtiepf is cut off with Ndel and Sail, and inserted into the Ndel and Xhol regions of the prepared vector pCOLAD-sAtiep, so that a recombinant vector pCOLAD-sAtiep-sAtiepf is prepared finally.

(24) The primers used for preparing the recombinant vector are summarized in Table 2, and information on the prepared recombinant vectors and sequences related thereto is summarized in Table 3.

(25) TABLE-US-00002 TABLE 2 Restriction enzyme Primer SEQ ID NO: region BsioIT-F2 SEQ ID NO: 7 SacI BsioIT-R SEQ ID NO: 8 BamHI BsioIF-F SEQ ID NO: 9 BamHI-NdeI BsioIF-R SEQ ID NO: 10 SaiI StioIT1-F2 SEQ ID NO: 11 EcoRI-NcoI StioIT1-R SEQ ID NO: 12 BamHI StioIT2-F SEQ ID NO: 13 BamHI-NdeI StioIT2-R SEQ ID NO: 14 SalI Atu5935-F2 SEQ ID NO: 15 EcoRI-NcoI Atu5935-R SEQ ID NO: 16 BamHI Atu2525-F SEQ ID NO: 17 BamHI-NdeI Atu2525-R SEQ ID NO: 18 SaiI sAtiep-TF SEQ ID NO: 19 BspHI sAtiep-TR SEQ ID NO: 20 SacI

(26) TABLE-US-00003 TABLE 3 Vector, Recombinant Genbank Accession No. (GI, NID, PID) Vector, Gene or References pACYDuet-1 Novagen pACYCD-BsioIT(F2) The present invention pACYCD-BsioIT-BsioIF(F2) The present invention pACYCD-StioIT1(F2) The present invention pACYCD-StioIT1-StioIT2(F2) The present invention pACYCD-Atu5935(F2) The present invention pACYCD-Atu5935-Atu2525(F2) The present invention BD171257-CDS1 BD171257 or GI: 27877069 BD171257-CDS2 BD171257 or GI: 27877069 sAtiep GenScript Inc. sAtiepf GenScript Inc. (containing restriction enzymes NdeI and SaiI) pCOLADuet-1 Novagen pCOLAD-sAtiep-sAtiepf The present invention

Exemplary Embodiment 2: Evaluation of the Activity of Myo-Inositol Transporter

(27) As prepared in the Exemplary Embodiment 1 above, each recombinant plasmid containing a myo-inositol transporter derived from B. subtilis, S. typhimurium and A. tumefaciens, pACYCD-BsiolT(F2), pACYCD-BsiolT-BsiolF(F2), pACYCD-StiolT 1 (F2), pACYCD-StiolT 1-StiolT2(F2), pACYCD-Atu5935(F2) and pACYCD-Atu5935-Atu2525(F2), are transformed in E. coli BL21(DE3) along with a recombinant plasmid pCOLAD-sAtiep-sAtiepf including inositol dehydrogenase and inosose isomerase coding genes, which convert from myo-inositol to D-chiro-inositol, so that a transformed strain is prepared.

(28) A 5 mL of the recombinant transformed strain prepared above is cultured in a glass test tube having 25 mm in diameter and 150 mm in height. When it comes to culture conditions, when a microbe reaches OD 0.6 under the conditions of 30° C. and 250 rpm by using an M9 minimum medium containing 1% (w/v) myo-inositol, 50 mg/L of chloramphenicol and 50 mg/L of kanamycin, 1 mM of IPTG is added to perform induction, and cultured for 48 hours. For the analysis of myo-inositol and D-chiro-inositol, a culture fluid is put into centrifugation, a 1 mL of culture supernatant thereof is taken out, boiled for 10 minutes, put into centrifugation again, and a 500 uL of supernatant thereof is taken out. The pre-treated culture supernatant is analyzed with HPLC (Shimadzu LCIOAvp) by using an RI detector under the conditions of Kromasil 5NH2 column (4.6 mm×250 mm), mobile phase 75% acetonitrile and column temperature 40° C. The HPLC chromatogram of myo-inositol, D-chiro-inositol and isomers thereof are shown in FIG. 2.

(29) The results of measuring the activity of each myo-inositol transporter are shown in FIG. 3. According to the results of FIG. 3, in case of pACYCD-StiolT1-StiolT2 containing both major and minor transporters StiolT 1 and StiolT2 derived from S. typhimurium in pACYCDuet-1, a conversion ratio of D-chiro-inositol is best. In the myo-inositol transporter derived from B. subtilis and A. tumefaciens, a conversion ratio of D-chiro-inositol is lowest. In case of S. typhimurium transporter having the best conversion ratio, about 1.2 g/L of D-chiro-inositol is converted from 10 g/L myo-inositol in 24 hours, while about 1.1 g of D-chiro-inositol is converted in 48 hours. Therefore, it is found that a conversion ratio does not increase in 24 hours later. When a gene is introduced, a microbe growth appears to slightly slow down. That's probably because it is located on a cell membrane due to the characteristics of transporter protein, thus exhibiting toxicity upon over-expression.

Exemplary Embodiment 3: Cloning of Inositol Dehydrogenase and Inosose Isomerase Genes and Preparation of a Recombinant Vector Including the Same

(30) Through continuous responses of Reaction Equations 1 to 3 below, D-chiro-inositol is converted from myo-inositol. The reactions of Reaction Equations 1 and 3 are catalyzed by inositol dehydrogenase and the reaction of Reaction Equation 2 is catalyzed by inosose isomerase.
Myo-inositol+NAD.sup.+←.fwdarw.2-keto-myo-inositol+NADH+H.sup.+  [Reaction Equation 1]
1-keto-D-chiro-inositol+NADH+H.sup.+←.fwdarw.D-chiro-inositol+NAD.sup.+   [Reaction Equation 2]
2-keto-myo-inositol←.fwdarw.1-keto-D-chiro-inositol  [Reaction Equation 3]

(31) A gene having the same function as inositol dehydrogenase gene (iolG) and inosose isomerase gene (ioll) is cloned from A. tumefaciens, B. subtilis, Corynebacterium glutamicum and Pantoea ananatis. Genes corresponding to iolG and ioll genes are amplified from a genome DNA of the strains above, a pair and a combination of the amplified iolG and ioll genes are inserted into pCOLADuet-1 expression vector (Novagen), so that six kinds of recombinant plasmid are prepared. Information on the iolG and ioll homologous genes listed above is shown in Table 4 below.

(32) TABLE-US-00004 TABLE 4 Genbank Accession No. (GI, NID, PID) Strain Gene SEQ ID NO: or References A. tumefaciens sAtiep SEQ ID NO: 21 BD171257_CDS1 or GI: 27877069 sAtiepf SEQ ID NO: 22 BD171257_CDS2 or GI: 27877069 AGRL628 SEQ ID NO: 23 AGR_L_628, GI: 159186216 AGRL627 SEQ ID NO: 24 AGR_L_627, GI: 159186217 B. subtilis BsioIG SEQ ID NO: 25 BSU39700, GI: 255767850 BsioII SEQ ID NO: 26 BSU39680, G1: 16081019 C. glutamicum CgioIG SEQ ID NO: 27 NCg10161 or GI: 19551414 CgioII0169 SEQ ID NO: 28 NCg10169 or GI: 19551422 P. ananatis Paidh SEQ ID NO: 29 PANA_3736, GI: 291619289 PaioII SEQ ID NO: 30 PANA_3268, GI: 291618821 C. glutamicum Cgiep SEQ ID NO: 31 NCgI2957 or GI: 19554252

(33) 3-1. Preparation of Recombinant Vector pCOLAD-sAtiep-sAtiepf

(34) A method for preparing recombinant vector pCOLAD-sAtiep-sAtiepf including iolG and ioll genes from Agrobacterium sp. AB10121 strain (WO 02/055715A).

(35) 3-2. Preparation of Recombinant Vector pCOLAD-AGRL628-AGRL627

(36) A recombinant vector including iolG and ioll genes derived from-A. tumefaciens is prepared through the following method. An AGRL628 gene corresponding to iolG is amplified from a genome DNA of Agrobacterium tumefaciens str. C58(taxid:176299; GenBank NID:NC_003062, ATCC33970) by using primer AGRL628-F and AGRL628-R. Then, the resulting one is cut off with restriction enzymes Pcil and SacI, and introduced into the Ncol and SacI regions of pCOLADuet-1 (Novagen), so that pCOLAD-AGRL628 is prepared. Also, AGRL_627 corresponding to ioll is amplified by using AGRL627-F and AGRL627-R primer. Then, the resulting one is cut off with restriction enzymes Ndel and Sail, and inserted into the Ndel and Xhol regions of pCOLAD-AGRL628, so that pCOLAD-AGRL628-AGRL627 recombinant vector is prepared.

(37) 3-3. Preparation of Recombinant Vector pCOLAD-BsiolG-Bsioll

(38) A recombinant vector including iolG and ioll genes derived from B. subtilis is prepared through the following method. BsiolG corresponding to iolG is amplified from a genome DNA of Bacillus subtilis subsp. subtilis str. 168 (taxid:224308; GenBank NID:NC_000964, ATCC23857) by using BsiolG-F and BsiolG-R primer. Then, the resulting one is cut off with restriction enzymes Pcil and Notl, and inserted into the Ncol and Notl regions of pCOLADuet-1, so that pCOLAD-BsiolG is prepared. Also, Bsioll corresponding to ioll is amplified by using Bsioll-F and Bsioll-R primer. Then, the resulting one is processed with restriction enzymes Ndel and Pacl, and inserted into the same restriction enzyme region of the plasmid pCOLAD-BsiolG constructed above, so that pCOLAD-BsiolG-Bsioll is prepared.

(39) 3-4. Preparation of Recombinant Vector pCOLAD-CgiolG-Cgioll0169

(40) A recombinant vector including iolG and ioll genes derived from C. glutamicum is prepared through the following method. CgiolG corresponding to iolG is amplified from a genome DNA of Corynebacterium glutamicum ATCC 13032 (taxid:196627; GenBank NID:NC_003450, ATCC13032) by using CgiolG-F and CgiolG-R primer, cut off with restriction enzymes BspHl and Notl, and inserted into the Ncol and Notl regions of pCOLADuet-1, so that pCOLAD-CgiolG is prepared. Also, CgiollOI69 corresponding to ioll is amplified by using Cgioll0169-F and Cgioll0169-R primer. Then, the resulting one is processed with Ndel and Pacl, and inserted into the same restriction enzyme region of the pCOLAD-CgiolG constructed above, so that pCOLAD-CgiolG-Cgioll0169 vector is prepared.

(41) 3-5. Preparation of Recombinant Vector pCOLAD-Paidh-Paioll

(42) A recombinant vector including iolG and ioll genes derived from Pantoea ananatis is prepared through the following method. Paidh corresponding to iolG is amplified from a genome DNA of Pantoea ananatis LMG 20103 (taxid:706191; GenBank NID:NC_013956, KCCM40419) by using Paidh-F and Paidh-R primer, cut off with restriction enzymes Pcil and Notl, and inserted into the Ncol and Notl regions of pCOLADuet-1, so that pCOLAD-Paidh is prepared. Also, Paioll corresponding to ioll is amplified by using Paioll-F and Paioll-R primer. Then, the resulting one is processed with Ndel and Pacl, and inserted into the same restriction enzyme region of the pCOLAD-Paidh prepared above, so that pCOLAD-Paidh-Paioll is prepared.

(43) 3-6. Preparation of Recombinant Vector pCOLAD-Cgiep-Paioll

(44) To prepare a recombinant vector in combination of iolG and ioll genes derived from different strains, first of all, an iolG homologous gene Cgiep (NCg12957 or G1:19554252) whose function has not been reported and which is less homogenous with BD171257_CDS 1 (GI:27877069), which is an iolG gene on the C. glutamicum genome, is cloned. Also, in case of a corresponding ioll gene, Paioll, which is ioll derived from P. ananatis whose function has not been reported and which is less homogenous with BD171257_CDS 2(GI:27877069), which is also an ioll gene, is used in combination. To describe in more detail, Cgiep is amplified from a genome DNA of Corynebacterium glutamicum ATCC 13032 (taxid:196627; Gen Bank NID:NC_003450, ATCC13032) by using primer Cgiep-F and Cgiep-R, processed and cut off with restriction enzymes BspHl and Notl, and inserted into the Ncol and Notl regions of the pCOLADuet-1, so that pCOLAD-Cgiep is prepared. Then, pCOLAD-Cgiep is processed and cut off with Ndel and Pacl, and Paioll, which is cut off with the same restriction enzyme, is inserted therein, so that pCOLAD-Cgiep-Paioll vector is finally prepared.

(45) In the Exemplary Embodiment 3, the primer used for preparing the recombinant vector is shown in the Table 5 below, and the recombinant vector prepared above is shown in the Table 6.

(46) TABLE-US-00005 TABLE 5 Restriction enzyme Primer SEQ ID NO: region AGRL628-F SEQ ID NO: 32 PciI AGRL628-R SEQ ID NO: 33 SacI AGRL627-F SEQ ID NO: 34 BamHI-NdeI AGRL627-R SEQ ID NO: 35 SaiI BsioIG-F SEQ ID NO: 36 PciI BsioIG-R SEQ ID NO: 37 NotI BsioII-F SEQ ID NO: 38 NdeI BsioII-R SEQ ID NO: 39 PacI CgioIG-F SEQ ID NO: 40 BspHI CgioIG-R SEQ ID NO: 41 NotI CgioII0169-F SEQ ID NO: 42 NdeI CgioII0169-R SEQ ID NO: 43 PacI Paidh-F SEQ ID NO: 44 PciI Paidh-R SEQ ID NO: 45 NotI PaioII-F SEQ ID NO: 46 NdeI PaioII-R SEQ ID NO: 47 PacI Cgiep-F SEQ ID NO: 48 BspHI Cgiep-R SEQ ID NO: 49 NotI

(47) TABLE-US-00006 TABLE 6 Genbank Accession No. (GI, NID, PID) Vector and Recombinant Vector or References pCOLADuet-1 Novagen pCOLAD-sAtiep-sAtiepf The present invention pCOLAD-AGRL628-AGRL627 The present invention pCOLAD-BsioIG-BsioII The present invention pCOLAD-CgioIG-CgioII0169 The present invention pCOLAD-Paidh-PaioII The present invention pCOLAD-Cgiep-PaioII The present invention

(48) 3-7. Homology of Cloned Genes

(49) A phylogenetic tree of the cloned inositol dehydrogenase (iolG) and inosose isomerase (ioll) genes is shown in the Tables 4 and 5, and the results of analyzing the homology of those genes are shown in the Tables 7 and 8.

(50) TABLE-US-00007 TABLE 7 Homology (%) of amino acid Genbank sequence of Accession Gene SEQ ID NO: SEQ ID NO: 21 No. (NID, PID, GI) Reference sAtiep SEQ ID NO: 21 100 BD171257 JP2001-006878, CDS-1, W002055715A1 G 1: 27877069 AGRL628 SEQ ID NO: 23 90.5 AGR_L_628, GI: 159186216 Paidh SEQ ID NO: 29 56.8 PANA_3736, GI: 291619289 BsioIG SEQ ID NO: 25 50.4 BSU39700, Yoshida et al. GI: 255767850 (AEM72, 2006) CgioIG SEQ ID NO: 27 35.4 NCg10161, G1: 19551414 Cgiep SEQ ID NO: 31 21.7 NCg12957, GI: 19554252

(51) TABLE-US-00008 TABLE 8 Homology (%) of amino acid Genbank sequence of Accession Gene SEQ ID NO: SEQ ID NO: 21 No. (NID, PID, GI) Reference sAtiepf SEQ ID NO: 22 100 BD171257 JP2001-006878, CDS-2, W002055715A1 G 1: 27877069 AGRL627 SEQ ID NO: 24 88.7 AGR_L_627, GI: 159186217 PaioII SEQ ID NO: 30 41.3 PANA_3268, GI: 291618821 BsioII SEQ ID NO: 26 16.9 BSU39680, Yoshida et al. G1: 16081019 (AEM72, 2006) CgioII0169 SEQ ID NO: 28 10.1 NCg10169, GI: 19551422

Exemplary Embodiment 4: Establishment of Optimal Culture Conditions for D-chiro-inositol-producing Strains

(52) Out of the recombinant plasmid prepared in the Exemplary Embodiment 3, pCOLAD-Cgiep-Paioll is introduced into E. coli BL21(DE3) strain along with pACYCD-StiolT1-StiolT2(F2), so that an attempt is made to optimize culture of the recombinant strain.

(53) 4-1. Survey of D-chiro-inositol Productivity According to Culture Temperatures

(54) A 50 mL is cultured in a baffled conical flask of 300 mL for about 40 hours under the culture conditions of 30° C., 37° C. and 180 rpm by using a terrific broth (TB) containing 15% (w/v) myo-inositol, 50 mg/L of chloramphenicol and 50 mg/L of kanamycin. For induction, IPTG is added at a concentration of 1 mM at OD 0.6. Out of the culture conditions, a concentration of myo-inositol added is determined in accordance with solubility (approximately 16-17% (w/v) at a corresponding temperature.

(55) For the analysis of myo-inositol and D-chiro-inositol, a culture fluid is put into centrifugation, 1 mL of culture supernatant is taken out, boiled for 10 minutes, put into centrifugation again, and 500 uL of the resulting supernatant is taken out. The pre-treated culture supernatant is analyzed with HPLC (Shimadzu LC10Avp). When it comes to analysis conditions, Kromasil 5NH2 column (4.6 mm×250 mm), mobile phase 75% acetonitrile, column temperature 40° C. and an RI detector are used. The results thereof are shown in FIG. 6.

(56) A carbon source glycerol out of a TB medium runs out in about 24 hours at both 30° C. and 37° C., and a microbe growth also comes to stop in about 24 hours accordingly. For 24 hours when the carbon source runs out and the microbe growth comes to stop, about 12 g/L of D-chiro-inositol is produced at 30° C. and about 14 g/L thereof is produced at 37° C., which is about 1.2 times higher than the former. Even after the microbe growth comes to stop, D-chiro-inositol is continuously converted so as to increase up to about 19 g/L at 37° C. in about 28 hours and at 30° C. in about 32 hours, respectively.

(57) Such conversion from myo-inositol into D-chiro-inositol is known as involving reaction equilibrium (Yoshida et al., AEM72, 2006). In other words, physicochemical reaction equilibrium of about 86:14 is involved in between myo-inositol and D-chiro-inositol, so the amount of D-chiro-inositol corresponding to 150 g/L of myo-inositol is about 20 g/L. This is experimentally confirmed in this exemplary embodiment, too. In other words, in case of culturing with D-chiro-inositol added therein, myo-inositol is produced close to a reaction equilibrium ratio (results are not indicated). As a result, 19 g/L of D-chiro-inositol prepared amounts to the maximum of culture with 150 g/L of myo-inositol added therein. At 37° C., a time required for reaching such reaction equilibrium is about four hours faster than that of 30° C. In result, as far as a culture temperature is concerned, it is confirmed that 37° C. is an appropriate culture temperature.

(58) 4-2. Productivity of D-chiro-inositol According to Induction Time

(59) The productivity of D-chiro-inositol according to induction time of E. coli BL21 (DE3) strain, in which pACYCD-StiolT1-StiolT2(F2) and pCOLAD-Cgiep-Paioll are introduced, is considered. Assuming that a group without an inducer IPTG added is a negative control group, such productivity is checked at 37° C. while injecting at an initial time 0, OD 0.6, OD 3 and OD 10, respectively. Other culture conditions are as shown in 4-1 above, and the results of culture are shown in FIG. 7.

(60) A very low productivity of D-chiro-inositol is shown in a group without IPTG-caused induction and a group in which IPTG is added to cause induction at OD 10 after growth comes to an end completely. In a group in which IPTG is injected upon the start of culture, it appears that a microbe growth is seriously hindered in the early stage of culture. When induction also occurs at OD 0.6 and OD 3, it seems that a microbe growth is immediately hindered. That's probably because a nitrogen source, a carbon source, etc., which should have been used for microbe growth, is actually consumed for protein synthesis while protein is expressed by means of IPTG, or probably because a membrane is damaged due to an inositol transporter protein. In result, it is confirmed that it is most preferable to inject an inducer when a microbe is adapted to a new medium environment at an early logarithmic phase and starts a logarithmic growth.

(61) 4-3. Confirmation of Productivity of D-chiro-inositol According to Inducer Types

(62) The productivity of D-chiro-inositol according to inducer types of E. coli BL21(DE3), in which pACYCD-StiolT1-StiolT2(F2) and pCOLAD-Cgiep-Paioll are introduced, is considered. As an inducer IPTG has a tendency to hinder cell growth, lactose, which can replace IPTG, is added as an inducer. Lactose is added by adjusting its time and amount of addition, and 0.5% (w/v) lactose is added at an early stage, OD 0.6 and OD 7, or 0.1% (w/v) lactose is added at OD 0.6. Other culture conditions are the same as shown in 4-1 above, and the results thereof are shown in FIG. 8. In case of IPTG, it is confirmed that an early growth is hindered as shown in the results of 4-2 above, but such hindrance is not found in a group with lactose added. When 0.5% lactose is added at an early stage, a production speed of D-chiro-inositol is fastest. When 0.5% lactose is added at OD 7, it is confirmed that D-chiro-inositol starts to produce late accordingly. Lactose is formed into allolactose by lacZ gene, which is expressed while a microbe grows, thus activating a T7 promoter of the inventive expression vector. In other words, only when a microbe starts to grow, adapting itself to myo-inositol of high concentration, that is, a high osmotic pressure environment, the promoter is activated. So, such expression by lactose seems to reduce toxicity to the microbe.

(63) According to the Exemplary Embodiment 4 above, the optimal culture condition is determined in such a way that lactose is added to a culture medium in advance by using a TB medium and cultured at 37° C. And such condition is used to perform a subsequent experiment.

Exemplary Embodiment 5: Comparison of Activity Between D-chiro-inositol Converting Genes

(64) Each of pCOLAD-sAtiep-sAtiepf, pCOLAD-AGRL628-AGRL627, pCOLAD-BsiolG-Bsioll, pCOLAD-CgiolG-Cgioll0169, pCOLAD-Paidh-Paioll and pCOLAD-Cgiep-Paioll, which are expression vectors for expressing the inositol dehydrogenase and inosose isomerase constructed in the Exemplary Embodiment 3, is introduced into E. coli BL21 (DE3) along with a pACYCD-StiolT1-StiolT2, an inositol transporter, whose transporter activity is confirmed in the Exemplary Embodiment 2, so that six kinds of recombinant strains are prepared. The recombinant strains prepared above are cultured by using the optimal fermentation conditions tested in the Exemplary Embodiment 4. The results of measuring culture are shown in FIG. 9.

(65) In terms of a production speed (panel C of FIG. 9) of logarithmic production time (about 8 to 24 hours) of D-chiro-inositol, a recombinant plasmid pCOLAD-AGRL628-AGRL627 shows the lowest production speed at 0.09, while pCOLAD-sAtiep-sAtiepf (WO 02/055715) also shows the lowest production at 0.14. In a gene selected in the present invention, it appears that myo-inositol dehydrogenase and inosose isomerase are converted into D-chiro-inositol at the fastest speed of a gradient 0.48 in combination with Cgiep and Paioll, respectively, that is, pCOLAD-Cgiep-Paioll. Such gradient value is about 5.3 times faster than the lowest speed of pCOLAD-AGRL628-AGRL627 and 1.8 times higher than the second speed of pCOLAD-CgiolG-Cgioll0169 (0.26). The concentration of D-chiro-inositol produced after a 40-hour culture is highest in the case of pCOLAD-Cgiep-Paioll. A final microbe growth speed comes to a pause phase before about 12 hours as a whole, and a microbe concentration is also lowest in pCOLAD-sAtiep-sAtiepf in proportionate to a production speed of D-chiro-inositol above. In result, when pCOLAD-Cgiep-Paioll is introduced, 22.7 g/L of D-chiro-inositol is converted from 150 g/L of myo-inositol and a conversion rations is about 15.1%. In case of pCOLAD-sAtiep-sAtiepf showing the lowest conversion ratio, about 12.5 g/L thereof is converted and a conversion ratio remains at about 8.3%. A fermentation experiment is performed in such a way that a recombinant E. coli strain, in which pCOLAD-Cgiep-Paioll is introduced from the results, is a producing strain.

Exemplary Embodiment 6: Increase in Production Through Fermentation of Recombinant Strains

(66) 1 L fermentation is performed by using the optimal recombinant strain obtained from the results of optimizing culture and selecting excellent genes performed above. Fermentation is performed in a 1 L capacity in a 3 L fermentation tank of MARADO-PDA [CNS Co., Ltd. Daejeon, Korea]. A 2YT medium is used for seed culture. In this culture, 15% myo-inositol and 0.5% lactose are added to a TB medium. A 50 mL of seed culture fluid, which was cultured up to OD 3, is inoculated into the fermentation tank, which was stabilized with a temperature, pH and OD, and then cultured for about 20 hours. A fermentation temperature is adjusted to 37° C. and pH is adjusted to 7.0 by using ammonia water. After OD is reduced to 40% or less, RPM is increased to maintain 40% or more. The results of fermentation are shown in FIG. 10. A microbe reaches up to about OD 18 within 18 hours. D-chiro-inositol reaches about 20 g/L, which is an equilibrium concentration within about 12 hours, and after then it is not increased any more.

Exemplary Embodiment 7: Fractionation and GC-MS Analysis of D-chiro-inositol Prepared

(67) Fraction of D-chiro-inositol prepared from microbes is performed by using HPLC (Shimadzu LCIOAvp). When it comes to analysis conditions, Kromasil 5NH2 Prep Column (10 mm×250 mm), mobile phase 75% acetonitrile, column temperature 40° C. and an RI detector are used. A 1004 of test sample is injected and flowed at a flow speed of 4 mL/min, so that an effluent for a corresponding peak is taken out. The effluent collected is concentrated and dried by using a vacuum condenser (EYELA, Japan), dissolved in 5 mL of distilled water, and GC-MS analysis is performed.

(68) GC-MS analysis is performed by using Shimadzu GCMS-QP2010. For treatment before analysis, 1 mg of sample is dissolved in a 1 mg of 1-trimethylsylyl-imidazole and pyridine mixed at a ratio of 1:1 and is reacted at 70° C. for 30 minutes so as to be used for analysis. For analysis conditions, HP-1 capillary column (30 m Length, 0.25 mm ID, 0.25 μm Film) is used and 1 μL of sample is injected into a split mode injector (1:50) and analyzed at a flow rate of 1 mL/min. A sample injection temperature is 280° C., an oven temperature is maintained at 150° C. for two minutes, increased up to 300° C. at a speed of 20° C./min, and maintained for two minutes. An MS profile for analysis results is compared with a profile of standard sample D-chiro-inositol (Sigma 468045, U.S.A.) and the results thereof are shown in FIG. 11. As a result, it is confirmed that the MS profile is accurately the same as the profile of standard sample.

(69) As the specific parts of the present invention have been described in detail above, such detailed description of the disclosed invention are only preferred examples of the present invention to the person with ordinary skill in the art, and it is clear that they are not used to limit the range of the present invention disclosed in the claims. Therefore, the actual range of the present invention is defined by the following claims and equivalents thereof.

(70) This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted herewith as the sequence listing text file. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. §1.52(e).