WO2004031393A1

WO2004031393A1 - A biosynthetic process of tdp-4-keto-6-deoxy-d-glucose as an intermediate of deoxy sugar

Info

Publication number: WO2004031393A1
Application number: PCT/KR2003/001058
Authority: WO
Inventors: Jin Suk Woo; Jong Min Oh; Byung Gee Kim; Jae Kyoung Sohng; Hei Chan Lee; Kwang Kyoung Liou; Sun Youp Kang; Dae Hee Kim; Dong Hyun Kim; Ji Young Yang; Young Soo Jung; Eui Bok Lee
Original assignee: Genechem Inc.
Priority date: 2002-10-07
Filing date: 2003-05-29
Publication date: 2004-04-15
Also published as: AU2003232656A1; KR20040031866A

Abstract

The present invention relates to a method for preparing TDP-4-Keto-6-deoxy-D-glucose, a biosynthetic intermediate of deoxy sugar, which plays a key role in the biological activity of antibiotics and anticancer drugs, effectively. More specifically, the present invention relates to a method for preparing TDP-4-keto-6-deoxy-D-glucose by adding thymine monophosphate kinase, acetate kinase, TDP-glucose synthase and TDP-D-glucose-4, 6-dehydratase into a reaction solution containing thymine monophosphate, ATP, acetyl phosphate and Glucose-1-phosphate. Based upon the present invention, high-priced TDP-4-keto-6-deoxy-D-glucose, a biosynthetic intermediate of deoxy sugar, could be prepared from low-priced thymine monophosphate just with one step reaction economically.

Description

A BIOSYNTHETIC PROCESS OF TDP-4-KETO-6-DEOXY-D-GLUCOSE AS AN INTERMEDIATE OF DEOXY SUGAR

FIELD OF THE INVENTION

The present invention relates to a method for preparing TDP-4-keto-6- deoxy-D-glucose, a biosynthetic intermediate of deoxy sugar, which has an important function in the biological activity of antibiotics and anticancer drugs, effectively.

BACKGROUND OF THE INVENTION

Generally, a sugar (glycone) in an organism has a great influence on the biological activities and functions of secondary metabolites in addition to as a main energy source. For example, many secondary metabolites having antibiotic and anticancer activity such as macrolides and anthracyclines contain deoxy sugar such as deoxyhexose, 2-deoxy sugar, 3-deoxy sugar, amino-deoxy sugar, nitro-deoxy sugar or the like in their configuration; and these deoxy sugar components of secondary metabolites are involved directly or indirectly in the biological activity and affinity of antibiotics and anticancer drugs, and are also known to take an important part in the tolerance of antibiotics.

Under these circumstances, many researchers are trying to develop new substances having more powerful efficacy and free from resistance problems than l conventional antibiotics by preparing new compounds having new activities or strengthened functions via a synthetic process of synthesizing compounds having new carbohydrate configurations and then combining the compounds with variously modified aglycones to prepare various derivatives. For example, Professor D. Kahn in Princeton University has synthesized a new compound with greater biological activity than conventional vancomycin through a glycosylation method by substituting a conventional glycone optionally with a new deoxy sugar, UDP-4-epi-vancosamin (Losey, HC et al., Biochemistry,; 40(15), 4745-55, 2001). However, in order to synthesize these new compounds, conventional organochemical methods have to proceed many complex steps, and therefore, the yield is very low, which causes a problem to be applied to a large scale of production. In order to overcome the above-described problems of the organochemical methods, a biological method using a biocatalyst was suggested. This biological method using a biocatalyst has such merits as reaction yield and stereo specificity to reaction substrate are high and generations of byproducts are very low. Thus, in this biosynthetic process, it is important to develop mass production processes for producing reaction substrates and biocatalysts used in the synthesis at low cost. Accordingly, microorganisms were extensively studied regarding the biosynthetic process of various antibiotic and anticancer substances, and as a result, it was discovered through the analysis of DNA sequencings of the microorganisms ' genes pool that many microorganisms producing antibiotics including 1-, 2-, or 3-deoxy sugar or the like in vitro commonly have the same biosynthetic route of TDP-4-keto-6-deoxy-D-glucose synthesis (Sohng, J K et al., J.Biochem. Mol. BIol., 31, 475-483, 1998).

TDP-4-keto-6-deoxy-D-glucose has the configuration represented by the following Formula 1 and is an important intermediate for biosynthesis of deoxy sugar, which can be transformed to bioactive deoxy sugar, 2-deoxy sugar, 3- deoxy sugar, amino-deoxy sugar, nitro-deoxy sugar or the like, by the 1-5 steps of enzyme reaction.

[Formula 1]

Although these deoxy sugar substances are important compounds directly or indirectly involved in the biological activity, affinity and resistance of secondary metabolites such as antibiotics and anticancer drugs, it is difficult or impossible to produce them by organochemical methods, and therefore, in order to develop new substances having such various activities, it is necessary to produce TDP-4- keto-6-deoxy-D-glucose, a biosynthetic intermediate of deoxy sugar, on a large scale effectively.

Accordingly, it has been reported that TDP-4-keto-6-deoxy-D-glucose is an important intermediate for the preparation of various deoxy sugar which works as a turning point in the biosynthetic routes, and therefore, mass production of TDP- 4-keto-6-deoxy-D-glucose became an important subject in the biosynthesis of novel deoxy sugar compounds.

In the beginning of the studies for preparing TDP-4-keto-6-deoxy-D-glucose, it was biosynthesized from TDP-D-glucose by using TDP-D-glucose-4,6- dehydratase (Stein, et al., Angew. Chem. Int. Ed, 34(16), 1995). However, because very expensive TDP-D-glucose is used as a substrate in this method, this method has a problem of low economic efficiency.

Therefore, many other researches have been performed to apply other substrates instead of TDP-D-glucose, and since then for example, Kula, a German research team, invented a method for biosynthesizing TDP-4-keto-6- deoxy-D-glucose from sucrose and TDP by using sucrose synthase (Stein, A et al., Glycoconj J., 15(2), 139-45, 1998). This method is more efficient and economical than the former method using TDP-D-glucose, but still has a problem that it uses expensive TDP.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide an effective method for preparing TDP-4-keto-6-deoxy-D-glucose, a biosynthetic intermediate of deoxy sugar, with lower cost and higher productivity, for the purpose of developing novel antibiotics and anticancer substances and producing them on a large scale,

To achieve the above-mentioned object, the present invention provides an effective method for preparing TDP-4-keto-6-deoxy-D-glucose, a biosynthetic intermediate of deoxy sugar, characterized as adding thymine monophosphate kinase, acetate kinase, TDP-glucose synthase and TDP-D-glucose-4,6- dehydratase into a reaction solution containing thymine monophosphate, ATP, acetyl phosphate and Glucose- 1 -phosphate, and reacting them.

The present invention uses thymine monophosphate (TMP) as a starting material, and uses four different enzymes of thymine monophosphate kinase, acetate kinase, TDP-glucose synthase and TDP-D-glucose-4,6-dehydratase in each step of the following reaction scheme 1, which are reacted simultaneously.

[Reaction Scheme 1]

dTDP- 4-keto- 6- deoxy-D-glucose

In the present invention, the enzymes are recombinant enzymes that are manufactured on a large scale from four different transformants transformed by using recombinant vectors containing enzyme genes extracted from microorganisms and purified.

The following is a detailed description of the manufacturing process of the above-mentioned enzymes.

First, thymine monophosphate kinase gene(tmk), acetate kinase gene(ack) and TDP-glucose synthase gene(rffH) were separated from the genome of E.coli K-12 strain and purified, and TDP-D-glucose-4,6-dehydratase gene(rfb'B') was separated from the genome of Salmonella typhimurium strain and purified. These enzyme genes were then inserted into expression vectors of pET15b, pET24ma, pET24ma and pRSETB respectively to prepare four recombinant vectors of ρET15b:tmk, pET24ma:ack, pET24ma:rffH and pRSETB.rfb'B'.

Each of the transformants containing the above-mentioned recombinant vectors were prepared by transforming the expression strains using these recombinant vectors respectively. As expression host for this method, E.coli XL 1 -Blue or E.coli BL21 may be used; E.coli XL 1 -Blue is excellent in the viewpoint of stability of strain and E.coli BL21 is excellent in the viewpoint of expression efficiency. Among the above transformants, E.coli XL 1 -Blue transformants obtained transforming E.coli XL 1 -Blue with recombinant vectors were deposited in an international depository authority, KCTC (Korean Collection for Type Cultures) on September 9, 2002, that is, E.coli XLl-Blue/pET15b:tmk was deposited with an accession No. KCTC 10334BP, E.coli XLl-Blue/pET24ma:ack with an accession No. KCTC 10331BP, E.coli XLl-Blue/pET24ma:rffH with an accession No. KCTC 10332BP, and E.coli XLl-Blue/pRSETB-rfb'B' with an accession No. KCTC 10333BP.

These transformed strains obtained above were separately cultured in large quantities to obtain aimed enzymes of thymine monophosphate kinase, acetate kinase, TDP-glucose synthase and TDP-D-glucose-4,6-dehydratase. These obtained enzymes were then added to reaction solution containing thymine monophosphate, ATP, acetyl phosphate and Glucose- 1 -phosphate and reacted simultaneously to produce the aimed product, TDP-4-keto-6-deoxy-D-glucose, on a large scale.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. la is a gene map of recombinant vector pET15b:tmk prepared by inserting an enzyme thymine monophosphate kinase gene(tmk) into expression vector pET 15b.

Fig. lb is a gene map of recombinant vector pET24ma:ack prepared by inserting an enzyme acetate kinase gene(ack) into expression vector pET24ma. Fig. lc is the gene map of recombinant vector pET24ma:rffH prepared by inserting an enzyme TDP-glucose synthase gene(rffH) into expression vector pET24ma.

Fig. Id is a gene map of recombinant vector pRSETB.rfb'B' prepared by inserting an enzyme TDP-D-glucose-4,6-dehydratase gene(rfb'B') into expression vector pRSETB. Fig. 2 is a picture showing an electrophoresis result of the crude extracts of transformed microorganisms, E.coli strains, obtained after enzyme-induced expression, in 12% SDS- polyacrylamide gel (lane 1 is a low molecular weight marker, lane 2 is a band of over-expressed thymine monophosphate kinase extracts, lane 3 is a band of over-expressed acetate kinase extracts, lane 4 is a band of over-expressed dTDP-glucose synthase extracts, lane 5 is a band of over- expressed dTDP-D-glucose-4,6-dehydratase extracts, lane 6 is a band of over- expressed E.coli BL21 extracts as the control).

Fig. 3 is a graph showing the effect of Mg²⁺ on the synthetic reaction of dTDP-4-keto-6-deoxy- glucose. Fig. 4 is a graph showing the effect of acetyl phosphate on the synthetic reaction of dTDP-4-keto-6-deoxy-glucose.

Fig. 5 is a graph showing the effect of Glucose- 1 -phosphate on the synthetic reaction of dTDP-4-keto-6-deoxy-glucose.

Fig. 6 is a graph of the result of high performance liquid chromatography showing the synthesis of dTDP-4-keto-6-deoxy-glucose performed in Example 4.

Fig. 7 is a graph of the result of high performance liquid chromatography showing gram level production of dTDP-4-keto-6-deoxy-D-glucose performed in the Example 5.

DETAILED DESCTIPTION OF THE INVENTION

The present invention will be described in more detail with the following examples and Examples, which are intended to illustrate the examples of the present invention, and therefore they should not be considered to restrict the scope of the present invention.

Example 1: Separation and Purification of whole chromosome DNA from E.coli K-12 and Salmonella typhimurium Strains of E.coli K-12 and Salmonella typhimurium were inoculated in 50m£ of sterilized LB media respectively, and incubated at 37 ^°C for 12 hours. The cultures were transferred to conic tubes and centrifuged for 10 minutes at 4,000 rpm to obtain precipitation pellets. 30m£ of Lysis buffer solution (150g of 15% sucrose, 12.5 ml of 2M tris-HCl and 50ml of 0.5M EDTA were contained therein, and which were filled up with distilled water to make the total volume 11) was added to each pellet obtained above to dissolve the pellet slowly, and again centrifuged for 10 minutes at 4,000 rpm. \0ml of the above lysis buffer solution was added to dissolve the cells again. Lysozyme (5mg/m#) was added thereto and reacted in a thermostatic bath of 37 ^°C for about an hour. 1.2m£ of 0.5M EDTA and 0.13m# of pronase solution were added and reacted for 5 minutes at 37 ^°C . 3ml of 10% SDS (Sodium dodecyl sulfate) were then added and mixed completely, then reacted for 10 minutes at 70 ^°C , and the reaction mixture was placed in ice water for 10 minutes. 3ml of 5M potassium acetate was added to the reaction mixture and the reaction mixture was again placed in ice water for 15 minutes. After adding phenol-chloroform (50:50) with the same amount as that of the contents in the tube, the mixture was shaken slowly, and stirred sufficiently then centrifuged at 4^°C with 4,000rpm for 30 minutes.

Then, the supernatant of the tube was moved to another conic tube, and RNA hydrolase was added with a ration of 50,(-g/m£ unit and reacted for an hour at 37 ^°C to remove RNA. 0.8 times the volume of isopropyl alcohol and 2.5 times the volume of ethanol were added thereto and stirred gently. Precipitated chromosomal DNA was collected and dried sufficiently to remove ethanol. By dissolving the above-obtained chromosomal DNA in 400 μl of TE buffer solution (prepared by mixing 5ml of 2M tris-HCl, 2ml of 0.5M EDTA and 993 ml of distilled water), the whole chromosomal DNAs of E.coli K-12 and Salmonella typhimurium were isolate, purify and finally aimed-product were obtained. These obtained genes were used as templates of PCR processes in the following Examples.

Example 2: Preparation of recombinant vectors and transformant containing genes for thymine monophosphate kinase, acetate kinase, TDP- glucose synthase and TDP-D-glucose-4,6-dehydratase

In order to prepare recombinant vectors and transformant containing genes of the aimed four different enzymes above, conventional PCR processes were conducted beforehand to amplify each gene as follows.

First, with reference to the public data of chromosomal genes' sequence, base sequences of thymine monophosphate kinase gene (tmk), acetate kinase gene (ack) and TDP-glucose synthase gene (rffH) from E.coli K-12 strain, and TDP-D-glucose-4,6-dehydratase (rfb'B') gene from Salmonella typhimurium strain were searched and confirmed, and then each set of the forward and reverse PCR primers for each enzyme gene was synthesized as follows.

Sequence 1 to 6: tmk

Forward primer: tmk-NcoI-CCATGGCAGTAAGTATATCGTCA Reverse primer: tmk-BamHI-GATCCTCATGCGCTCCAACTCCTTCACCCAG

ack Forward primer: ack-Ndel-CATATGTCGAGAGTAAGTTAGTACTGGTTCTG Reverse primer: ack-EcoRI-GAATTCTCAGGCAGTCAGGCGGCTCGCGTC

rffH Forward primer: rfϊH-Ndel-CATATGAAAGGTATTATCCTGGCGG Reverse primer: rfϊH-EcoRI-GAATTCTCAATACTGGCGCGGACGGG

rfb'B'

Forward primer: rfbB-Ndel-CCAAGTCGATATGCTAGCAGTGCACTGG Reverse primer: rfbB-Hindm-CGGGACGAAGCTTACGAACGGTT

Using the full chromosomal DNAs of E.coli K-12 and Salmonella typhimurium strains obtained in the Example 1 as templates and using the above primers, general PCRs were executed. Specifically, 6μl of 10XPCR buffer solution [500mM KC1, lOOmM tris-Cl

(pH 8.3 at room temperature), 15mM MgCl₂, 0.1% gelatin], 6μl of dNTP (lOmM for each), Iμl of forward primer, \ μJl of reverse primer, 2.5μl of Taq DNA polymerase and OΛμJl of DNA template of Example 1 were added together, and distilled water was poured into this buffer solution to make a total volume of βOμi to prepare PCR solution, and this PCR solution was put into a PCR machine designed to undergo the steps of: initial denaturation at 95 ^°C ; annealing at 40 ^°C ; and extension at 72 ^°C . The above process was repeated 30-40 times.

After the above-described PCR process, the above-obtained PCR products underwent agarose gel electrophoresis to observe the bands of the each of the aimed enzyme genes. Then, the aimed 4 enzyme gene bands obtained above were taken, and inserted into the split expression vectors digested with adequate restriction enzymes for each gene as shown in table 1 to obtain recombinant vectors pET15b:tmk, pET24ma:ack, pET24ma:rfffl and pRSETB:rfb'B', each of which contains thymine monophosphate kinase, acetate kinase, TDP-glucose synthase and TDP-D-glucose-4,6-dehydratase respectively. Gene maps showing the recombination process of each of the four enzyme genes are described in Fig. la to Fig. Id.

After the recombination process, the above- obtained four recombinant vectors were transformed into E.coli XL 1 -Blue to produce E.coli XL 1 -Blue/ ρET15b:tmk, E.coli XLl-Blue/ρET24ma:ack, E.coli XLl-Blue/pET24ma:rffH, and E.coli XLl-Blue/pRSETB:rfb'B^*.

The details regarding the production of recombinant vectors and transformants for the four enzymes are described briefly in table 1.

[TABLE 1]

Digestion of vectors with restriction enzymes and manufacturing of recombinant vectors and transformants were carried out with reference to the general experimental method of molecular biology(Joseph Sambrook et al., Molecular Cloning., A laboratory Manual, 3rd ed., 2001). In addition, with the same way, E.coli BL21, as expression hosts, were transformed by using the above four recombinant vectors to produce E.coli BL21/pET15b:tmk, E.coli BL21/pET24ma:ack, E.coli BL21/pET24ma:rffH and E.coli BL21/pRSETB:rfb'B'.

Example 3: Enzyme expression of thymine monophosphate kinase, acetate kinase, TDP-glucose synthase and TDP-D-glucose-4,6-dehydratase and observation thereof

In this example, the aimed enzymes were induced to express their activities in the transformants obtained in Example 2 and then electrophoresis was performed to confirm their expression degrees.

First, in order to obtain thymine monophosphate kinase, acetate kinase, TDP-glucose synthase and TDP-D-glucose-4,6-dehydratase from the transformants, E.coli BL21/pET15b:tmk, E.coli BL21/pET24ma:ack, E.coli BL2 l/pET24ma:rffH and E.coli BL2 l/pRSETB:rfb'B' were inoculated into liquid LB media, and cultured overnight at 37 ^°C to perform seed culture. Then, 5% (v/v) amount of the seed culture was inoculated into a new 50m£ of liquid LB media and cultured at 37^°C for about 2 hours as a main culture. Adequate concentration of IPTG (Isopropyl β-D-Thiogalacto pyranodside) for each enzyme was added to each media as shown in table 2 to induce the expressions of enzymes.

[TABLE 2]

Cell culture media, wherein final OD is 3.5 at 600nm (OD₆₀₀ = 3.5), was transferred to a new conic tube with 50ml of capacity, and centrifuged with 4,000 rpm at 4°C for 10 minutes. After removing the supernatant, the media components on the cell surfaces were washed with 20ml of 50mM tris-HCl buffer solution (pH=7.5), and the remainders were centrifuged again with 4,000 rpm at 4°C for 10 minutes. After Removing supernatant again, 4ml of sonicated buffer solution [prepared by adding ImM EDTA(pH 8.5) and ImM PMSF (phenyl methyl sulfunyl fluoride) as pronase inhibitor; ImM DTT (DL- dithiothreitol) to inhibit disulfide bonding; and ImM MgCl₂ for enzyme activity; to 50mM tris-HCl buffer solution (pH 7.5) containing 10% glycerol] was added thereto to re-suspend the cells, then the cells were crushed with a sonicator. The crushed cells were centrifuged with 15,000 rpm at 4^°C for 30 minutes, then the supernatant was transferred to an Ependorf tube. The above-obtained enzyme solution was stored at a temperature below -40 ^°C . The above obtained underwent electrophoresis in 12% SDS-polyacrylamide gel to observe the degree of over expression.

As the result of the electrophoresis, a picture illustrated in Fig. 2 was obtained. In Fig. 2 showing the result of electrophoresis, lane 1 represents a low molecular weight marker, lane 2 represents the band of over-expressed thymine monophosphate kinase extracts, lane 3 represents the band of over- expressed acetate kinase extracts, lane 4 represents the band of over-expressed TDP-glucose synthase extracts, lane 5 represents the band of over-expressed TDP-D-glucose-4, 6-dehydratase extracts, and lane 6 represents the band of over- expressed E.coli BL21 extracts as the control.

From Fig. 2, four enzymes of thymine monophosphate kinase, acetate kinase, TDP-glucose synthase and TDP-D-glucose-4,6-dehydratase were certified as the forms of protein bands in the gel, which proves that the transformants prepared in the present invention - E.coli BL21/pET15b:tmk, E.coli BL21/ρET24ma:ack, E.coli BL21/pET24ma:rffH and E.coli BL21/ pRSETB:rfb'B' - are able to produce thymine monophosphate kinase, acetate kinase, TDP-glucose synthase and TDP-D-glucose-4,6-dehydratase on a large scale repectively.

In addition, by performing the same experimental process to other transformants E.coli XLl-Blue/pET15b:tmk, E.coli XLl-Blue/pET24ma:ack, E.coli XLl-Blue/pET24ma:rffH and E.coli XLl-Blue/pRSETB:rfb'B', similar results as to those of Fig. 2 were obtained.

Example 4: Synthesis of TDP-4-keto-6-deoxy-D-gIucose using thymine monophosphate kinase, acetate kinase, TDP-glucose synthase and TDP-D- glucose-4,6-dehydratase

In this Example, TDP-4-keto-6-deoxy-D-glucose was synthesized from the starting material, thymine monophosphate, with just one step reaction using the four enzymes, thymine monophosphate kinase, acetate kinase, TDP-glucose synthase and TDP-D-glucose-4,6-dehydratase prepared in Example 3.

Beforehand, adequate concentration of substrate and reaction time for the optimum reaction condition was determined (Figs. 3-5) by executing the synthetic reactions using reactants with various concentrations at each step. As shown in the Reaction Scheme 1 above, ATP, the reactant of the first step, was recycled by acetate kinase to improve economical and reaction efficiencies of the reaction.

The reaction was conducted in a thermostat with 37^°C . First, IμJt of 1M ATP, Iμl of 2M MgCl₂ 6H₂0, lOμ of 1M acetyl phosphate, 32μl of 0.5mM Glucose- 1 -phosphate and 4μi of 50mM Tris buffer solution were added to 20 - of 1M thymine- 5 '-monophosphate as the starting material. As the enzymes for this reaction, I2μi of TDP-D-glucose-4,6-dehydratase, 12μJ of TDP-glucose synthase, 4μl of acetate kinase and 4μl of thymine monophosphate kinase were then added thereto in order (reverse order to reaction) to make the total volume as lOO ϋ, and reacted for 120 minutes. After reaction, the degree of reaction process was observed by HPLC.

RP-HPLC (Reverse Phase High Performance Liquid Chromatography) is known to be one of the most effective ways to analyze nucleotide or sugar nucleotide (Albermann, C. et al., Glycobiology, 10(9), 875-81, 2000); and in the present example, C₁₈(4.6X 150 mm KC-PACK ODS-A, KYOTO CHROMATO) was used as an analysis column; lOOmM of 90% KH₂P0₄/ K₂HP0₄, 8mM of tetrabutylammonium hydrogen sulfate(pH 7.0) and 5% methanol were used as an effluent; and elution was performed with a flow rate of 1 ml/mm. Results are illustrated in Fig. 6. As confirmed by the graph of Fig. 6, the aimed product dTDP-4-keto-6- deoxy-D-glucose was effectively produced by one step reaction using the four enzymes of the present invention, thymine monophosphate kinase, acetate kinase, TDP-glucose synthase and TDP-D-glucose-4,6-dehydratase.

Example 5: Synthesis, isolation and purification of TDP-4-keto-6-deoxy-

D-glucose with gram unit

Since the biological activities of the enzymes for each step were confirmed with the result of example 4, a reaction to obtain the final aimed product with gram level was executed in this example.

The present reaction was conducted with 100m£ volume in a 500m£ flask; substrates and enzyme extracts were added with the amounts correspondingly increased with the ratio same as that of Example 4, and reacted.

672.4mg of 20mM thymine- 5 '-monophosphate, 60mg of ImM ATP, 407mg of 20mM MgCl₂-6H₂0, 1.84g of lOOmM acetylphosphate and 4.5g of 150mM Glucose- 1 -Phosphate were added to 80m£ of 50mM tris-HCl buffer solution, then stirred to dissolve the substrates. 4ml of TDP-D-glucose-4,6-dehydratase, 4ml of TDP-glucose synthase, 2ml of acetate kinase, and 2ml of thymine monophosphate kinase were added in order thereto while stirring at 37^°C for 80 hours. After the reaction, the degree of reaction process was observed with HPLC as done in example 4.

The results are illustrated in Fig. 7. After reaction, the obtained reaction solution was heated in 100^°C boiling water for 3 minutes to inactivate the enzymes, then centrifuged with 15,000 rpm for 30 minutes, and the supernatant was freeze-dried (lyophilized) for concentration. This condensed reaction solution was injected into the upper part of a Dowex-l X2, Cl^'-form column of anion exchange resin to separate the solution according to concentration gradient of salt. The eluted solution containing aimed product was concentrated again and salt removed by Sephadex G-10 gel filtration chromatography method. The final product with a high purity was obtained through these steps, and was concentrated by lyophilization (freeze-dry). 81% yield of the final product was obtained after purification. The results of 1H-NMR, ¹³C-NMR and MALDI- TOF analysis to the final purified product were described as follows.

1H -NMR(D₂0, 500MHz): 5.56 ppm(dd, J=3.53, [6.73]), IH, hexose 1"), 3.64 ppm(ddd, J = 9.95, [3.03]), IH, hexose 2"), 3.81 ρpm(d, J - 9.95, IH, hexose 3"), 4.11 ppm(q, J = 6.36, IH, hexose 5"), 1.24 ppm(d, J = 6.40, IH, hexose 6"), 6.36 pρm(t, J= 6.86, IH, ribose l¹), 2.42-2.37 ppm(dd+m, 2H, ribose 2'), 4.66 ppm(mc, IH, ribose 3'), 4.20 ppm(m, IH, ribose 4', 2H, ribose 5'), 7.76 ppm(s, IH, base 6), 1.95ppm (s, 3H, base CH₃) ¹³C -NMR(D₂0, 500MHz): 94.10 ppm(hexose 1"), 73.02 ppm(hexose 2"),

74.11 pρm(hexose 3"), 206.3 lppm(hexose 4"), 71.41 ρρm(hexose 5"), 12.33 ppm(hexose 6"), 85.23 ppm(ribose 1'), 38.97 ppm(ribose 2'), 71.41 ppm(ribose 3'), 86.28 ppm(ribose 4'), 65.80 ppm(ribose 5^*), 152.14 ppm(base 2), 166.99 ppm(base 4), 112.16 ppm(base 5), 137.65 ppm(base 6), 11.67 ppm(base CH3) [M⁺²Na-H] 591.1 m/z [M^+jNa-2H] 613.1 m/z

INDUSTRIAL APPLICABILITY

The present invention provides a method for synthesizing an expensive compound, TDP-4-keto-6-deoxy-D-glucose, an intermediate of deoxy sugar, from the starting material, low-priced thymine monophosphate, with just one step reaction using four kinds of recombinant enzymes, which is more economical and efficient than the conventional synthesis method.

Conventionally, it was difficult to prepare deoxy sugar intermediate by organochemical synthetic method and took much cost to prepare the deoxy sugar intermediate conventional biosynthetic method, however according to the present invention, it is possible to prepare the deoxy sugar intermediate easily on a large scale with low cost; therefore, it is possible to invent antibiotics or anticancer drugs with new or strengthened activities through various deoxy sugar derivatives prepared by using deoxy sugar intermediate, and to improve productivity due to low priced mass production.

Regarding the development of new medical drugs such as antibiotics and anticancer drugs, the importance of deoxy sugar and its biosynthetic intermediate, TDP-4-keto-6-deoxy-D-glucose, is highlighted day by day, but it is not easy to synthesize this substance and the cost of production is excessively high with the current method. Considering these, because the present invention can provide TDP-4-keto-6-deoxy-D-glucose on a large scale from TMP, which costs about one thirtieth (1/30) of TTP used in the conventional, the present invention has great effective value and far-reaching effects, and therefore is expected to be used for various purposes in relevant fields.

Claims

WHAT WE CLAIMED IS:

1. A method for preparing TDP-4-keto-6-deoxy-D-glucose, an intermediate of deoxy sugar, by adding thymine monophosphate kinase, acetate kinase, TDP- glucose synthase and TDP-D-glucose-4,6-dehydratase to a reaction solution containing thymine monophosphate, ATP, acetyl phosphate and Glucose- 1- phosphate, and reacting them.

2. The method according to Claim 1, wherein the thymine monophosphate kinase, acetate kinase and TDP-glucose synthase are derived from E.coli K-12.

3. The method according to Claim 1, wherein the TDP-D-glucose-4,6- dehydratase is derived from Salmonella typhimurium.

4. The method according to Claim 1, wherein the thymine monophosphate kinase is obtained from a culture of microorganism transformed by a recombinant vector pET15b:tmk.

5. The method according to Claim 1, wherein the acetate kinase is obtained from a culture of microorganism transformed by a recombinant vector pET15ma:ack.

6. The method according to Claim 1, wherein the TDP-glucose synthase is obtained from a culture of microorganism transformed by a recombinant vector pET24ma:rffH.

7. The method according to Claim 1, wherein the TDP-D-glucose-4,6- dehydratase is obtained from a culture of microorganism transformed by a recombinant vector pRSETB:rfb'B\

8. A microorganism transformed by a recombinant vector pETl 5b:tmk.

9. The microorganism according to Claim 8, wherein the microorganism is E.coli XLl-Blue/pET15b:tmk (KCTC 10334BP) transformed by recombinant vector pET 15b: tmk.

10. A microorganism transformed by a recombinant vector pET15ma:ack.

11. The microorganism according to Claim 10, wherein the microorganism is E.coli XLl-Blue/pET24ma:ack (KCTC 1033 IBP) transformed by recombinant vector pETl 5ma:ack.

12. A microorganism transformed by a recombinant vector pET24ma:rffH.

13. The microorganism according to Claim 12, wherein the microorganism is E.coli XLl-Blue/pET24ma:rffH (KCTC 10332BP) transformed by recombinant vector pET24ma:rffH.

14. A microorganism transformed by a recombinant vector pRSETB:rιb'B'.

15. The microorganism according to Claim 14, wherein the microorganism is E.coli XLl-Blue/pRSETB:rfb'B' (KCTC 10333BP) transformed by recombinant vector pRSETB÷rfb'B'.