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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose

Definitions

• the present invention relates to a novel recombinant E. coli, a fermentation process and a enzymatic conversion process for tagatose production using such recombinant E. ⁇ li harboring vector containing the gene of L-arabinose isomerase, and the promoter controlled artificially.
• D-Tagatose is one of ketohexoses as well as one of D-galactose isomers. D-Tagatose is reported as the sweetener having the taste most similar to that of sucrose (92%). In addition, D-tagatose does not show laxative effect, which other polyols generally do. Due to such reasons, D-tagatose has been recently regarded as a non-caloric sweetener substituted by sucrose [Zehener, L. R., D-Tagatose as a low-calorie carbohydrate sugar and bulking agent, EP 257626 (1988) ; Marzur, A. W., Functional sugar substitutes with reduced calories, EP 341062 (1989)].
• D-Galactose could be converted into D-tagatose in the presence of a calcium catalyst [Beadle, J. R., Saunders, J. P., and Wajda, T. J., Process for manufacturing tagatose, WO 92/12263 (1992)].
• a calcium catalyst Beadle, J. R., Saunders, J. P., and Wajda, T. J., Process for manufacturing tagatose, WO 92/12263 (1992)
• the chemical synthesis is economical, this process also requires disadvantageous high temperature and high pressure.
• the biological process has been researched with interest as an environmentally clean process. As a consequence, the biological production of D-tagatose has been intensively studied recently.
• L-arabinose isomerase (AraA) could convert galactose into tagatose.
• Cheetham reported that L-arabinose isomerase from Mycobacterium and Lactobacillus catalyzes the conversion from D-galactose to D-tagatose as well as that of from L-arabinose to L-ribulose, because of the similar substrate configuration [Cheetham, P. S. J., and Wootton, A. N., "Byconversion of D-galactose into D-tagatose", Enzyme Microbiol. Technol, 15, 105-108 (1993)].
• Enterobacter agglomerans could also produce tagatose from galactose during the growth on the arabinose pre-induced medium. Such implies the fact that arabinose isomerase could mediate the conversion of tagatose [Kim, S. Y., Roh, H. J., and Oh, D. K., "D-Tagatose production from D-galactose by Enterobacter agglomerans TY-25", Kor. ]. Appl. Microbiol. Biotechnol, 25, 490-494 (1997)].
• the object of the present invention is to provide a recombinant E.coli having a new metabolic pathway for producing tagatose from galactose.
• Another object of the present invention is to provide new method for tagatose production by using said recombinant E.coli.
• the further object of the present invention is to provide a new method for tagatose production by using the L-arabinose isomerase originated from said recombinant E.coli.
• the present invention relates to a recombinant E.coli (KCTC-0603BP) harboring vector comprising i ) the gene of L-arabinose isomerase (EC 5.3.1.4; araA) and ii ) the promoter controlled artificially (pTClOl) for tagatose production.
• araA is originated from the group consisting of E.coli, Bacillus, Salmonella, Enterobacter, Klebsiella, Pseudomonas, Lactobacillus, Zymononas, Ghiconobacter, RJtizobium, Acetobacter, RJwdobacter, Agrobacterium, and other microorganisms. Further, araA is integrated into the host chromosome.
• the another aspect of the present invention relates to a fermentation process for tagatose production wherein the medium comprises 10-300 g/L of galactose, 7-13 g/L of yeast extract, 2-4 g/L of KH 2 P0 4 5-7 g/L of Na 2 HP ⁇ 4 and 1-3 g/L of ammonium chloride.
• L-arabinose isomerase from said strain mediates galactose to tagatose conversion.
• the L-arabinose isomerase can be immobilized for the recycle.
• the conversion medium comprises 10-500 g/L of galactose, 2-4 g/L of KH2PO 4 , and 5-7 g/L of Na 2 HP0 4 .
• Fig. 1 is a chemical structure of D-tagatose and D-galactose.
• Fig. 2 is a schematic diagram of bioconversion by L-arabinose isomerase.
• Fig. 3 is a photograph which represents PCR product of L-arabinose isomerase from E.coli. Arrow indicates the PCR product of Ara A from E.coli (1.5 kb). Lane 1 shows the size marker (lkb DNA Ladder, NEB, USA).
• Fig. 4 is a plasmid map of pTClOl, which harboring L-arabinose isomerase.
• Fig. 5 is a photograph which represents EcoRl-Hindl ⁇ double digest of pTClOl. Arrow indicates the pTClOl. Lane 1 shows the size marker (lkb DNA Ladder, NEB, USA).
• Fig. 6 is a profile of tagatose production by purified L-arabinose isomerase.
• D-Tagatose is a potential bulking agent in food as a non-caloric sweetener.
• plasmids harboring the L-arabinose isomerase gene (araA) from Esdierichia coli is constructed because L-arabinose isomerase has been suggested as an enzyme that mediates the bioconversion of galactose to tagatose as well as that of arabinose to ribulose.
• the constructed plasmids has been named as pTClOl, which contains the araA from E.coli.
• tagatose has been produced from galactose in 9.0—11.0 % yields.
• the purified L-arabinose isomerase of E.coli with the plasmid pTClOl has also produced 25—35 g/L of tagatose from 100 g/L of galactose for 150 —180 hours bioconversion.
• the enzyme extract of E.coli with the plasmid pTClOl has been immobilized into alginate bead. By using an immobilized enzyme system, 11 —14 g/L of tagatose has been produced from 10 % galactose for 24 hours.
• E.coli is a Gram negative facultative anaerobic rod-form bacteria [Holt J. G., et. al, Bergey's Manual of Systematic Bacteriology 9th ed., 179, Williams & Wilkins (1994)]. It shows simple nutrient requirement, fast life cycle (about 20 min) in the optimal condition (pH 7.0, 37°C) [Stanier R. Y., et. al, The Microbiol World, 5th ed., 439 - 452(1986)].
• E.coli is the most well known organism in physiology and genetics. Therefore, metabolism of E.coli can be easily controlled by the methods of genetic engineering and metabolic engineering.
• the gene of L-arabinose isomerase (araA) of E.coli W3110 has been cloned by using a PCR technique, inserted into vector pKK223-3, which yields pTClOl.
• the constructed plasmids harboring araA from E.coli has been used to transform E.coli JM105 (E.coli JM105/pTC101).
• ImM IPTG was added to medium.
• AraA L-Arabinose isomerase
• the normal E.coli does not express AraA in the medium for tagatose production because the medium contains galactose as a sole carbon source.
• the AraA could be artificially induced by adding an IPTG.
• E.coli JM105/pTC101 was deposited in Korean Collection for Type Cultures in Korea Research Institute of Bioscience and Bioengineering, in accession number of KCTC-0603BP on April 26, 1999 under Budapest Treaty.
• Step 1 PCR cloning of araA and Insertion into Expression Vector
• the template for PCR of araA was chromosome of a wild type E.coli W3110 [Genetic stock center collection number (CGSC) 4474],
• the each primer used in PCR contained the part of araA terminal sequence and restriction enzyme site (EcoRI and Hi ⁇ rflll, respectively).
• FORWARD 5'-GACGAATTCATGACGATT-3' (SEQ ID NO. 1)
• BACKWARD 5'-TGCAAGCTTTTAGCGACG-3' (SEQ ID NO. 2)
• PCR product sequence (SEQ ID NO. 3) is as follows.
• the constructed plasmid was transformed into E.coli JM105 [Roh, H. J., Kim, P., Park Y. C, and Choi, J. H., Biotechnol and Appl. Biochem., BA99/65, in press] (Fig.4).
• Step 2 Strain Selection
• the transformed strains were selected on the plate containing ampicillin (50g/ml) on the base of ampicillin-resistance. The colonies were further confirmed by restriction enzyme which digests the harbored plasmids, which shows 1.5 kb + 4.5 kb DNA fragments at the time of digestion by EcoRI and HmdIII (Fig. 5). The finally selected strain was named as E.coli JM105/pCT101, and said E.coli JM105/pTC101 was deposited in Korean Collection for Type Cultures in Korea Research Institute of Bioscience and Bioengineering, in accession number of KCTC-0603BP on April 26, 1999 under Budapest Treaty.
• the E.coli JM105/pTC101 was pre-cultured in the LB-medium, and then transferred into main culture medium.
• the main medium contained following components (Table 1).
• Tagatose and galactose were estimated by using a HPLC (alliance 2690, Waters, USA) with RI detector (RID410, Waters, USA). The separation was made by C18-amine column (KR100-10NH 2 , Kromasil, Bohus, Sweden) eluted isocratically by 80% acetonitrile (35°C, 2 ml/min).
• recombinant E.coli expressing L-arabinose isomerase could convert galactose into tagatose, where control strain could not.
• a crude extract of AraA from E.coli was prepared as follows. Actively growing cells (50 ml, O.D. 600 2.0) in the LB-medium containing IPTG (1 mM) were prepared by centrifugation (7,000 g, 4°C). The cell pellet was resuspended in a 5 ml of phosphate buffer (50 mM, pH 7.0). The cells were disrupted by using an ultrasonic processor (50-watt Model, Cole-Parmer) at 40% output for 10 min in the ice. The crude arabinose isomerase solution was obtained after removal of cell-debris by centrifugation of the cell-disrupted suspension at 7,000 g and 4"C for 15 min.
• a crude extract of AraA was fractionated by ammonium sulfate salting out at 40-60% saturation, and pelleted by centrifuge (10,000g, 4°C). The pellet was further dialyzed overnight at 4°C using a cellulose membrane tubing (MWCO : 12,000, Sigma, USA) to remove ammonium sulfate. Column chromatography was further carried out using DEAE (Sigma, USA) and Q-Sepharose (Sigma, USA) as anion exchange resins. The final purified L-arabinose isomerase showed 43 U/ml, where one unit defined as the amount of enzyme which produced 1 g-tagatose per minute at 35 °C, pH 7.0.
• the reaction mixtures consisted of 9 ml of galactose (1 g) dissolved in 50mM phosphate buffer (pH 7.0) and 1 ml of enzyme solution mentioned the above.
• the bioconversion for tagatose production was performed at 35 °C, pH 7.0 for 168 hrs. As shown in Fig. 6, the final tagatose concentration was reached 29.5 g/L. Remained galactose was 70.5 g/L.

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