KR100320269B1 - New 2-Ketoaldonate Reductase Gene and 2-Keto-L-Glonate Production Using It - Google Patents

Abstract

The present invention determines the gene sequence of the novel E. coli-derived 2-ketoaldonate reductase and deletes the genes of these enzymes to inhibit the intracellular metabolism of intermediates during vitamin C production by glucose metabolism. To the production of 2-keto-L-gulonate, a vitamin C precursor in high yield, from phosphoglycerate dehydrogenase and hydroxypyruvate reductase from Escherichia coli PCR cloning of the yiaE gene showing similarity with (hydropyruvate reductase) and investigating the substrate specificity of the enzymatic protein product of the cloned yiaE gene to verify that the yiaE gene is a gene encoding 2- ketoaldonate reductase. after determining that a defect in the gene coding for the verification yiaE a 2-keto aldose reductase carbonate, and then E. coli I As a result of measurement of 2-keto-ALDO carbonate reductase activity is expressed in recombinant E. coli, 2-keto reductase The aldose carbonate yiaE a gene defect caused by 2-keto-organisms in Escherichia coli-El-production of the glow carbonate Prevents the intracellular metabolism of 2-keto-di-gluconate, 2,5-diketo-di-gluconate, and the final product, 2-keto-L-glonate, which are intermediates in There is an excellent effect of improving the production yield of keto-L- glonate.

Description

Novel 2-ketoaldonate reductase gene and 2-keto-L-glonate production using the same

The present invention relates to a gene sequence of the novel 2-ketoaldonate reductase derived from E. coli and the production of 2-keto-L-glonate using the same. More specifically, the present invention relates to the efficient bioconversion of 2-keto-L-glonate by genetic engineering of the novel E. coli derived 2-ketoaldonate reductase.

2-keto-L-Glonate, a vitamin C precursor using bioconversion process

bioconversion process by microorganisms of gulonate) from glucose to gluconate, 2-keto-D-gluconate and 2,5-diketo-di-gluconate Glucose pathway that produces (2,5-diketo-D-gluconate) and then converts to 2-keto-el-glonate. This route has only recently entered commercial pilot production, but the information is limited to the research of Shionogi and Genentech in Japan. Since the raw material price is 40% of sorbitol, the research has been conducted since the beginning of the first stage production by recombinant strains in the early 80s. Observing the physiological and metabolic aspects of the glucose pathway, glucose is oxidized in periplasm by glucose dehydrogenase to be converted into gluconate and then membrane-bound gluconate, 2-keto-di- Converted to 2,5-diketo-di-gluconate by gluconate dehydrogenase. All of the enzymes involved in these dehydrogenation reactions are known as membrane-bound enzymes. As the enzyme reaction from glucose in the medium to 2,5-diketo-di-gluconate proceeds in the periplasm, 2,5 -Diketo-di-gluconate is produced in high yield (Matsushita et al.,Adv. Microb. Physiol, 26, 247-301, 1994). Membrane-bound glucose dehydrogenase, a coenzyme of these enzymes, is known to be PQQ-dependent and gluconate, 2-keto-di-gluconate dehydrogenase, is FAD-dependent. 2,5-diketo-di-gluconate converted from glucose through three stages of dehydrogenation was converted to 2-keto-el-glonate via cytoplasmic 2,5-diketo-di-gluconate reductase Must be reduced. However, until now, microorganisms producing 2,5-diketo-di-gluconate are known to have no 2,5-diketo-di-gluconate reductase activity. Conversion to 2-keto-L-glonate. The possibility that this pathway could be used for the production of 2-keto-L-glonate is described by Sonoyama in 1982 from 2-keto- from 2,5-diketo-di-gluconate using Corynebacteria. It was confirmed by producing L-glonate (Sonoyama et al.,Appl. Environ. Microbiol, 43, 1064-1069, 1982; Sonoyama et al., US Pat. No. 3,998,697), after which a number of microorganisms are known which can convert 2,5-diketo-di-gluconate to 2-keto-l-glonate (Sonoyama et al., JP 4,543,331). The 2,5-diketo-di-gluconate reductases known to date are known to be NADPH-dependent. In fact, most of the acetic acid bacteria and aeroniaErwinia), Pseudomonas (Pseudomonas) It is known that a variety of microorganisms can produce 2,5-diketo-di-gluconate from glucose, but it is 2,5- because it does not have 2,5-diketo-di-gluconate reductase activity. Securing microorganisms capable of converting iketo-di-gluconate to 2-keto-el-glonate is an important factor in the production of 2-keto-l-glonate via the glucose pathway (Sonoyama et al.,Agric. Biol. Chem, 51, 2003-2004, 1987).

In 1982, Sonoyama et al. Produced 2,5-diketo-di-gluconate from Erwinia sp. And mutated 2-keto-el-glo using strains of Corynebacterial microorganisms. It was reported that a two-step bioconversion process to convert to Nate could yield 106 g / L 2-keto-L-glonate at a yield of about 85% for 100 hours of fermentation (Sonoyama et al. , Appl. Environ.Microbiol , 43, 1064-1069). They studied the process of mixed culture of Eurnia microorganisms and Corynebacteria microorganisms and searched for new 2,5-diketo-di-gluconate producing bacteria. Most 2,5-diketo-di-gluconate producing strains and 2-keto-el-glonate converting strains have high metabolism of 2,5-diketo-di-gluconate and produced 2-keto- Since el-glonate can be reduced and metabolized again by idonate, it is reported that breeding of a new strain having a related variation in enzymatic activity is required. In 1987, Sonoyama et al. Described 2-keto-l-glonate when converting 2,5-diketo-di-gluconate to 2-keto-l-glonate using a Corynebacterial microorganism. Metabolic pathways of 2,5-diketo-di-gluconate of the genus Corynebacteria were analyzed in order to solve the accumulation of 2-keto-di-gluconate, which is an optical isomer of (Sonoyama et al . , Agric. Biol. Chem ., 51, 3039-3047, 1987). According to their results, a problem that may occur when converting 2,5-diketo-di-gluconate to 2-keto-l-glonate is that 2,5-diketo-di-gluconate is 2 Under the action of 2-ketoaldonate reductase and 2,5-diketo-di-gluconate reductase, simultaneously with 2-keto-di-gluconate and 2-keto-el-glonate Is to be converted. In addition, 2-keto-L-glonate converted from 2,5-diketo-di-gluconate is converted to idonate by 2-ketoaldonate reductase and then 5-keto-di-gluconate ( 5KDG), it was confirmed that it can be converted to gluconate and metabolized by 5-keto-di-gluconate reductase. In order to solve this problem, 2-keto-di from 2,5-diketo-di-gluconate was induced by mutants that lost the activity of 2-ketoaldonate reductase and 5-keto-di-gluconate reductase. A mutant strain that produced 2-keto-L-glonate without the accumulation of -gluconate was obtained, yielding 90.5 mole% yield, but production of byproduct idonate could not be prevented.

Anderson et al. Isolated the 2,5-diketo-di-gluconate reductase gene from Corynebacterial microorganisms ( Erwinia herbicola ) to establish a one-step production process (Anderson et al., Science , 230, 144-148, 1985). The initial yield was very low, but the experimental results for steady yield improvement yielded 100 g / L 2-keto-L-glonate in about 100 hours. An enzyme with similar properties was cloned from the same strain and published a higher (20-30 g / L) process in Greenley et al. (Glindley et al. , Appl. Environ. Microbiol ., 54, 1770). -1775, 19988). The reason for the low yield of the above recombinant strains using the glucose pathway is firstly, the necessity of NADPH regeneration required for intracellular 2,5-diketo-di-gluconate reductase, and secondly, 2,5-diketo- Intracellular transfer of di-gluconate, third secretion of 2-keto-L-glonate formed intracellularly, fourth of 2,5-diketo-di-gluconate and 2-keto-el-glonate It is analyzed to be due to degradation by host cells. Yield improvement was attempted to block byproduct idonate production pathways and metabolic pathways of 2,5-diketo-di-gluconate and 2-keto-el-glonate degradation. Anderson et al. Found that the two-ketoaldonate reductase of Erwinia is gluconate at 2-keto-di-gluconate at 2,5-diketo-di-gluconate and 2-keto-di-gluconate. Attempted to prevent the production of by-product idonate by depleting the gene encoding 2-ketoaldonate reductase, revealing that it catalyzes the conversion of all idonates in 2-keto-el-glonate. I didn't see any progress. This was confirmed to be due to the presence of an enzyme system that functions the same as the 2-ketoaldonate reductase already found in the Erwinia strain.

The biggest problem of the above bioconversion method or metabolic method is that the microorganism with oxidative metabolic process must have metabolic pathway using intermediate or final product in the cell. By simply blocking the previously known metabolic pathways can prevent the production of by-products, but caused many problems in the growth of microorganisms and smooth metabolic processes.

We consist of three subunits of Erwinia cypripedii and convert membrane gluconate dehydrogenase (GADH) to convert gluconate to 2-keto-di-gluconate (GADH) ( Yum et al., J. Bacteriol . 179, 6566-6572, 1997), were cloned and expressed in E. coli and converted to high yield. However, during the conversion experiment from gluconate to 2-keto-di-gluconate using recombinant E. coli, 2-keto-di converted to gluconate after gluconate was completely consumed in the medium as shown in FIG. Gluconate little by little disappeared from the culture. Of course, the 2-keto-di-gluconate bioconversion is not a problem because the gluconate is rapidly converted to 2-keto-di-gluconate in high yield, but as mentioned above, 2-keto, a vitamin C precursor using glucose metabolism The cause of the E. coli was used as a host cell in the L-glonate bioconversion. In this process, the present inventors have no choice but to convert 2-keto-di-gluconate to gluconate if 2-keto-di-gluconate is metabolized, and thus, 2-keto using the cell extract of E. coli as a coenzyme solution. NADPH-dependent reductase activity of reducing di-gluconate to gluconate was measured. As a result, 2-keto-D-glucosidase enzyme activity was observed with the carbonate for reducing as gluconate This Corynebacterium (Corynebacterium), Brevibacterium (Brevibacterium), control Winiah (Erwinia), acetonitrile bakteo (Acetobacter), Microorganisms such as Gluconobacter , Serratia , Pseudomonas , etc. (Sonoyama et al. , Agric. Biol. Chem. 51, 3039-3047, 1987; Truesdell, et al. , J. Bacteriol . 173, 6651- 6656, 1991; Yum, et al ., Biosci. Biotechnol. Biochem . 62, 154-156 1998) showed that metabolic processes related to ketogluconate may also be present in E. coli. In microorganisms such as Erwinia, Acetobacter, Gluconobacter, Serratia and Pseudomonas, glucose is 2-keto-di-gluconate, 5-keto-di-gluconate, 2,5-diketo-di-gluconate In order to oxidize to ketogluconate, the reaction proceeds by a membrane-bound dehydrogenase linked to an electron transport chain as described above (Ameyama et al ., Agric. Biol. Chem . 51, 2943-2950, 1987; Sonoyama et al ., Agric . Biol. Chem. 52, 667-674 , 1988). Ketogluconate or phosphorylated ketogluconate is only possible after reduction by NADPH-dependent reductase to enter the metabolic process (Sonoyama et al. , J. Ferment. Technol . 65, 311-317, 1987; Truesdell, et al., J. Bacteriol. 173, 6651-6656, 1991). Convert 2-keto-di-gluconate to gluconate, 2,5-diketo-di-gluconate to 5-keto-di-gluconate, and 2-keto-l-glonate to idonate NADPH-dependent 2-ketoaldonate reductases, known to catalyze the reaction of scavenging reactions, have been incorporated into Brevibacterium ketosoreductum (Yum, et al ., Biosci. Biotechnol. Biochem . 62, 154-156, 1998). Biochemical properties have been reported by pure or partial purification in Erwinia herbicola (Truesdell, et al., J. Bacteriol . 173, 6651-6656, 1991). In addition, although the substrate specificity to 2,5-diketo-di-gluconate was not investigated, acetic acid 2-keto-di-gluconate reductase also reacted to 2-keto-L-glonate, iodonate, 2- It is known to convert keto-di-gluconate to gluconate (Ameyama and Adachi, CARBOHYDRATE METABOLISM. PART D. Wood, WA ed. 89, 198-202, 1982). Metabolic pathways involved in the use of more detailed ketogluconates are Corynebacterium (Sonoyama et al ., Agric. Biol. Chem . 51, 3039-3047, 1987) and Trusdell, et al ., J. Bacteriol . 173, 6651-6656, 1991). Ketogluconate metabolic pathways of these two microorganisms convert 2,5-diketo-di-gluconate into 5-keto-di-gluconate in the genus of Erwinia, but to gluconate in the corynebacterium microorganisms. It has considerable similarity, except that it is converted to 2-keto-di-gluconate before it is. Continuous conversion of ketogluconate to gluconate is accomplished by soluble NAD (P) H-dependent reductases, which are phosphorylated to become 6-phosphogluconate followed by pentose phosphate Metabolized via the pentose phosphate pathway.

We not only reduce the 2-keto-di-gluconate described above to gluconate but also convert 2,5-diketo-di-gluconate to 5-keto-di-gluconate, and 2-keto-el Determining the presence of the 2-ketoaldonate gene of Escherichia coli encoding 2-ketoaldonate reductase catalyzing the reduction of glonate to idonate and from these results there may be other ketogluconate reductases present in E. coli Using the genome base database of E. coli, which was completed in 1999, related enzymes were searched among genes whose products were not yet identified, and the potential genes were isolated by PCR, the gene products were purified, and the substrate specificity was examined. Genes encoding aldonate reductase have been found in E. coli. 2-ketoaldonate reductase is an enzyme that can affect the final yield by inducing the metabolism of intermediate conversion products when the glucose metabolic pathway is introduced into E. coli.

Accordingly, an object of the present invention is to provide a gene sequence of the novel E. coli-derived 2-ketoaldonate reductase. Another object of the present invention is to delete the gene of the novel E. coli-derived 2-ketoaldonate reductase, 2-keto-di-gluconate, 2,5-diketo which is an intermediate in the production of vitamin C by glucose metabolism It inhibits the intracellular metabolism of di-gluconate and the final product 2-keto-el-glonate to produce 2-keto-el-glonate, a vitamin C precursor, in high yield. Another object of the present invention is to transform glucose into a deficiency gene lacking the novel E. coli-derived 2-ketoaldonate reductase gene. To provide a transformed host cell to convert to.

The object of the present invention is to PCR-cloned yiaE gene showing similarity with phosphoglycerate dehydrogenase and hydroxypyruvate reductase from E. coli and purifying enzyme protein of this gene product. After determining the amino acid sequence and investigating substrate specificity to verify that the cloned yiaE gene is a gene encoding 2- ketoaldonate reductase, a kanamycin resistance gene is added to the yiaE gene encoding 2- ketoaldonate reductase. Depletion of 2-ketoaldonate reductase gene by binding, introduced into E. coli, and measuring 2-ketoaldonate reductase activity expressed in recombinant E. coli to investigate the loss of function of the enzyme and the ketogluconate metabolic pathway Achieved by writing

1 is a graph showing the enzymatic activity of converting gluconate into 2-keto-di-gluconate using recombinant E. coli with the cloned membrane-bound gluconate dehydrogenase gene.

Figures 2a and 2b shows the nucleotide sequence and amino acid sequence of the 2-ketoaldonate reductase gene derived from E. coli.

Figure 3 shows the metabolic process of ketogluconate in other strains, including E. coli.

The present invention cloned yiaE , a 2- hydroxydehydrogenase gene showing similarity to phosphoglycerate dehydrogenase and hydroxypyruvate reductase by PCR, and transformed Escherichia coli with a pHD2 plasmid inserted with the yiaE gene. By measuring the 2- ketoaldonate reductase activity of the cell extract obtained from the recombinant Escherichia coli and then recombining to verify that the yiaE gene is a gene encoding 2- ketoaldonate reductase; After attaching His-Tag to the COOH terminal of the gene product of yiaE , the 2- ketoaldonate reductase gene, and performing PCR, the yiaE gene was cut, inserted into a plasmid, introduced into E. coli, cultured, and the enzyme protein Ni-NTA. Obtaining only an enzyme protein by purifying with resin; Measuring a protein molecular weight of the purified enzyme protein and determining an amino acid sequence; Investigating optimal activity and substrate specificity of the purified enzyme protein; 2-keto reductase genes of aldose carbonate yiaE by inserting a gene fragment containing the Km resistance gene of Tn903 pUC4K in the middle of the gene of 2-keto aldose carbonate defect yiaE gene :: Km produced the gene and the gene is inserted into the strain Comparing the 2- ketoaldonate activity of the strain with the yiaE gene; Comprising the step of preparing the ketogluconate metabolic pathway based on the substrate specificity of 2-ketoaldonate reductase.

Hereinafter, the detailed description of the present invention will be described step by step with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1: 2-ketoaldonate reductase gene cloning produced by Escherichia coli and 2-ketoaldonate reductase obtained by expressing a mutant gene

In this example, the E. coli Genome Sequencing Project (Blattner et al., Science , 277, 1453-1462 1997; Sofia et al., Nucleic Acids Res . 22, 2576-2586, 1994) was completed in late 1997. Therefore, to find a gene encoding an enzyme that converts 2-keto-di-gluconate to gluconate, an estimated putative hydroxyacid reductase or dehydrogenase (E. coli gene) has not yet been identified. Searching for genes encoding proteins thought to be dehydrogenases, located at 80.1 minutes on E. coli chromosome, located between bisC - cspA genes, phosphoglycerate dehydrogenase and hydroxypyruvate reductase ( 2-hydroxyacid dehydrogenase ( yiaE ) showing similarity with hydroxypyruvate reductase (Blattner et al., Science , 277, 1453-1462 1997; Sofia et al., Nucleic Acids Res . 22, 2576-2586, 1994).

First step: yiaE Gene cloning

In this step, to identify the protein encoded by the yiaE gene, E. coli W3110 chromosomal DNA was cloned by PCR. At this time, PCR primers [2KRA-5 ′] as shown in FIGS. 2A and 2B based on the yiaE sequence (GenBank accession No. AE000432) (Blattner et al., Science , 277, 1453-1462 1997) stored in a GeneBank. ; 5'ACGGGTGGTCACGACCTGAACAT3 ', the forward primer and oligobase sequence 2KRA-3';5'ATGAACGGTTCGCTGGGTGTGCT3', the reverse primer] was designed and used for PCR cloning. PCR was performed using Perkin Elmer's GeneAmp PCR system 2400 for 30 seconds of denaturation at 95 ° C, annealing at 65 ° C for 30 seconds, and enzymatic polymerization for 2 minutes at 72 ° C. Minute extension reactions were carried out at 72 ° C. The PCR reaction product was digested with the restriction enzyme Bcl I, the 1.5 kb DNA fragment was digested with the same restriction enzyme, linked to the CIP-treated pUC19 vector plasmid, and transformed into E. coli DH5α. The recombinant plasmid in which the PCR DNA fragment was inserted was named pHD2 and finally confirmed yiaE gene cloning through DNA sequencing. That is, the 2-ketoaldonate reductase activity of the recombinant E. coli cell extract into which the pHD2 plasmid into which the cloned yiaE gene was inserted was introduced was measured. As a result, the enzyme activity was 10 times higher than that of E. coli DH5α having no plasmid. This activity not only converts 2-keto-di-gluconate to gluconate but also converts 2,5-diketo-di-gluconate to 5-keto-di-gluconate, 2-keto-el-glonate It was found that the yiaE gene encodes 2- ketoaldonate reductase by idonate conversion.

Escherichia coli DH5α recombined with the recombinant plasmid pHD2 obtained in this step was named E. coli DH5α / pHD2, and the strain was deposited with the KCTC 0507BP at the Gene Bank of the Korea Institute of Science and Technology.

Second step: yiaE Purification of 2-ketoaldonate reductase protein, a gene product

In this stepyiaETo more precisely determine whether the gene product is 2-ketoaldonate reductase, His-tagged by metal-chelate affinity chromatography using a Ni-NTA column. Enzyme protein was purified. In order to isolate and purify 2-ketoaldonate reductase from E. coli,yiaERecombinant plasmids were prepared to attach 6 His-Tags to the COOH terminus of the gene product. That is, using 2KRA5 'used for cloning as the forward primer in the first step and reverse primer 5'GGGgaattcAGTGATGGTGATGGTGATG GTCCGCGACGTGCGGATTCACAC3 '[human-made restriction enzymeEcoRI recognition parts are shown in lowercase letters and underlined partsyiaEPCR was performed under the same reaction conditions as that of cloning, showing the base sequence complementary to the COOH terminus of the gene, the additional 6 His-coding portions, and the stop codon and tcA complementary to each other. . PCR product restriction enzymesBclI andEcoAfter digestion with RI, ligation with pUC19 digested with the same restriction enzyme was carried out and transformed into E. coli DH5α to finally prepare pUCHisC. Through sequencingyiaEIt was confirmed whether the gene is inserted. 6His-tag fusion enzyme protein was purified in recombinant E. coli using Ni-NTA resin from QIAGEN. Cell cultures of 500 ml of recombinant E. coli DH5α (pUCHisC) were centrifuged to collect the cells, suspended in 20 ml of Na-phosphate (pH 8.0), 0.3 M NaCl solution and sonicated on ice. 16,000 cell debrisgThe supernatant was directly passed through a column containing 1.6 ml of Ni-NTA resin by centrifugation at. The column was washed with 30 ml 50 mM Na-phosphate (pH 8.0), 0.3 M NaCl, 10% glycerol buffer and C-terminal 6 His-tagged 2-ketoaldonate reductase was 50 mM Na Recovered by eluting with citrate buffer (pH 6.0).

Third Step: Amino Acid Sequencing of Purified 2-Ketoaldonate Reductase

Purity of the protein purified in the second step was performed by SDS-PAGE according to the method of Laemmli (Laemmli, 1970). The purified enzyme protein was determined to be 74,000 Daltons by Sephacryl S-200 gel filtration. The molecular weight determined by SDS-PAGE was deduced amino acid sequence inferred to 36,000 Daltons. It is consistent with the determined molecular weight. This indicates that the enzyme protein is present as a dimer. The molecular weight and subunit structure of E. coli 2-ketoaldonate were determined by the inventors of 2-keto isolated from B. ketosoreductum (Yum et al ., Biosci. Biotechnol. Biochem . 62, 154-156,1998). Similar to aldonate. In order to determine the amino-terminal amino acid sequence of the purified enzyme protein, SDS-PAGE purified protein was purified using poly-vinylidene difluoride (PVDF) for 1 hour at 100 V using a Bio-Rad Trans-Blot apparatus. ) To the membrane. PVDF membranes were stained with dye solution (0.1% Coomassie Blue R-250 in 50% (v / v)) and decolorized with decolorizing solution (10% (v / v) acetic acid in 50% methanol) for 3 minutes. The amino acid sequence was determined by cutting the stained enzyme protein band (band) with an Applied Biosystems model 470A sequencer. The amino terminal amino acid sequence of the purified protein is NH ₂ -Met-Lys-Pro-Ser-Val-Ile-Leu-Tyr-Lys-Ala-Leu-Pro-Asp-Asp-Leu-Leu-Gln-Arg-Leu- Determined by Gln-Glu-His, this amino acid sequence is consistent with the deduced amino acid residues from 1 to 22 in FIG. 2A. This proved that the transcription initiation site was not the ATG of nt 355-357, but the ATG following the ribosome-binding sequence and GGAG (nt 357-360) as described above.

Fourth Step: Biochemical Properties of 2-keto-aldonate Reductase

In order to investigate the biochemical properties of the enzyme of the present invention, the optimum pH and substrate specificity of the enzyme activity were investigated. As a result, the optimum pH of enzyme activity was 7.5 and NADPH was used as electron donor rather than NADH as coenzyme. The 2-ketoaldonate reductase of the present invention is characterized in that 2,5-diketo-di-gluconate is converted to 5-keto-di-gluconate and 2-keto-di-gluconate is shown in Table 1. Nate has been shown to catalyze the enzymatic reaction of reducing 2-keto-L-glonate to idonate.

Substrate Specificity of Escherichia Coli 2-ketoaldonate Reductase Quality Relative Activity (%) ^α 2-keto-di-gluconate (2KDG) 100 2,5-diketo-di-gluconate (25DKG) 46 2-keto-l-glonate (2KLG) 55 5-keto-D-gluconate (5KDG) 0 D-fructose 0 L-Solbos 0

Step 5: preparation of 2-ketoaldonate reductase with missing genes

In this step, when the membrane-bound gluconate dehydrogenase (GADH) gene is expressed in recombinant E. coli to produce 2-keto-di-gluconate, as described in step 4 above, 2- To prevent the loss of keto-di-gluconate, E. coli host cells lacking the 2- ketoaldonate gene ( yiaE ) were made by depleting the yiaE gene on the chromosomal using homologous recombination. . To this end, a plasmid pHD-Km having a kanamycin resistance gene inserted into the yiaE gene was prepared. Gene yiaE raised to insert a Bam HI fragment Km resistance 1.2kb containing the Tn903 gene of pUC4K (Pharmacia) in the middle nucleotide (oligonucleotide) 5'TGCGCACGT G AT G CC AGCGCCAT3 'and 5'CTTTGGCTTCAACATGCCCATCCTC3' (respectively nucleotides 838-860 And 861-885, the underline indicates the Bam HI restriction enzyme recognition site and the dark font indicates the substituted nucleotide to artificially make the restriction enzyme recognition site) as a primer, restriction of new Bam HI by PCR An enzyme recognition site was made. The PCR template was pHD2 and was ligation after polynucleotide kinase treatment. The prepared plasmid pHD-Bam was digested with restriction enzyme Bam HI and ligation with 1.4 kb Bam HI fragment (Km) of pUC4K. Plasmids with the insertion mutation of the yiaE gene were used to prepare mutations on E. coli chromosomes. Restriction method of a plasmid, such as the pHD-Km Winans digested with Sca I (Winans, etc., J. Bacteriol. 161, 1219-1221, 1985) in accordance with by transforming the JC7623 (recBC, sbcBC) Km ^r colonies of (colony ) And DNA fragments containing Km resistance genes were integrated into chromosomes by P1-mediated transduction (Miller, Experiments in molecular genetics. Cold Spring Harbor Laboratory, 1972). .

Example 2: 2-ketoaldonate reductase activity assay with gene deletion

PCR of the 2KRA-5 'and 2KRA-3' primers used for cloning the yiaE gene to determine whether the 2-ketoaldonate reductase, in which the gene was deleted in Example 1, finally formed a yiaE :: Km deletion on the chromosome to the identified and the W3110 strain chromosome (chromosome) yiaE the gene function is not lost if the JC7623 as shown in Table 2 (yiaE :: Km) and W3110 (yiaE :: Km) the enzymatic activity of the strains lost 2KR It was confirmed that. The pHD2 plasmid was introduced into the W3110 ( yiaE :: Km ) mutant strain to finally confirm whether the loss of 2KR enzymatic activity of the deleted mutant strain is from the yiaE gene loss. As a result, 2-ketoaldonate reductase activity of the strain W3110 ( yiaE :: Km ) (pHD2) was restored, and it was finally confirmed that the yiaE gene encodes a 2- ketoaldonate reductase protein.

Enzyme Activity Analysis of yiaE Gene Deficient Mutant Strain ^a 2KR active ^b (protein U / mg) Conversion of 2-keto-di-gluconate to gluconate JC7623 0.042 + JC7623 ( yiaE :: Km) 0 - W3110 0.009 + W3110 ( yiaE :: Km) 0 - W3110 ( yiaE :: Km) (pHD2) 0.390 + A strain was incubated at 37 ° C. for 18 hours using LB medium, and enzyme activity was measured using 2-keto-di-gluconate as a substrate in cell extracts.

Example 3: Ketogluconate Metabolism Pathway

As described in Example 1 and Example 2, the present invention detected the yiaE gene, proved that the gene product was ketogluconate reductase, and confirmed the substrate and the reaction product of the enzyme. Therefore, based on this fact, the metabolic pathway of E. coli ketogluconate is shown in FIG. That is, 2,5-diketo-di-gluconate reductase is metabolized after conversion to 5-keto-di-gluconate in the genus Erwinia and 2-keto-di-gluconate in the genus Corynebacterium. Metabolized after conversion to E. coli can be converted to gluconate via 2-keto-di- gluconate, 5-keto-di- gluconate, 2-keto-L- glonate. Gluconate produced via the above pathway is fully metabolized via the Entner-Doudoroff (ED) pathway. Routes through 2-keto-el-glonate, iodonate, 5-keto-di-gluconate of 2,5-diketo-di-gluconate have recently been described by Bausch et al. (Bausch et al., J Bacteriol 180, 3704). -3710, 1998) revealed the presence of IA dehydrogenase (5KR (I)), and thus the route is possible. Finally, membrane-bound gluconate dehydrogenase (E. coli) is used as a production host microorganism. Membrane bound 2-keto-di-gluconate dehydrogenase to produce 2-keto-di-gluconate or 2,5-diketo-di-gluconate or 2,5-diketo-di-gluconate To prevent ketogluconate from being metabolized intracellularly by expressing the 2,5-diketo-di-gluconate reductase gene from nate to produce a ketogluconate such as 2-keto-el-glonate, a vitamin C precursor It is possible by defective genes to yiaE It was confirmed.

The present invention has the effect of cloning the gene of 2- ketoaldonate reductase derived from Escherichia coli as described through the above Examples and Experimental Examples and determining and providing the base sequence of the yiaE gene encoding the reductase . Depletion of the yiaE gene, which is a gene of 2- ketoaldonate reductase, based on the metabolic pathways of these enzymes, results in the production of 2-keto-L- glonate , an intermediate in the production of 2-keto-L- glonate by E. coli bioconversion. By preventing intracellular metabolism of gluconate, 2,5-diketo-di-gluconate and the final product 2-keto-el-glonate, the yield of the production of vitamin C precursor 2-keto-l-glonate was It is a very useful invention for the biological industry because it has an excellent effect of improving.

Claims (6)

A gene derived from Escherichia coli and encoding the 2-ketoaldonate reductase, which comprises the following gene. ATGAAGCCGTCCGTTATCCTCTACAAAGCCTTACCTGATGATTTACTGCAACGCCTGCAA 60 GAGCATTTCACCGTTCACCAGGTGGCAAACCTCAGCCCACAAACCGTCGAACAAAATGCA 120 GCAATTTTTGCCGAAGCTGAAGGTTTACTGGGTTCAAACGAGAATGTAAATGCCGCATTG 180 CTGGAAAAAATGCCGAAACTGCGTGCCACATCAACGATCTCCGTCGGCTATGACAATTTT 240 GATGTCGATGCGCTTACCGCCCGAAAAATTCTGCTGATGCACACGCCAACCGTATTAACA 300 GAAACCGTCGCCGATACGCTGATGGCGCTGGTGTTGTCTACCGCTCGTCGGGTTGTGGAG 360 GTAGCAGAACGGGTAAAAGCAGGCGAATGGACCGCGAGCATAGGCCCGGACTGGTACGGC 420 ACTGACGTTCACCATAAAACACTGGGCATTGTCGGGATGGGACGGATCGGCATGGCGCTG 480 GCACAACGTGCGCACTTTGGCTTCAACATGCCCATCCTCTATAACGCGCGCCGCCACCAT 540 AAAGAAGCAGAAGAACGCTTCAACGCCCGCTACTGCGATTTGGATACTCTGTTACAAGAG 600 TCAGATTTCGTTTGCCTGATCCTGCCGTTAACTGATGAGACGCATCATCTGTTTGGCGCA 660 GAACAATTCGCCAAAATGAAATCCTCCGCCATTTTCATTAATGCCGGACGTGGCCCGGTG 720 GTTGACGAAAATGCACTGATCGCAGCATTGCAGAAAGGCGAAATTCACGCTGCCGGGCTG 780 GATGTCTTCGAACAAGAGCCACTGTCCGTAGATTCGCCGTTGCTCTCAATGGCCAACGTC 840 GTCGCAGTACCGCATATTGGATCTGCCACCCATGAGACGCGTTATGGCATGGCCGCCTGT 900 GCCGTGGATAATTTGATTGATGCGTTACAAGGAAAGGTTGAGAAGAACTGTGTGAATCCG 960 CACGTCGCGGACTAA 975 Recombinant plasmid pHD2 comprising the gene of claim 1. E. coli DH5α / pHD2 strain transformed with the recombinant plasmid pHD2 of claim 2 (Accession No. KCTC 0507BP). Recombinant strain having a plasmid pHD-Km in which the kanamycin-resistant gene is inserted in the middle of the gene of claim 1 and then introduced into E. coli bacteria, lacking the activity of 2-keto-L-glonate reductase. A method of preparing 2-keto-di-glunate comprising culturing the recombinant strain of claim 4. 2-ketoaldonate reductase derived from Escherichia coli and having the following characteristics. a) Composition and molecular weight: The molecular weight is 74kDa, consisting of homodimer, b) amino acid sequence: the amino acid sequence is shown as follows, MKPSVILYKALPDDLLQRLQEHFTVHQVANLSPQTVEQNAAIFAEAEGLLGSNENVNAAL 60 LEKMPKLRATSTISVGYDNFDVDALTARKILLMHTPTVLTETVADTLMALVLSTARRVVE 120 VAERVKAGEWTASIGPDWYGTDVHHKTLGIVGMGRIGMALAQRAHFGFNMPILYNARRHH 180 KEAEERFNARYCDLDTLLQESDFVCLILPLTDETHHLFGAEQFAKMKSSAIFINAGRGPV 240 VDENALIAALQKGEIHAAGLDVFEQEPLSVDSPLLSMANVVAVPHIGSATHETRYGMAAC 300 AVDNLIDALQGKVEKNCVNPHVAD 324 c) Substrate specificity and activity: 2,5-diketo-D-gluconate to 5-keto-D-gluconate, 2-keto-D-gluconate to gluconate, 2-keto-L-gulonate to idonate To switch, d) coenzyme: NADPH.