KR20050038694A - Function and sequence of gene and amino acid of kasugamycin biosynthetic enzyme kasc - Google Patents

Abstract

The present invention relates to the gene sequencing and amino acid sequence of the enzyme for the synthesis of gas sugar mycin, dienepi glucose-4, 6- dehydratase (dNDP-) derived from Streptomyces kasugaensis KACC 20262 glucose-4,6-dehydratase) has the effect of determining the gene sequence and amino acid sequence of KasC. Dehydrogenation of hydrogen at position 6 causes dTDP-4-keto-6-deoxy-glucose, dUDP-4-keto-6-deoxy-glucose, dGDP-4-keto-6-deoxy-glucose, dADP-4-keto By producing -6-deoxy-glucose, it is an important enzyme that produces intermediate sugars that are essential for antibiotic biosynthesis. Biosynthesis-related enzymes, which function similar to conventional KasCs, mainly produce only dTDP-4-keto-6-deoxy-glucose, while KasC is able to produce various intermediate sugars. In addition, dTDP-4-keto-6-deoxy-glucose, a product of this enzyme, is currently produced by a domestic venture company (Genechem Inc.) and sold at a high price (10 mg, 240,000 won), dUDP-4-keto-6- Deoxy-glucose, dGDP-4-keto-6-deoxy-glucose, and dADP-4-keto-6-deoxy-glucose will be produced and sold at higher prices. Therefore, KasC, a dNDP-glucose 4,6-dehydratase, is an important enzyme in the production of aminoglycoside antibiotics, and its products and enzymes are sold as high value-added products. Can be.

Description

Nucleotide sequence and amino acid sequence of enzyme Cassche gene involved in biosynthesis of Gasshumaicin and its function {Function and Sequence of gene and amino acid of kasugamycin biosynthetic enzyme KasC}

The present invention relates to the gene sequence and amino acid sequence of the enzyme for the synthesis of gas sugar mycin, and the function of the enzyme. More specifically, the present invention relates to the gene sequence and amino acid sequence of DNDP-glucose-4,6-dehydratase KasC for biosynthesis of the antibiotic gashgamycin. It is about.

Indiscriminate abuse of antibiotics has led to the emergence of bacteria, fungi, and viral cancer cells that are resistant to conventional antibiotics, and actually reported by the US Centers for Disease Control and Prevention (CDC, USA) in 1,300 hospitals in 1992. Patients were reported to have died from bacterial diseases that were highly resistant to antibiotics, which further increased the demand for renal antibiotics to combat them. To date, about 10,000 antibiotics have been separated from microorganisms, and only about 100 of them are commercialized. Over time, the success rate and commercialization rate of new functional antibiotics have been significantly reduced. In order to solve this problem, in the case of aminoglycoside antibiotics, a method of neutralizing resistance mechanisms of resistant bacteria by synthesizing and attaching an artificial side chain to an existing antibiotic structure This has been widely done. Therefore, semi-synthetic products such as amikacin developed by nexstar and amplimed, debekacin and arbekacin from meiji Seika, Japan, and ispamycin from Schering-Plough, etc. It has led the market for lycoside antibiotics. However, since these semisynthetic products are targeted only to the target antibiotic, for example kanamycin for amikacin, and gentamicin for ispamicin, It is limited and inevitably increases the unit price in the process due to the synthetic product rather than the natural fermentation. Therefore, in order to overcome the above problems, instead of synthesizing side chains, the focus is on the development of new materials that modify the structure of existing antibiotics by introducing useful genes using genetic engineering techniques and actinomycetes vectors. At some point, antibiotic biosynthesis studies, which underlie these studies, are becoming increasingly important.

The present inventors first isolated a gene population for biosynthesis of gashugamycin, and among these, dNDP-glucose-4,6-dehydratase, which occupies an important position for gashgamycin biosynthesis. The nucleotide sequence and amino acid sequence of the KasC gene were identified and its function was identified. This enzyme is an important enzyme commonly involved in the biosynthesis of most aminoglycoside antibiotics. Its products are dTDP-4-keto-6-deoxy-glucose, dUDP-4-keto-6-deoxy-glucose, and dUDP-. 4-keto-6-deoxy-glucose, dGDP-4-keto-6-deoxy-glucose, and dADP-4-keto-6-deoxy-glucose are not only used as important substrates for biosynthesis research, but also similar to conventional KasC. While functioning biosynthetic enzymes mainly produce dTDP-4-keto-6-deoxy-glucose, KasC is able to produce a variety of intermediate sugars. In addition, dTDP-4-keto-6-deoxy-glucose, a product of this enzyme, is currently produced by a domestic venture company (Genechem Inc.) and sold at a high price (10 mg, 240,000 won), dUDP-4-keto-6- Deoxy-glucose, dGDP-4-keto-6-deoxy-glucose, and dADP-4-keto-6-deoxy-glucose will be produced and sold at higher prices. Therefore, if the enzyme is used to study antibiotic biosynthesis as well as to produce high value-added substrates and enzymes, its industrial value is great.

It is an object of the present invention to provide endogenous glucose-4, 6-dehydratase (dNDP) inserted as part of a cosmid clone into which a population of gas sugar may be derived from Streptomyces kasugaensis KACC 20262. The present invention provides the nucleotide sequence of KasC gene having the function of -glucose-4,6-dehydratase) and the amino acid sequence translated therefrom, as well as revealing its function.

The object of the present invention is to predict the PCR product after PCR amplification of the gene library prepared using the genome of S. kasugaensis KACC 20262, which is a Gashugamycin producing strain, using KS4 and KS5 primers. Clones containing five cosmids pJY168, pJY191, pJY194, pJY328, and pJY341 that were positive in bands of size were isolated, and these were inoculated into LB medium containing gas sugar mycin (1 mg / ml) Was detected, and the nucleotide sequence of about 20 kb obtained from these cosmid clones was determined, and the amino acid sequence of the gene was compared and homologous to about 1.0 kb of dieendi-4,6-dihydratase. This was achieved and achieved by analyzing its function.

 Hereinafter, the configuration and operation of the present invention.

The present invention is to prepare a gene library using the genome of S. kasugaensis KACC 20262, a production strain of Gashgamycin, and then biosynthesis of sugar Gashgamine, a 6-deoxyhexose of Kashugamycin. PCR was amplified using primers KS4 (5'-GTGTCCGTGATCTCGAGCTCG-3 ') and KS5 (5'-AACAAGCCGGTGCTGTTCTAC-3'), which can effectively amplify the TDP-glucose synthase that must exist for the test. Isolation of the clones comprising the five cosmid clones pJY168, pJY191, pJY194, pJY328, pJY341, which were inoculated into LB medium containing gasgamycin (1 mg / ml) Investigating that the five cosmids pJY168, pJY191, pJY194, pJY328, pJY341 are involved in the biosynthesis of gashgamycin, the five cosmids pJY168, pJY191, pJY194, pJY328 , subcloning all BamHI fragments contained in pJY341 into E. coli plasmid pBluescrip KS (+) and determining the base sequence of about 20 kb fragments by bidirectional sequencing, wherein the region coding for KasC exists in the gene group. KasC reveals amino acid homology with the dNDP-glucose-4,6-dehydratase gene by comparing the amino acid sequence translated from the sequencing and determining the sequence: KasC gene is transformed into E. coli based on the identified homology. Conversion to overexpression and then enzymatic reaction to identify the product.

      Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1 Streptomyces Gasuensis Streptomyces kasugaensis  Genetic Library of KACC 20262)

Streptomyces spectabilis ATCC 27741 was incubated in GYM medium, pure chromosomal DNA was isolated according to Hopwood's method, and the chromosomal DNA was partially cut with restriction enzyme Sau3AI, and partially The chromosome DNA of 30-40 kb in size was linked to a cosmid vector for producing a BamHI digested gene using T4 DNA ligase, and then strapped using a lambda packaging kit from STRATAGENE. Myses Gasugaensis Gene Library was created.

Example 2: Cloning of the Gasugamycin Biosynthesis Gene and Screening for Resistance to Gasugamycin

10000 colonies of the Streptomyces Gasuensis gene library prepared in Example 1 were inoculated in a 1.5 ml Eppendorf tube containing 1 ml of LBA liquid medium using a yellow tip and then incubated for one day. The cultures were then transferred to new Eppendorf tubes at 30 ul. Eventually 50 samples were transferred to one Eppendorfpu tube and the number of samples transferred to the new tube was reduced from the original 10,000 to 200. Plasmids were extracted from the collected 200 samples, and PCR reactions were performed with DNAs from 200 samples. The PCR primer used at this time was TDIP-glucose synthase, which is involved in the initial synthesis of sugars for biosynthesis of all antibiotics with sugars derived from 6-deoxyhexose in the antibiotic structure, such as gashugamycin. KS4 (5'-GTGTCCGTGATCTCGAGCTCG-3 ') and KS5 (5'-AACAAGCCGGTGCTGTTCTAC-3') primers developed for (TDP-glucose synthase) were used. As a result of the PCR reaction, PCR bands were identified in 5 samples out of 200 samples. Then, plasmids were extracted from 250 samples (1 ml) used to prepare 5 samples, and PCR was performed again. The final five recombinant cosmid clones were selected. Using the isolated recombinant cosmid was incubated in a solid medium containing 1 mg / ml of gas sugar. As a result, all the cosmid clones were indirectly confirmed to be biosynthetic genes because they were resistant to gasshumycin.

Example 3: Cloning of dNDP-glucose-4,6-dehydratase gene kasC from cosmid fragments and gene and amino acid sequencing and similarity search

     In this embodiment, since enzymes involved in antibiotic biosynthesis generally exist in a certain region of the genome, five cosmid clones 30-40 kb in which there is a population of Gashgammycin biosynthesis genes are also aminoglycoside-based. Since there will be a dTDP-glucose synthase gene used for antibiotic production, all BamHI fragments contained in the five cosmids pJY168, pJY191, pJY194, pJY328, and pJY341 will be added to E. coli plasmid pBluescrip KS (+). After subcloning, the sequencing of the 20 kb fragment could be determined by bidirectional sequencing.

The base sequence of the KasC gene and the amino acid sequence translated from this gene base sequence are shown in SEQ ID NO: 1. Using the FramePlot 2.3.2 program (ishikawa, J. and Hotta, K. FEMS Microbiol. Lett. 174: 251-253, 1999), the portion encoding the protein was searched based on the primary DNA sequence results. Experimental results show that there is an open reading frame encoding proteins in this region.

The protein, named KasC, consists of 330 amino acids starting with the start codon ATG and ending with the stop codon TGA. Also, as a result of searching for amino acid homology through the database, KasC was found to be 33% with GilE of S. griseoflavus, 35% with galE4 of Streptomyces avermitilis , and Streptomyces carzinostaticus subsp. It showed 37% homology with NDP-hexose-4,6-dehydratase of, and at least 30% homology with genes encoding dTDP-glucose-4,6-dehydratase of other actinomycetes.

Example 4: Checking the function based on homology of gene KasC

The kasC gene was isolated from pJY168 by PCR and then digested with EcoRI-HindIII and linked to pET28a + digested with the same type of enzyme to construct pETKasC. pETKasC was transformed into E. coli BL21 and incubated for 14 hours in 3 ml LB broth medium containing 50 ug / ml kanamycin. Each cultured E. coli transformant was inoculated with 1% in 250 ml LB broth (50 ug / ml kanamycin), incubated from OD 600 to 0.5, and added to a concentration of 1 mM IPTG to induce expression. Cell growth was grown to 1 at OD 600 and then cultured cells were collected by centrifugation at 5,000 rpm for 15 minutes. The collected cells were pulverized by ultrasonication, and cell free extracts were separated by centrifugation. KasC proteins were solublized with uria and refolded with sephacryl S-300 column. Enzyme reaction to confirm KasC function was carried out by adding 160 mM Tris-HCl (pH7.9), 8 mM NAD +, 1.6 mM dTDP-glucose, and KasC cell extract to 300 ㎕ of the total reaction mixture. After reacting for 60 minutes, it was immediately stored at -20 ° C until analysis. The enzyme reaction product of KasC was confirmed by measuring absorbance at Spectrophotometer, 320 nm. To determine substrate specificity, the enzyme reaction product was analyzed after the same reaction as above using dUDP-glucose, dGDP-glucose, and dADP-glucose instead of dTDP-glucose.

As described above, the present invention isolates a gene population for biosynthesis of gasuamycin derived from Streptomyces kasugaensis KACC 20262, and occupies an important position for biosynthesis. The sequence of the dNDP-glucose-4,6-dehydratase gene and the sequence of the protein to be derived were identified and its function was identified. This enzyme is an important enzyme that is commonly involved in biosynthesis of other aminoglycoside antibiotics, as well as gas sugars. DTDP-4-keto-6-deoxy-glucose, dUDP-4-keto-6-deoxy-glucose, dGDP-4-keto-6-deoxy-glucose and dADP-4-keto-6-deoxy- Glucose is used as an important substrate for antibiotic biosynthesis research. dTDP-4-keto-6-deoxy-glucose is currently produced by a domestic venture company (Genechem Inc.) and sold at a high price (10 mg, KRW 240,000), dUDP- 4-keto-6-deoxy-glucose, dGDP-4-keto-6-deoxy-glucose and dADP-4-keto-6-deoxy-glucose will be produced and sold at a higher price. Therefore, the use of this enzyme is of great value because it can be used to produce not only antibiotic biosynthesis research but also high value-added products and enzymes.

       Figure 1 shows a restriction map of the gene encoding the dieendi-glucose-4,6-dihadradase (KasC) of the present invention in the gas sugar gamma biosynthetic gene population.

       Figure 2 is a homology search result compared with the US GenBank data based on the amino acid sequence translated from the gene of dNDP-glucose-4,6-dehydratase of the present invention.

Figure 3 quantifies the product through the enzymatic reaction of the enzyme and dTDP-glucose, dUDP-glucose, dGDP-glucose, dADP-glucose and the like obtained by expressing dNDP-glucose-4,6-dehydratase of the present invention in E. coli Picture.

<110> SUH, Joo Won Myong Ji University <120> Function and sequence of gene and amino acid of kasugamycin biosynthetic enzyme KasC <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 990 <212> DNA <213> Artificial Sequence <220> <223> Sequence of gene of kasugamycin biosynthetic enzyme KasC <400> 1 atgtcaccca cgaccacgca ctgggcagga cgccaggtgc tcgtcacggg agccgacggt 60 ttcatcggtt cgcatctcac cgagaccctg gtgagccgcg gcgcccgagt caccgcggtc 120 gtccgacgcg tctcggccgc acaggtgacg caccggctcc gcaatctctc cgccgcgacc 180 gtcgacgccc tcgaacgggt ggtgcacgtg gacctcgcgg gaccttcggc ggtcgacgtc 240 ctcggccggc tggaggccga cacctggttc cacctcgccg cggacgccta cgtaccggca 300 tcgctggacc agcccgccga tgtggtgcgc accaatgtga tgtccactct ccacgtcctg 360 ctggcagccc agcagcggca gcccgcgcac ctcctggtga cgagttcgag cgaggtctac 420 ggcagccagc cggacgcgat caccgaacgg catccgctgg aaccggccac gccctacgcg 480 gcgtccaagg tggcctgcga ccgcctggcc tggtcctggc accacaccta cggcctgccg 540 ctcaccatcg tccggccgtt caacagctac gggccccgcc acgtctacga cgcggtaccg 600 ctcttcctgg ccagagcgct gcggggcgag ccgatcacca tcaacggcag cggtgagcag 660 acgcgcgacc tcaccttcgt ggccgacacc gtcgcggggt tcctcgccct ggcggagctg 720 ccggccaccg gcgagacgta caacatcggc accggcacgg accaccgcat catcgacgtg 780 gcccgcgcca tcgtggccct gaccgggtcg cagagcgaga tcgtgcacgg cccaccgcgc 840 tccggcgaag tgctcaagct gcaggccgat ccggcgaaac tcaccgaggc caccgggtgg 900 cgtgccgagt acgacctggc caggggcctg gcggacaacc tggtctggat gcgggaacac 960 gtggagacgg tatggccgac acgatcctga 990 <210> 2 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Sequence of amino acid of kasugamycin biosynthetic enzyme KasC <400> 2 Met Ser Pro Thr Thr Thr His Trp Ala Gly Arg Gln Val Leu Val Thr 1 5 10 15 Gly Ala Asp Gly Phe Ile Gly Ser His Leu Thr Glu Thr Leu Val Ser 20 25 30 Arg Gly Ala Arg Val Thr Ala Val Val Arg Arg Val Ser Ala Ala Gln 35 40 45 Val Thr His Arg Leu Arg Asn Leu Ser Ala Ala Thr Val Asp Ala Leu 50 55 60 Glu Arg Val Val His Val Asp Leu Ala Gly Pro Ser Ala Val Asp Val 65 70 75 80 Leu Gly Arg Leu Glu Ala Asp Thr Trp Phe His Leu Ala Ala Asp Ala 85 90 95 Tyr Val Pro Ala Ser Leu Asp Gln Pro Ala Asp Val Val Arg Thr Asn 100 105 110 Val Met Ser Thr Leu His Val Leu Leu Ala Ala Gln Gln Arg Gln Pro 115 120 125 Ala His Leu Leu Val Thr Ser Ser Ser Glu Val Tyr Gly Ser Gln Pro 130 135 140 Asp Ala Ile Thr Glu Arg His Pro Leu Glu Pro Ala Thr Pro Tyr Ala 145 150 155 160 Ala Ser Lys Val Ala Cys Asp Arg Leu Ala Trp Ser Trp His His Thr 165 170 175 Tyr Gly Leu Pro Leu Thr Ile Val Arg Pro Phe Asn Ser Tyr Gly Pro 180 185 190 Arg His Val Tyr Asp Ala Val Pro Leu Phe Leu Ala Arg Ala Leu Arg 195 200 205 Gly Glu Pro Ile Thr Ile Asn Gly Ser Gly Glu Gln Thr Arg Asp Leu 210 215 220 Thr Phe Val Ala Asp Thr Val Ala Gly Phe Leu Ala Leu Ala Glu Leu 225 230 235 240 Pro Ala Thr Gly Glu Thr Tyr Asn Ile Gly Thr Gly Thr Asp His Arg 245 250 255 Ile Ile Asp Val Ala Arg Ala Ile Val Ala Leu Thr Gly Ser Gln Ser 260 265 270 Glu Ile Val His Gly Pro Pro Arg Ser Gly Glu Val Leu Lys Leu Gln 275 280 285 Ala Asp Pro Ala Lys Leu Thr Glu Ala Thr Gly Trp Arg Ala Glu Tyr 290 295 300 Asp Leu Ala Arg Gly Leu Ala Asp Asn Leu Val Trp Met Arg Glu His 305 310 315 320 Val Glu Thr Val Trp Pro Thr Arg Ser *** 325 330

Claims (2)

A gene sequence encoding dNDP-glucose 4,6-dehydratase (KasC) shown in SEQ ID NO: 1 derived from Streptomyces kasugaensis KACC 20262. Amino acid sequence shown in SEQ ID NO: 1 translated from the nucleotide sequences of claim 1 and its function.