KR20000066751A - Gene and amino acid sequences of enzyme used in spectinomycin biosynthesis - Google Patents

Abstract

PURPOSE: Provided are base sequence and amino acid sequence of enzyme used for synthesizing spectinomycin. In particular, provided is gene sequencing of methyltransferase and glycosyltransferase originated from Streptomyces spectabilis ATCC 27741 for synthesizing antibiotic, spectinomycin. CONSTITUTION: A base sequence and amino acid sequence of methyltransferase and glycosyltransferase are determined by following steps of; (a)performing PCR amplification of gene library using genome of S. spectabilis ATCC 27741 and isolating clone having two cosmids pHCG121 and pHCG171; (b)transforming Streptomyces lividans, which is sensitive to spectinomycin, with two cosmids and examining two cosmids having an effect on biosynthesis of spectinomycin by identifying resistance of transformant against spectinomycin; (c)subcloning all the BamHI fragments of two cosmids into pBluescript KS(+) and identifying both directions of base sequence, thereby methyltransferase gene is identified in recombinant plasmid pHCG1714 and also, area coding spcG and spcM is identified in BamHI inserting fragment of the recombinant plasmid pHCG1714; (e)sequencing both spcG and spcM; and (f)identifying amino acid homology between SpcG and glycosyltransferase and amino acid homology between SpcM and methyltransferase.

Description

Gene and amino acid sequences of enzyme used in spectinomycin biosynthesis}

The present invention relates to gene sequences and amino acid sequences of enzymes for spectinomycin biosynthesis. More specifically, the present invention relates to the gene sequence and amino acid sequence of the methyltransferase enzyme and glycosyltransferase enzyme for biosynthesis of the antibiotic spectinomycin.

Indiscriminate abuse of antibiotics has led to the emergence of bacteria, fungi, and viral cancer cells that are resistant to conventional antibiotics, and in fact, reported by the US Centers for Disease Control and Prevention (CDC, USA) in 1992, 1,300 people were admitted 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 10000 kinds of antibiotics are separated from microorganisms, and only about 100 of them are commercialized. As time passes, the success rate and commercialization rate of new functional antibiotics are 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 Isepamicin from Schering-Plough, etc. It has led the market for lycoside antibiotics. However, since these semisynthetic compounds are targeted only to the target antibiotic, for example kanamycin for amikacin, and gentamicin for isepamicin, It is limited and has a disadvantage in that the unit price is high in the process due to the synthetic product rather than the natural fermentation product.

Therefore, the present inventors did not synthesize side chains in order to overcome the above problems, but by using genetic engineering techniques and actinomycetes vectors, methyltransferase genes and spectinos are known to form methyl groups of spectinomycin. Streptomycin, tobramycin, and apramycin were isolated by glycosyltransferase genes attached to actinamine, a mycin aminocyclitol, and actinospectose, a 6-deoxyhexose sugar. It was intended to directly isolate renal functional antibiotics from the cultures of the transformants by introducing them into strains producing all aminoglycoside antibiotics such as kanamycin, gentamicin, sisomycin, and micronomycin. Many of the conventional semi-synthetic products artificially synthesized side chains in the amino group, which is the aminocyclitol target position, which is isolated from the spectinomycin producing strain. The methyltransferase gene supports the function of attaching a side chain to an amino group of aminocyclitol which is necessarily present in the structure of all aminoglycoside antibiotics including the antibiotics described above. Glycosyltransferases also have the ability to attach actinamine, a spectinomycin aminocyclitol, and actinospectose, a 6-deoxyhexose sugar. The enzymes can be used to attach spectose to actinamine-like structures or to attach actinamine-like structures to develop compounds of a completely new structure that have not yet been identified in nature. That is, antibiotics having a new structure that does not exist in nature and have a completely different biological activity by introducing and transforming a methyl transferase gene or glycosyltransferase gene into existing aminoglycoside antibiotic producing strains. It is possible to develop [also known as actino-streptomycin and methyl-aminoglycoside antibiotics]. Actinamine, the aminocyclitol of spectinomycin, is composed of 2-deoxystreptamine (2-DOS), which forms aminocyclitol of most aminoglycoside antibiotics. The amino group 1 and 3 of the similar 2-DOS structure is substituted with a methyl group.

Aminoglycoside antibiotics have been widely used for the treatment of infectious diseases due to their broad antimicrobial spectrum and strong bactericidal action. In general, streptidine is shown as shown in the following formula (I) according to structural differences of aminocyclitol. (streptidine), 2-deoxystreptamine, and antibiotics including actinamine, etc. are classified into 6-deoxyhexose for each aminocyclitol. The structure is attached to the sugar.

During the biosynthesis of aminocyclitol, the aminotransferase enzyme, which produces two amino groups, was found to have amino acid homology in most aminoglycoside antibiotic-producing strains. In view of the structural similarity between 2-deoxystreptamine, actinamine, and streptidine, the pathway between them is considered to be similar.

The present inventors use methyltransferase in the spectinomycin biosynthesis gene as shown in the following formula (II) to form 2-deoxystreptamine constituting aminocyclitol of at least 90% aminoglycoside antibiotics, respectively. The introduction of methyl group into 2-amino group of 2-deoxystreptamine to develop a novel functional aminoglycoside antibiotic, and also the actinamine methyl group and streptidine guanidine group We tried to develop streptidino-spectacin and actino-streptomycin, respectively. Glycosyltransferase, which is present in the vicinity of the methyltransferase enzyme, attaches actinspectose, a 6-deoxyhexose sugar of spectinomycin, to the actinamine structure during the production of spectinomycin. As it has a function of giving, it can be used for the development of a compound of new structure by artificially attaching the actinamine structure to the actinamine structure as well as the actinospactose and other sugar structures similar to the actinospactose. You can also attach the original Actinosspectose sugar to the structure.

An object of the present invention is methyltransferase and glycosyltransfer partially inserted into two cosmid clones into which a population of spectinomycin biosynthetic genes derived from Streptomyces spectabilis ATCC 27741 is inserted. The base sequence of the glycosyltransferase gene is determined and provided. Another object of the present invention is to provide an amino acid sequence translated from the gene sequence of the methyltransferase and glycosyltransferase. It is another object of the present invention to provide a recombinant plasmid comprising a DNA fragment having the gene sequence.

The object of the present invention is to predict a PCR product after PCR amplifying a gene library prepared using the genome of S. spectabilis ATCC 27741, a spectinomycin producing strain using AG3 and AG4 primers. Clones containing two positive cosmids, pHCG121 and pHCG171, were isolated and then transformed into spectinomycin-sensitive Streptomyces lividans with these cosmids. Subsequently, all the BamHI fragments contained in the two cosmids were subcloned into E. coli plasmid pBluescript KS (+), and the DNA fragment containing the methyltransferase gene was determined by bidirectional sequencing. The isolated pHCG1714 recombinant plasmid was isolated and the entire nucleotide sequence of the nearby region was determined. Next, the entire gene sequence confirmed that the coding region of two proteins spcG and spcM existed in the BamHI insertion fragment of the recombinant plasmid pHCG1714, and the amino acid sequence of these proteins was compared and analyzed, and the protein SpcG was a glycosyltransferase gene. This was achieved by revealing homology with amino acids and protein SpcM homology with methyltransferase.

Hereinafter, the configuration and operation of the present invention.

1 is a photograph showing electrophoresis by cutting two cosmids pHCG121 and pHCG171 into which a spectinomycin biosynthetic gene group is inserted, with restriction enzyme BamHI.

Figure 2 shows a restriction map of recombinant plasmid pHCG1714 in which the methyltransferase and glycosyltransferase genes of the present invention are inserted into an E. coli plasmid.

3 is a homology search result compared to US GenBank data based on amino acid sequences translated from the glycosyltransferase gene of the present invention.

4 is a homology search result compared to US GenBank data based on amino acid sequences translated from the methyltransferase gene of the present invention.

The present invention provides a gene library using the genome of S. spectabilis ATCC 27741, a spectinomycin-producing strain, and then the sugar activinospecto sugar 6-deoxyhexose of spectinomycin. Using primers AG3 (5'-CTSGCSGGCGGCWSSGGSACSMGSCTSYACCC-3 ') and AG4 (5'-RYGTCSGTGATCTCSAGCTCGCCSCG-3'), which can effectively amplify TDP-glucose synthase, which is essential for biosynthesis of oz PCR amplification to isolate a clone comprising two cosmid clones, pHCG121 and pHCG171, which are positive in a band of expected size; The two cosmids, pHCG121 and pHCG171, were transformed with spectinomycin-sensitive Streptomyces lividans using the two cosmids, pHCG121 and pHCG171, thereby confirming that the transformants were resistant to spectinomycin. Investigating the involvement of mycin biosynthesis; All the BamHI fragments contained in the two cosmids, pHCG121 and pHCG171, were subcloned into the E. coli plasmid pBluescript KS (+), and then methyltransferase originally targeted to the pHCG1714 recombinant plasmid into which approximately 2.7 kb fragments were inserted by bidirectional sequence determination. Confirming the presence of the gene and determining the entire base sequence of the enzyme and its neighboring region to confirm that there are regions encoding two proteins spcG and spcM in the BamHI insertion fragment of the recombinant plasmid pHCG1714; By determining the base sequences of the two proteins spcG and spcM and comparing the amino acid sequences translated therefrom, SpcG reveals amino acid homology with the glycosyltransferase gene and SpcM is homologous to methyltransferase. It consists of a step of revealing.

In the present invention, in order to confirm that the two separated cosmids pHCG121 and pHCG171 are involved in spectinomycin biosynthesis, the transformants are transformed into spectinomycin-sensitive streptomycin lividans with the cosmid. Resistance to mycin was searched. This is based on the fact that the genome of 20 to 30 enzymes that are responsible for antibiotic biosynthesis are usually gathered in parts and resistance genes exist in these biosynthetic gene populations to prevent their harm from antibiotics they produce. As a result, it was confirmed that Streptomyces lividans transformed with cosmid pHCG171 can survive at 60 ug / mL of spectinomycin.

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 spectabilis  Gene library of ATCC 27741)

Streptomyces spectabilis ATCC 27741 was incubated in R2YE medium, pure chromosomal DNA was isolated according to Hopwood's method, and the chromosomal DNA was partially cut with restriction enzyme Sau3A, Stratagene company's lambda packaging after connecting 30-to-40 kb of chromosomal DNA cut partially with BamHI and HpaI to cosmid vectors for replication in E. coli and actinomycetes. Streptomyces spectabilis gene library was prepared using the kit (λ packaging kit).

Example 2: Cloning of Spectinomycin Biosynthetic Genes and Screening for Spectinomycin Resistance in Streptomyces Lividans

1000 colonies from the Streptomyces spectabilis gene library prepared in Example 1 were inoculated in a 1.5 mL Eppendorf tube containing 500 uL of LBA liquid medium using a yellow tip and then incubated for one day. The cultures were then transferred to a new Eppendorf tube at 30 uL. Eventually 50 samples were transferred to a single Eppendorf tube and the number of samples transferred to a new tube was reduced from the first 1000 to 20. Plasmids were extracted from the collected 20 samples, and PCR reactions were performed with the DNAs of the 20 samples. The PCR primers used were TDP-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 spectinomycin. AG3 (5'-CTSGCSGGCGGCWSSGGSACSMGSCTSYACCC-3 ') and AG4 (5'-RYGTCSGTGATCTCSAGCTCGCCSCG-3') primers developed for TDP-glucose synthase were used. As a result of the PCR reaction, PCR bands of two sizes among 20 samples were identified, plasmids were extracted from 100 samples (470uL) used to prepare two samples, and PCR was performed again. Recombinant cosmid clones were selected. Using recombinant cosmid, Streptomyces lividans ATCC 1326, which is not resistant to aminoglyco antibiotics, was transformed according to Hopwood's method, and then the antibiotic marker of pDW103 The cells were cultured in a solid medium containing 20 µg / mL thiostrepton and 60 ug / mL spectinomycin. As a result, it was confirmed that the biosynthetic genes were indirectly by selecting transformants having thiostrepton and spectinomycin resistance, and pHCG171 recombinant cosmid confirmed the resistance to the two antibiotics. FIG. 1 shows the results of electrophoresis of the cosmid pHCG121 clone, which showed positive resistance in a PCR reaction using AG3 and AG4 primers, but did not show resistance, and electrophoresis with restriction enzyme BamHI. Is not visible, but the two clones overlapped each other.

Example 3: Cloning of Methyltransferase and Glycosyltransferase Genes in Cosmid Fragments

In this embodiment, since enzymes involved in antibiotic biosynthesis generally exist in a certain region of the genome, two cosmid clones 30 to 40 kb in which the spectinomycin biosynthetic gene population exists are aminoglycoside antibiotics in the insertion fragment. Since there will be a methyltransferase gene used for material generation, the two cosmid clones were digested with restriction enzyme BamHI, and then all fragments were subcloned into E. coli plasmid pBluescript KS (+) and bidirectional sequencing was performed by 800bp. As a result, it was confirmed that the target methyltransferase gene exists in the pHCG1714 recombinant plasmid into which the 2.7kb fragment was inserted, and the entire nucleotide sequence of the enzyme and its neighboring enzyme was determined. The BamHI insertion fragment of pHCG1714 had a size of 2.7 kb and the presence of a region coding for two proteins named spcG and spcM.

Example 4: Determination of the base sequence and amino acid sequence of the spcM gene and spcG gene

In the present embodiment, to determine the base sequence and amino acid sequence of the spcM gene and spcG gene, a subclone of which the base sequence is easily determined by treating the insertion fragment of the pHCG1714 recombinant plasmid with restriction enzymes ApaI, BamHI, SmaI, NaeI After nucleotide sequence was determined. Figure 2 shows the restriction map and gene location of pHCG1714 containing the spcM gene and spcG gene subcloned in pBluescript KS (+). The base sequences of the spcM and spcG genes and the amino acid sequences translated from the base sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2. The CODON PREFERENCE program (Bibb, M. J. et al., Gene, 1984) was used to search for portions encoding proteins based on primary DNA sequence results. Experimental results show that there is an open reading frame encoding two proteins in this region. The first protein, named SpcG, was composed of 293 amino acids with ATG of base sequence 285 as the start codon and TGA with base sequence 1166 as the stop codon, so the molecular weight was expected to be 32,829 Daltons (Da). According to the codon usage of this protein, the total G + C content was 71.3%, and the G + C content of the third codon was 90.5%, similar to that of the typical actinomycetes codon. In addition, the amino acid homology was searched through a database. As a result, glycosyltransferase (Mol. Miocrobiol. 1995. 15: 917-933) and Escherichia coli clanic acid required for the biosynthesis of Erwinia amylovora Amylovora acid) Glycosyltransferases required for biosynthesis (J. Bacteriol. 1996. 178: 4885-4893), Glycosyltransferases involved in the production of lipopolysaccharides of Burkholderia pseudomallei (Mol. Microbiol. SpcG also acts as a glycosyltransferase that acts when a sugar called actinospectose is attached to actinamine during the production of spectinomycin. It was judged as (Fig. 3). The second protein, named SpcM, was composed of 267 amino acids consisting of GTG having a base sequence of 1163 as an initiation codon and a terminating codon of TGA having a base sequence of 1966. Thus, the molecular weight was expected to be 29,285 Daltons (Da). In the codon usage of this protein, the total G + C content was 72.3%, of which the third codon was 92.2%, which was similar to the typical actin codon usage. In addition, the amino acid homology search through the database showed that the site-specific DNA methyltransfer of Citrobacter freundii, PvuII DNA methyltransferase (Eur J. Biochem. 1997. 247: 1009-1018) of Proteus vulgaris. Site-specific DNA methyltransferase (Nucleic.Acids Res. 1989. 17: 9823-9832) and methylase (Pseudomonas alcaligenes) (GenBank, U880881) show high amino acid homology with spcM gene It was determined that methyltransferase enzyme plays a role of attaching a methyl group in the final step in the production of actinamine, an aminocyclitol (FIG. 4).

As described above, the present invention provides a protein base sequence and amino acid sequence of methyltransferase and glycosyltransferase derived from Streptomyces spectabilis ATCC 27741. The methyltransferase has a function of adding or substituting a methyl group to an amino group in the actinamine structure constituting the aminocyclitol of the aminoglycoside antibiotic, and the glycosyltransase has the actinospec on the actinamine structure. The methyltransferase and glycosyltransferase genes are amino-glucose, because they have the function of artificially attaching other sugar structures similar to toose and actinospeptose, or attaching actinospeptose to actinamine-like structures. In addition to the side antibiotic production strain And because there is an excellent effect capable of directly separating the new antibiotic in transformant obtained from it it would be very useful inventions the Biopharmaceutical Industry.

Claims (6)

A gene sequence encoding glycosyltransferase (spcG) isolated from Streptomyces spectabilis ATCC 27741 and shown in SEQ ID NO: 1. Gene sequence encoding the methyltransferase (spcM) shown in SEQ ID NO: 2 isolated from Streptomyces spectabilis ATCC 27741. An amino acid sequence shown in SEQ ID NO: 1 by translating from the gene base sequence of claim 1. An amino acid sequence shown in SEQ ID NO: 2 by translating from the nucleotide sequence of claim 2. It is derived from Streptomyces spectabilis ATCC 27741 and has spectinomycin resistance in transformation of Streptomyces lividans, and has the glycosyltransferase (spcG) gene of claim 1 and the description of claim 2. Recombinant cosmid pHCG 121, pHCG 171, containing the methyltransferase (spcM) gene of. Recombinant plasmid pHCG1714 comprising the glycosyltransferase spcG gene of claim 1 and the methyltransferase spcM gene of claim 2.