KR20040047041A - Industrial Applications and Genetic Information of Salbostatin and Trehalose biosynthesis related enzyme SalA, SalB, SalC, SalD, SalE, SalF, SalG, SalH, SalI, SalJ, SalK, SalL, SalM, SalN, SalO, SalP, SalQ - Google Patents

Abstract

PURPOSE: Enzymes related to salbostatin and trehalose biosynthesis and the use thereof are provides, which enzymes are useful as probe for producing a DNA chip isolating microorganisms producing bio-stimulating materials having valienamine structure, and are useful for producing a primer for isolating the corresponding biosynthesis gene pool. CONSTITUTION: The enzymes related to salbostatin and trehalose biosynthesis are SalA, SalB, SalC, SalD, SalE, SalF, SalG, SalH, SalI, SalJ, SalK, SalL, SalM, SalN, SalO, SalP, SalQ having the amino acid sequences set forth in SEQ ID NO: 18 to SEQ ID NO: 34, respectively, wherein the genes encoding the enzymes SalA, SalB, SalC, SalD, SalE, SalF, SalG, SalH, SalI, SalJ, SalK, SalL, SalM, SalN, SalO, SalP, SalQ have the nucleotide sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 17.

Description

Industrial Applications and Genetic Information of Salbostatin and Trehalose biosynthesis related enzyme SalA, SalB, SalC, SalD, SalE, SalF, SalG, SalH, SalI , SalJ, SalK, SalL, SalM, SalN, SalO, SalP, SalQ}

The present invention relates to gene sequences and amino acid sequences of enzymes for salbostatin biosynthesis and their industrial applications. More specifically, the present invention relates to N-acetylglucosamine-6-phosphate deacetylase homology enzyme SalA, sugar phosphorylation homology enzyme SalB, trehalose phosphate synthesis for spastatin and trehalose biosynthesis. Trehalose-phosphate synthase SalC, Transporter enzyme SalD, Trehalose-6-phosphate phosphatase SalE, ADP-glucose synthase SalF, regulator enzyme) SalG, Glycosyltransferase SalH, Trehalose phosphorylase SalI, Reductase SalJ, Transporter SalK, Ballylon kinase (2-epi-5-epi- valiolone 7-kinase) SalL, Cyclitol dehydrogenase SalM, Cyclitol oxidoreductase S alN, SEQ ID NO: 1 of Valiolone-7-phosphate 2-epimerase SalO, Hydrolase SalP, 2-epi-5-epi-valiolone synthase SalQ Gene sequence up to No. 17 and amino acid sequences from SEQ ID NO: 18 to SEQ ID NO: 34, and their industrial applicability.

Bioactive substances with valienamine structure include Salbostatin, Validamycin, acarviosin, Acarbose, Adiposin, Trestatin, etc. The above compounds act as α-glucosidase inhibitors such as Trehalase. It is a widely used or important compound under development. Therefore, to isolate and secure gene sources that biosynthesize such valienamine structures is applied to PCR primers or DNA chip probes using the above genetic information to isolate microorganisms that produce bioactive substances with valienamine structure for microorganisms. It can be of great value because it can provide an opportunity to analyze and secure the structure of the compound.

The present inventors first isolated a gene group that biosynthesizes Salbostatin, which has a valienamine structure and can be used as an agricultural pesticide. N-acetylglucosamine-6-phosphate deacetylase homologous enzyme SalA, sugar kinase homokinase SalB, trehalose-phosphate synthase SalC, transporter enzyme SalD, trehalose phosphate phosphatase Trehalose-6-phosphate phosphatase SalE, ADP-glucose synthase SalF, regulator enzyme SalG, glycosyltransferase SalH, trehalose phosphorylase SalI , Reductase SalJ, Transporter SalK, 2-epi-5-epi-valiolone 7-kinase SalL, Cyclitol dehydrogenase SalM, Cyclitol oxidoreductase SalN, Ballylon phosphate epimerase The gene sequences and amino acid sequences of Valiolone-7-phosphate 2-epimerase SalO, Hydrolase SalP, and 2-epi-5-epi-valiolone synthase SalQ were identified. Among these enzymes are the 2-epi-5-epi-valiolone 7-kinase SalL, Cyclitol dehydrogenase SalM, Cyclitol oxidoreductase SalN, Ballylon phosphate Epimerase SalO, Hydrolase SalP, and 2-epi-5-epi-valiolone synthase SalQ are important enzymes commonly involved in Valienamine biosynthesis. Its industrial application is also great.

An object of the present invention is N-acetylglucosamine-6-phosphate deacetylase inserted as part of a cosmid clone into which a salvostatin and trehalose biosynthetic gene population derived from Streptomyces albus KCTC9015 is inserted. ) Homologous Enzyme SalA, Sugar Phosphorylation Homogenase (Sugar kinase) SalB, Trehalose-phosphate synthase SalC, Transporter SalD, Trehalose phosphate phosphatase Enzyme (Trehalose-6-phosphate phosphatase) ) SalE, ADP-glucose synthase SalF, regulator enzyme SalG, glycosyltransferase SalH, trehalose phosphorylase SalI, reductase SalJ , Transport SalK, Between-epi-5-epi-valiolone 7-kinase SalL, Between Cytolitol dehydrogenase SalM, Cyclitol oxidoreductase SalN, Baliolone-7-phosphate 2-epimerase SalO, Hydrolase SalP, Ballylon Synthetase (2-epi-5-epi-valiolone synthase) SalQ gene sequence and amino acid sequence are provided.

The object of the present invention is to predict the PCR product after PCR amplification using Val-F and Val-R primers of a gene library prepared using the genome of Streptomyces albus ( S. albus KCTC 9015), a salvostatin producing strain. Clones containing four cosmids pSH2101, pSH2102, pSH2103, and pSH2104 that react positively with a band of the same size were isolated. After the production of shotgun libeary, 400 libreys were sequenced using T7 and T3 primers to determine the base sequence of about 20 kb. As a result, it was confirmed that 18 coding regions exist and the amino acid sequence of this gene was determined. By comparative analysis, SalA is N-acetylglucosamine-6-phosphate deacetylase, Sal is glycosylation homology enzyme (Sugar kinase), SalC is Trehalose-phosphate synthase, SalD is Transporter enzyme, SalE is Trehalose-6-phosphate phosphatase, SalF is ADP-glucose synthase), SalG is a regulator enzyme, SalH is a glycosyltransferase, SalI is Trehalose phosphorylase, SalJ is a reductase, SalK is a transporter, SalL is 2-epi-5-epi-valiolone 7-kinase, SalM is Cyclitol dehydrogenase, SalN is Cyclitol oxidoreductase, SalO is balliolone Phosphate Epimerase SalP Hydrolase and SalQ reveal homology with the 2-epi-5-epi-valiolone synthase. Achieved.

Hereinafter, the configuration and operation of the present invention.

1 is a genetic map containing 20 kb of salvostatin and trehalose biosynthetic gene group, N-acetylglucosamine-6-phosphate deacetylase, Sugar kinase, Trehalose-phosphate synthase, Transporter, Trehalose-6-phosphate phosphatase, ADP of the present invention. -glucose synthase, regulatory enzyme, Glycosyltransferase, Trehalose phosphorylase, reductase, Transporter, 2-epi-5-epi-valiolone 7-kinase, Cyclitol dehydrogenase, Cyclitol oxidoreductase, Valiolone-7-phosphate 2-epimerase, Hydrolase, and 2-epi It contains the -5-epi-valiolone synthase gene.

The present invention is a gene library using the genome of Streptomyces albus ( S. albus KCTC9015), a salvostatin-producing strain, 2-epi-5-epi-valiolone necessarily present for biosynthesis of salvostatin Valienamine Four cosmids pSH2101, pSH2102, pSH2103, which showed positive reaction with PCR amplification using primer Val-F of SEQ ID NO: 35 and primer Val-R of SEQ ID NO: 36, which can effectively amplify synthase The clone containing pSH2104 was isolated. Among them, the cosmid gene of pSH2104 was partially cut into 1-2 kb and subcloned into plasmid pBluescript KS (+) to make a shotgun libeary, and 400 libreys were used as bases using T7 and T3 primers. Determining a nucleotide sequence of about 20 kb by sequencing: confirming that there are 18 coding regions from SalA to SalQ in the gene group; and Compared to the amino acid sequences translated from the sequence determination, SalA is N-acetylglucosamine-6-phosphate deacetylase, SalB is sugar kinase homozyme (Sugar kinase), SalC is trehalose phosphate synthase ( Trehalose-phosphate synthase, SalD is a transporter enzyme, SalE is Trehalose-6-phosphate phosphatase, SalF is ADP-glucose synthase, and SalG is a regulator (regulatory enzyme), SalH is glycosyltransferase, SalI is trehalose phosphorylase, SalJ is reductase, SalK is transporter, SalL is balion kinase (2 -epi-5-epi-valiolone 7-kinase), SalM is Cyclitol dehydrogenase, SalN is Cyclitol oxidoreductase (Cyclitol oxi) doreductase), SalO is a Valorone-7-phosphate 2-epimerase SalP hydrolase, and SalQ is homologous to the vialonone synthase (2-epi-5-epi-valiolone synthase) It consists of confirming that there is.

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 albus (salvostatin producing strain) Streptomycesalbus Genetic library of KCTC9015)

Streptomyces albus KCTC9015, a salvostatin producing strain, was cultured in R2YE medium, chromosomal DNA was purified according to Hopwood's method, and the chromosomal DNA was partially cut with restriction enzyme Sau3A. Stratagene, a 30-40 kb chromosomal DNA fragmented with BamHI and HpaI, was linked to a cosmid vector for replication in E. coli and actinomycetes. Streptomyces albus ( S. albus KCTC9015) gene library was prepared using the Lambda packaging kit.

Example 2: Cloning of Salvostatin and Trehalose Biosynthesis Genes

1000 colonies of the Streptomyces albus gene library prepared in Example 1 were inoculated into a 1.5 ml Eppendorf tube containing 500 l of LBA liquid medium using a yellow tip, followed by incubation in an incubator for one day. This culture was then transferred to new Eppendorf tubes at 30 ul. Eventually 50 samples were transferred to one Eppendorf tube and the number of samples transferred to the new tube was reduced from the first 1000 to 20. Plasmids were extracted from the collected 20 samples, and PCR reactions were performed using the DNA of 20 samples. The PCR primer used was acbC (2-epi-5-epi-valiolone synthase) among acarbose biosynthetic genes with a valienamine structure in the compound structure, such as salvostatin, and 3, which is thought to have similar biochemical mechanisms. Primer Val-F of SEQ ID NO: 35 and Primer Val-R of SEQ ID NO: 36 developed by comparative analysis with the -dehydroquinate synthase gene were used. As a result of the PCR reaction, PCR bands were identified in 4 samples out of 20 samples, plasmids were extracted from 200 samples (470 ul) used to prepare 2 samples, and PCR was performed again. Recombinant cosmid clones were selected.

Example 3: Gene and Amino Acid Sequencing and Similarity Search of 20kb Fragments Containing 2-epi-5-epi-valiolone synthase Gene in Cosmid Fragments

In this embodiment, since enzymes involved in biosynthesis of bioactive substances are generally present in a certain region of the genome, four cosmid clones 30-40 kb containing salvostatin biosynthetic gene populations are involved in salvostatin biosynthesis in the insertion fragment. It is expected that most of the enzyme genes will be present, and pSH2104 of the 4 cosmids will be partially cleaved to 1-2kb and subcloned into plasmid pBluescript KS (+) to make shotgun libeary. A nucleotide sequence of about 20 kb was determined by nucleotide sequence determination using a primer. The base sequence of 18 enzyme genes from SalA to SalQ 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 protein coding part was searched based on the primary DNA base sequence results. Experimental results show that there is an open reading frame encoding proteins in this region.

The protein named SalA consists of 381 amino acids starting with start codon GTG and ending with stop codon TGA, SalB consists of 306 amino acids starting with ATG and ending with stop coton TGA, and SalC begins with ATG Consists of 490 amino acids ending with stop codon TGA, SalD consists of 384 amino acids starting with ATG and ending with stop codon TGA, SalE consists of 293 amino acids starting with ATG and ending with stop codon TGA SalF consists of 389 amino acids starting with GTG and ending with stop codon TGA, SalG consists of 212 amino acids starting with ATG and ending with stop codon TGA, and SalH begins with GTG and ends with stop codon TGA. Consists of 422 amino acids, SalI consists of 442 amino acids starting with ATG and ending with stop codon TGA SalJ consists of 252 amino acids starting with ATG and ending with stop codon TGA, SalK consists of 393 amino acids starting with ATG and ending with stop codon TAG, and SalL starts with GTG and stop codon TGA. Consisting of 313 amino acids ending with SalM, consisting of 365 amino acids starting with ATG and ending with stop codon TGA, SalN consisting of 253 amino acids starting with ATG and ending with stopcoating TGA, SalO consists of ATG Consists of 267 amino acids starting with and ending with stop codon TGA, SalP consists of 224 amino acids starting with ATG and ending with stop codon TGA and SalQ consists of 410 amino acids starting with ATG and ending with stop codon TAG It was.

In addition, as a result of the amino acid homology search through the database, SalA has an N-acetylglucosamine-6-phosphate deacetylase and e-value of Streptomcyes coelicolor has a homology of about 3e-69, SalA is of Streptomcyes coelicolor N-acetylglucosamine -6-phosphate deacetylase and e-value have a homology of 3e-69, SalB has a sugar kinase of Streptomcyes coelicolor and a homology of 2e-28 of e-value, SalC has trehalose of Streptomcyes coelicolor -Phosphate synthase and e-value have homology of about 8e-25, SalD has a homology of 6e-06 with transporter enzyme of Rhodobacter sphaeroides , and SalE has trehalose-6 of Streptomcyes coelicolor . -Phosphate phosphatase and e-value have a homology of 3e-05, SalF has a homology of Geobacillus ADP-glucose synthase and e-value of 7e-46, and SalG has a homology with Brucella's regulatory gene. e-val ue has a homology of about 7e-08, SalH has no similar amino acid sequence, but the amino acid MOTIF sequence present in the glycotransferase existed, and SalI had a trehalose phosphorylase and e- of Sulfolobus . The value is about 2e-42 homology, SalJ has about 2e-17 homology with Shewanella reducatse, and SalK is about 1e-12 with transporter enzyme of Streptomcyes coelicolor SalL has a homology of 2-epi-5-epi-valiolone 7-kinase and e-value of 4e-68 which is involved in acarbose biosynthesis, and SalM is Cyclitol involved in acarbose biosynthesis. Dehydrogenase and e-value have homology about e-106, SalN has Cyclitol oxidoreductase, which is involved in acarbose biosynthesis, and e-value has homology of 2e-61, and SalO is involved in acarbose biosynthesis. -7-phosphate 2-epimerase and e-value have a homology of 3e-36, SalP has a homology of Brucella's Hydrolase and e-value of 7e-31, and SalQ has 2-epi involved in acarbose biosynthesis. The -5-epi-valiolone synthase and e-value had homology about e-126.

As described above, the present invention separated the trehalose biosynthetic gene group from the gene population and the neighboring region of the biosynthesis of salvostatin derived from Streptomyces albus KCTC9015, the base sequence of 20kb equivalent Analysis and analysis include N-acetylglucosamine-6-phosphate deacetylase, sugar kinase homokinase, trehalose-phosphate synthase, transporter enzyme, Trehalose-6-phosphate phosphatase, ADP-glucose synthase, regulator enzyme, glycosyltransferase, trehalose phosphorylase , Reductase, transporter, bagoron kina (2-epi-5-epi-valiolone 7-kinase), Cyclitol dehydrogenase, Cyclitol oxidoreductase, Valorone phosphate epimerase (Valiolone-7-phosphate) 2-epimerase, hydrolase, and 2-epi-5-epi-valiolone synthase gene sequences and derived protein sequences were identified. Among these enzymes are the ballyon kinase (2-epi-5-epi-valiolone 7-kinase), cyclitol dehydrogenase, cyclitol oxidoreductase, and the ballyon phosphate epimerase (Valiolone-7-phosphate 2-epimerase), hydrolases, and balliolone synthase (2-epi-5-epi-valiolone synthase) are common to salvostatin as well as to biosynthesis of bioactive substances with valienamine structures. It is an important enzyme involved. Therefore, this genetic information is highly valuable because it can be applied to gene probe for DNA chip production and biosynthetic gene population to separate microorganisms producing bioactive substance with valienamine structure.

Claims (2)

N-acetylglucosamine phosphate deacetylase, N-acetylglucosamine-6-phosphate deacetylase shown in the sequence listing derived from Streptomyces albus KCTC 9015, Sugar kinase, Trehalose phosphate synthase ( Trehalose-phosphate synthase, Transporter enzyme, Trehalose-6-phosphate phosphatase, ADP-glucose synthase, regulatory enzyme, glycotransferase (Glycosyltransferase), Trehalose phosphorylase, Reductase, Transporter, Ballylon kinase (2-epi-5-epi-valiolone 7-kinase), Cycitol dehydrogease Cyclitol dehydrogenase, Cyclitol oxidoreductase, Valorone phosphate epimerase (Valiolone-7-ph Gene sequences encoding osphate 2-epimerase, Hydrolase, and Ballyon synthase. A DNA chip for the isolation of valinenamne-containing bioactive substance-producing strains using the amino acid sequence shown in the sequence listing by translating from the nucleotide sequences of claim 1 and its application to the preparation of a primer for separating the biosynthetic gene population.