JPH01137979A - Glucose isomerase gene, recombinant having said gene and microorganism containing said recombinant - Google Patents

Abstract

PURPOSE:To enable mass-production of glucose isomerase, by separating a gene coding a glucose isomerase originated from actinomycetes and clarifying the structure of the gene. CONSTITUTION:A polymeric DNA of an actinomycete cell is completely digested with a restriction enzyme Bam H1 and a DNA fragment fraction having a chain length of about 5.7kb is separated by neutral sucrose density-gradient centrifugal separation process. The fragment is linked to Bam H1 site of a pUC vector using a ligase and transformed to E.coli to obtain a DNA library. The glucose isomerase gene is selected from the library by using a probe consisting of an oligonucleotide labeled with <32>P.

Description

[Detailed Description of the Invention] [Industrial Application Field] Glucose isomerase (also referred to as xylose isomerase) is an enzyme that isomerizes glucose, which is an aldose, and fructose, which is a ketose. Isomerization increases sweetness by about 1.5 times. Currently, the total production of isomerized sugar is 90% by immobilizing the enzyme and continuously isomerizing it.
It has reached more than 10,000 tons (yearly).

The present invention relates to a novel recombinant that has DNA containing a so-called structural gene encoding this enzyme and a so-called regulatory gene region that controls its expression, and a new microorganism that expresses the gene.

[Prior art and its problems] Glucose isomerase derived from actinomycetes is currently used for the production of high-fructose sugar. However, until the present invention, its structure had not been elucidated, and the relationship between enzyme activity and function had not been elucidated at all.

[Means for Solving the Problems] The present inventor has discovered that Streptomyces s
p, for example S, griseofuscus S-
41), and succeeded in elucidating the structure and isolation of the gene encoding glucose isomerase (including regulatory genes and structural genes). This has made it possible to produce glucose isomerase in actinomycetes that originally do not have the enzyme gene. Furthermore, by replacing the regulatory gene region with a regulatory gene that can be expressed in the corresponding host, the enzyme can be expressed in other microorganisms. These are meant to provide a system for elucidating the relationship between enzyme function and structure, and offer the possibility of transforming enzyme function using protein engineering techniques.

The present invention corresponds to glucose isomerase, which is present on the chromosome of actinomycetes (Zenda Uta Muni sp.), and which is a restriction enzyme based on the restriction enzyme Ba
m) Corresponding to glucose isomerase, a gene contained in the 5.7 kb fragment fraction cut out by II, the 5.7 kb-Baml of the chromosome of Streptomyces (one fragment fraction contains the promoter region and the complete translation region of the enzyme) and a recombinant with DNA containing a part of the xylulokinase gene polycistronically expressed with the enzyme gene and glucose isomerase, obtained from Streptomyces and completely digested with BaIIH1 by sucrose density gradient centrifugation. .7kb
- The present invention relates to a microorganism having a vector in which a DNA containing most of the enzyme structural gene including a promoter region is integrated into a DNA fragment obtained from a DNA fragment fraction and excised by restriction enzyme 1Ith1.

The gene of the present invention has the base sequence shown in FIG.
A structural gene that encodes three amino acids and the initiator “methionine” but does not encode a leader sequence and its 5′
- Consists of upstream regulatory genes.

More specifically, the present invention deals with glucose isomerase, obtained from actinomycete cells, and derived from nuclei and nuclear fragments.
A nuclear-derived genetic fragment that is NA and contains oligonucleotides corresponding to the primary structure or partial structure of glucose isomerase and a region identified by Southern blot hybridization, and also contains a promoter region, initiator, and terminator. , relates to a recombinant having the gene and a microorganism expressing the gene.

The nuclear gene fragment according to the present invention can be produced by extracting and separating it from actinomycete cells having glucose isomerase activity and amplifying it by genetic engineering techniques.

In order to isolate the nuclear gene fragment corresponding to glucose isomerase from actinomycete cells, it is desirable to extract high molecular DNA. The stage of the actinomycete is not critical, but late logarithmic growth is preferable.

Examples of methods for extracting high-molecular nuclear DNA from actinomycete cells include the method reported by Murray and Thompson [Nucleic Acids Res, 8.
4321 (1980)]. In order to identify the DNA fragment containing the glucose isomerase gene from the prepared Streptomyces macromolecular DNA, the DNA fragment cut with an appropriate restriction enzyme is ligated to an appropriate vector using a ligase, and these are used to transform the DNA fragment. From the converted cells,
Selection may be performed by colony hybridization using an oligonucleotide synthesized based on the determination of the partial amino acid sequence of glucose isomerase as a probe. The vector used is ptlc when E. coli is used as the host.
The λ-phage system and the λ-phage system are easy to use. However, the most efficient method for preparing the gene is as shown in the example, after complete digestion of actinomycete polymeric DNA with Bam) 11, a DNA fragment with a chain length of approximately 5.7 kb is obtained by neutral sucrose density gradient centrifugation. The fractions were collected and the Bam of pUc vector was added using ligase.
After binding to the H1 site, it is transformed into E. coli to create a DNA library. Search for glucose isomerase genes from this library can be performed by labeling the above oligonucleotide with 32p and using probe b.

Plasmids are extracted and separated from the bacterial cells obtained by mass culturing the thus obtained transformants by a conventional method, such as the alkaline-SDS method, and then subjected to cesium chloride density gradient ultracentrifugation, gel filtration, and agarose gel electrophoresis. A nuclear-derived gene fragment corresponding to glucose isomerase can be obtained by methods such as methods.

In order to confirm that the nuclear gene obtained as described above is the desired one, it is necessary to partially determine the amino acid sequence of glucose isomerase of the obtained nuclear gene fragment, and synthesize it based on the partial determination of the amino acid sequence of glucose isomerase. Southern blot hybridization may be performed using the obtained oligonucleotide as a probe.

The greatest use of the glucose isomerase nuclear genes obtained in this way is to incorporate these nuclear genes into vectors of microorganisms, plants, and animals to produce glucose isomerase or this improved enzyme in microorganisms, plants, and animals. It's about getting used to it. Here, microorganisms include prokaryotes such as Escherichia coli and eukaryotes such as Saccharomyces cerevisiae.

Furthermore, another use is that by improving the promoter region of glucose isomerase, a vector for high expression in actinomycetes can be created.

Integration of the glucose isomerase nuclear gene into a vector can usually be carried out in vitro as follows.

Both ends of the DNA of the glucose isomerase nuclear gene are treated with exonuclease, if necessary, and NAs are connected to each end as necessary, or a plurality of bases in combinations that can be annealed are polymerized. Thereafter, this is incorporated into the target vector. The method of integration involves cleaving the vector with an appropriate restriction enzyme and, if necessary, polymerizing an appropriate linker or a plurality of annealing-enabled combinations of bases. The double-stranded DNA thus processed and vector DNA are mixed and ligated using ligase.

The obtained recombinant DNA is introduced into vector hosts such as microorganisms, plant cells, and animal cells.

There are various hosts that can be used here, including bacteria of the genus Escherichia such as Escherichia coli, bacteria of the genus Bacillus such as Bacillus subtilis, yeasts of the genus Saccharomyces such as Saccharomyces cerevisiae, tobacco, and eggplants such as petunia. Animal cultured cells such as Ba1bIC373 are suitable. Vectors used for these hosts are illustrated below.

EK-based plasmid vector (stringent type) p
scroll, pRK353. pRK646. pR
248. pDF41, etc., EK-based plasmid vectors (relaxed type) Ca1El, pVH51, pA
c105. R5F2124. pcRl.

pMB9. BR313, pBR322, pBR324,
pBR325, pBR327゜pBR3211, pKY
228J pKY27Qo, pKNB(1, pK
C7゜pKB158. pMK2004. pAc
Ycl, pAcYc184. λdu1 etc., λ
gt-based phage vector λgt/λC1λgt/λ
B, λWES・λB′, λZJvir・λB′
, LO-λB to λ.

λWES-Ts622. λDam et al., Sharon 4A, Sharon 3A, Sharon 16A of Sharon vector.

Sharon 13A, Sharon 14A, Sharon 15A.

Sharon 8. Sharon 10. Sharon 17. Sharon 2
0 etc., Tiollais group vector (DL512. λZEQS, λZYV5φ,
λZUV φ2゜λZUVφ3. λYEQSφ1
, λYEQSφ, λYEQSφ3゜λBam,
λSst, etc., Bacillus subtilis plasmid vector pTA101
5. pLs15. pTA1020. pL528.
pLs13. pTA1050, pTA1060.
pTA1030. Staphylococcus-derived plasmid vector pT127, such as pTA1031.

pc194. pc221. pC223, pUB11
2. pLIBlo.

psAO501, ps8 100. pE1!14.
pTP4. pJD as a yeast vector such as pTI'5
B219. YEp13. YRp7. Ylpl.

pyc, pTC2, various vectors derived from Ti plasmids and various vectors derived from cauliflower mosaic virus (including binary vectors) as plant vectors, pSVK derived from sv4° as animal vectors,
pI-11β-1pSV)I, n-1-H+, pβ
2X, psXβ+, etc. However, in the case of a Ti plasmid-derived plant vector, the obtained recombinant DNA
-H Agrobacterium tumefaciens T37 etc. and co-cult the wood-modified microorganism into plant cells.
Recombinant DNA can be introduced into a host plant by infection, such as by ure.

That the glucose isomerase obtained as a wood recombinant DNA product is structurally the same as that of the natural type can be confirmed by techniques such as immunoprecipitation or immunoplot of the product.

[Example] Next, the present invention will be explained in detail with reference to examples.

Example 1 Stretomyces griseofu
scus S-41) was cultured to the late stage of logarithmic growth according to a conventional method, centrifuged under cooling, and its wet weight (5z, 8gr) was immediately frozen in liquid nitrogen. Add 15% sucrose to this, 20m
MEDTA, 30mM Tris-HCj (+)
H8,0), 0.7% SOS was added and ground, and then kept at 50°C for 30 minutes, after which Murray and Tho
mpson method [Nucleic Ac1ds Re
s, 8.4321 (1980)].
A was extracted. This DNA had a molecular weight of 25 kb or more and showed a single band on agarose gel electrophoresis, with no degradation observed. 500 μg of this high molecular DNA was
The mixture was completely digested with 100 ml of water, treated with phenol, and then precipitated with ethanol according to a conventional method. Then, it was layered on a 10% to 40% neutral sucrose density gradient and centrifuged at 26.00 rpm for 26 hours. The length of DNAMi contained in each fraction was examined by fractionating 800 μβ and subjecting a portion of the fraction to agarose gel (0.4%) electrophoresis. As a result, the average DNA chain length of fraction 31 was 5.8 kb and 8.25μ
The amount of DNA was 1.5 g. Approximately 1 ug of this DNA fraction was taken, and 1 ug of Bam t11 completely digested pUc13 was taken and treated with gauze. The reaction was performed using T4 DNA ligase (20
The test was carried out using U) at t4°C for 14 hours.

Next, the mixture was transformed into Escherichia coli NM522 according to a conventional method, and the resulting transformed product was cultured to obtain about 2000 colonies.

On the other hand, when glucose isomerase was purified from actinomycetes and the amino acid sequence of its N-terminal region was analyzed, it was found that 5-
F-Q-P-T-P-ED-ni-F-T-F-G-L
-W-T-V-Q-4 was obtained, so oligonucleotides were synthesized for the less degenerate region. That is, when colony piperidization was performed using the above 23mar as a probe, nine positive colonies were obtained. Since the inserted DNA of all clones was the same, 5.7 kb, one of them, pGI-32, was used for the following research. From the results of restriction enzyme mapping and Southern plot hybridization, it was found that most of the glucose isomerase gene was located within the 2.7 kb region excised by Sphl. Therefore, it can be used as a vector to facilitate structural analysis and expression of the glucose isomerase gene in organisms other than actinomycetes! 1UC119 (and pUc11
8) using the method of 5teven Hen1koff [
Gene, 28.351 (1984)] and Yani
The method of sch-Perron et al. [Gene, 33°1
03 (1985)] derived from pGI-32.
LI-GI-32 and pals-GI-65 (抛1
Plasmids were created in which the insertion site of 2.7 kb was inserted into pUc119 (clone) to various extents, and cloned in E. coli. Using a series of these defective plasmids, the complete structure of the glucose isomerase gene was revealed by the Sanger method (see Figure 1). As a result, it was revealed that Streptomyces glucose isomerase is a protein consisting of 393 amino acids and having a molecular weight of 43,843. The structure of the precursor protein also revealed that the enzyme is not secreted because it lacks a leader sequence after the initiator methionine.

Example 2 In order to express actinomycete glucose isomerase in a heterologous microorganism, ptl-GI-32 produced in Example 1 above was used.
A series of deletion plasmids, 5teven He
The method of n1koff [Gene, 28.351 (1
984)] and the method of Yanisch-Perron et al. [Gene, 33.103 (1985)]. Of course, there are other methods for producing deletion plasmids, such as a method using BaR31, as long as they are appropriate. The purpose here was to delete approximately 2 kb of the 5'-flanking region of the gene. As a result, one of the deletion mutants showed that plasmid pU-GI-32
A clone with -68 was obtained. This plasmid was deleted up to the first letter of the glutamine codon following the initiator methionine of the glucose isomerase gene shown in FIG. 1 (see I). Therefore, a synthetic double-stranded oligonucleotide, 5'-
d[CAGCCATGGGCTTCC] was ligated with ligase. After cutting this with Ncol, it was ligated to the Nco1 site of the E. coli expression vector pKK233-2,
After cutting the 3'-terminal fHl-in, it was circularized using ligase. This expression vector was used in Escherichia coli N, an IacIQ strain.
Transformed into M522. It was confirmed by immunoplotting and activity measurement of the disrupted bacterial cell supernatant that this expression plasmid produced glucose isomerase within the bacterial cells upon addition of IPTG. This bacterium has been deposited with the National Institute of Microbial Technology, and its deposit number is FERM P-RI τIT.

[Effects of the Invention] The present invention provides a gene encoding actinomycete glucose isomerase, a recombinant having the gene, and a novel microorganism expressing the gene. It has become possible to clarify the relationship between the activity and structure of glucose isomerase by making full use of protein engineering techniques, which will lead to the creation of a better enzyme and will also help increase the production efficiency of glucose isomerase. It can make a big contribution.

[Brief Description of the Drawings] Figure 1 shows the base sequence of the glucose isomerase gene. Patent Applicant Acting Director, Food Research Institute, Ministry of Agriculture, Forestry and Fisheries Attorney Patent Attorney Kubo

Claims (1)

[Claims] 1) Corresponding to glucose isomerase,
eptomces sp. ) and is included in the 5.7 kb fragment fraction cut out with the restriction enzyme Bam Hl. 2) The gene according to claim 1, wherein the gene has the base sequence shown in FIG. 3) Corresponding to glucose isomerase, the 5.7 kb-Bam Hl fragment fraction of the Streptomyces chromosome contains the promoter region and the complete translated region of the enzyme, as well as part of the xylulokinase gene that is polycistronically expressed with the enzyme gene. A recombinant that has DNA containing parts. 4) The recombinant according to claim 3, wherein the DNA is a prokaryotic or eukaryotic vector. 5) The recombinant according to claim 3, wherein the gene has the base sequence shown in FIG. 6) Corresponding to glucose isomerase, obtained from actinomycetes, obtained by sucrose density gradient centrifugation into a 5.7 kb DNA fragment fraction completely digested with Bam Hl, and containing the restriction enzyme Sph
A microorganism having a vector in which DNA containing most of the enzyme structural gene including the promoter region is integrated onto the DNA fragment excised by l. 7) The microorganism according to claim 6, wherein the microorganism is Escherichia coli, Bacillus subtilis, or Saccharomyces cerevisiae.