JPH0544278B2 - - Google Patents

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, to fructose, which is a ketose. Isomerization increases sweetness by about 1.5 times. Currently, the total production of isomerized sugar is 900,000 tons (per year) by immobilizing the enzyme and continuously isomerizing it.
The above has been reached. 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 developed a gene (regulatory gene and structural gene) encoding glucose isomerase derived from Streptomyces sp., for example, S. griseofuscus S-41. ) and elucidated its structure. This has made it possible to produce glucose isomerase in actinomycetes, which 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 exists on the chromosome of Streptomyces griseofuscus S-41 and is excised with the restriction enzyme Bam H1.
The gene contained in the 5.7kb fragment fraction corresponds to glucose isomerase, and it corresponds to the chromosome of the actinomycete mentioned above.
Compatible with recombinant and glucose isomerase having DNA in the 5.7kb-Bam H1 fragment fraction that includes the promoter region, the complete translation region of the enzyme, the enzyme gene and a part of the xylulokinase gene expressed in polycistronic enzymes. Bam was obtained from the above-mentioned actinomycetes by sucrose density gradient centrifugation.
On the DNA fragment obtained from the H1 complete digestion 5.7kb-DNA fragment fraction and cut out with the restriction enzyme Sph 1,
It relates to a microorganism having a vector into which DNA containing most of the enzyme structural gene including the promoter region has been integrated. The gene of the present invention has the base sequence shown in FIG. 1, and consists of a structural gene that encodes 393 amino acids and an initiator methionine, and does not encode a leader sequence, and a regulatory gene in its 5'-upstream region. More specifically, the present invention deals with glucose isomerase, which is obtained from Streptomyces griseofuscus S-41 and is derived from the nucleus and nuclear fragments.
A nuclear-derived gene fragment of DNA that contains a region identified by Southern blot hybridization with an oligonucleotide corresponding to the primary structure or partial structure of glucose isomerase, 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 is extracted from actinomycete cells having glucose isomerase activity.
It can be produced by separating and amplifying it using genetic engineering techniques. In order to isolate the nuclear gene fragment corresponding to glucose isomerase from Streptomyces griseophuscus S-41, 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 Streptomyces griseophuscus S-41 include:
Examples 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 actinomycete polymer DNA, it was necessary to cut it with an appropriate restriction enzyme etc.
DNA fragments are ligated to appropriate vectors using ligase, and cells transformed with these are transformed by colony hybridization using oligonucleotides synthesized based on partial amino acid sequencing of glucose isomerase as probes. All you have to do is select. When using E. coli as a host, pUC and lambda phage vectors are easily used. However, the most efficient method for preparing the gene is as shown in the example, after complete digestion of the actinomycete polymeric DNA with Bam H1, DNA fragments with a chain length of approximately 5.7 kb are obtained by neutral sugar-blocked density gradient centrifugation. Collect the fractions and cleave the pUC vector using ligase.
After binding to the Bam 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 ³² P and using it as a probe. 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 further subjected to cesium chloride density gradient ultracentrifugation, gel filtration, and agarose gel electrolysis. A nuclear gene fragment corresponding to glucose isomerase can be obtained by electrophoresis or the like. 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. Microorganisms include prokaryotes such as Escherichia coli and Satucharomyces.
There are eukaryotic organisms such as S. 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 necessary DNA is connected to each end, or a plurality of bases in combinations that can be annealed are polymerized.
Thereafter, this is incorporated into the target vector.
The integration method 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 processed in this way and vector DNA are mixed and connected 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 Satucharomyces such as Satucharomyces cerevisiae, tobacco, and eggplants such as Petiunia. Animal cultured cells such as BalbIC3T3 and the like are suitable. Vectors used for these hosts are illustrated below. EK-based plasmid vectors (stringy end type) pSC101, pRK353, pRK646, pRK248,
pDF41, etc., EK-based plasmid vectors (relaxed type) CalE1, pVH51, pAC105, RsF2124,
pCR1, pMB9, BR313, pBR322, pBR324,
pBR325, pBR327, pBR328, pKY2289,
pKY2700, pKN80, pKC7, pKB158,
pMK2004, pACYC1, pACYC184, λdu1, etc.
λgt・λc, λgt・λB of λgt-based phage vectors,
λWES・λB′, λZJvir・λB′, λALO・λB,
λWES・Ts622, λDam, etc., Syaron Vector's Syaron 4A, Syaron 3A, Syaron 16A, Syaron 13A, Syaron 14A, Syaron 15A, Syaron 8, Syaron 10, Syaron 17, Syaron 20, etc.
Tiollais group vector
L512, λZEQS, λZYV5φ, λZUV φ2, λZUV
φ3, λYEQSφ1, λYEQSφ, λYEQSφ3,
Bacillus subtilis plasmid vectors such as λBam and λSst
pTA1015, pLS15, pTA1020, pLS28, pLS13,
pTA1050, pTA1060, pTA1030, pTA1031, etc.
Plasmid vector derived from Staphylococcus
pT127, pC194, pC221, pC223, pUB112,
pUB110, pSA0501, pSA2100, pE194, pTP4,
pTP5 etc., pJDB219 as yeast vector,
YEp13, YRp7, YIp1, 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 SV ₄₀ , pI-11β as animal vectors ^- ,
There are pSVH _io +K ⁺ , pβ2X, pSXβ+, etc. However, in the case of Ti plasmid-derived plant vectors,
The obtained recombinant DNA is once introduced into Agrobacterium tumefaciens T37, etc., and the recombinant microorganism is co-cultured into plant cells to infect the host plant.
DNA can be introduced. That the glucose isomerase obtained as the recombinant DNA product is structurally the same as that of the natural type can be confirmed by techniques such as immunoprecipitation or immunoblotting of the product. [Example] Next, the present invention will be explained in detail with reference to Examples. Example 1 Streptomyces griseofuscus S-41
was cultured until late logarithmic growth according to a conventional method, centrifuged under cooling, and its wet weight (52.8 gr) was immediately frozen in liquid nitrogen. Add 15% sucrose, 20mM EDTA,
After adding 30mM Tris-HCl (PH8.0) and 0.7% SDS, and then incubating at 50℃ for 30 minutes, Murray
and Thompson's method [Nucleic Acids Res.8,
4321 (1980)]. this
The 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 completely digested with Bam H1, 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 26,000 rpm for 26 hours. The DNA chain length contained in each fraction was examined by fractionating 800 μm and subjecting a portion to agarose gel (0.4%) electrophoresis. As a result, the average DNA chain length of fraction 31 was 5.8 kb, and 8.25 μg
The amount of DNA was Approximately 1 μg of this DNA fraction was taken and treated with 1 μg of Bam H1 completely digested pUC13 and ligase treatment. Reaction is T4 DNA ligase (20U)
The test was carried out at 4°C for 14 hours. Next, E. coli
It was transformed into 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, S-F-Q-P-T-P-E-D-K-
Since the result was F-T-F-G-L-W-T-V-Q-W, oligonucleotides were synthesized for the less degenerate region. That is, ^5'GTA GAAT CTTA GTCT CTCIGGIGTIGG ³ ' (I=
When colony hybridization was performed using the above 23mer 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 blot hybridization, it was found that most of the glucose isomerase gene was located on the 2.7 kb region excised by Sph1 . Therefore, to facilitate structural analysis and expression of the glucose isomerase gene in organisms other than actinomycetes, we used pUC119 (and
pUC118) using Steven Henikoff's method [Gene, 28 , 351 (1984)] and Yanisch-Perron
[Gene, 33 , 103 (1985)]
pU−GI−32 and pUS−GI−65 derived from −32 ( Spt
Plasmids in which the insertion site of the 2.7 kb clone according to No. 1 was inserted into pUC119) were deleted to various degrees were prepared and cloned in E. coli. Using a series of these defective plasmids, we revealed the complete structure of the glucose isomerase gene by the Sanger method (see Figure 1). As a result, Streptomyces glucose isomerase
It was revealed that it is a protein with a molecular weight of 43,643 consisting of 393 amino acids. The structure of the precursor protein 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, pU-
A series of deletion plasmids of GI-32
Steven Henikoff's method [Gene, 28 , 351
(1984)] 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 Bal31, as long as they are appropriate. Here, we aimed to delete about 2 kb of the 5'-flanking region of the gene. As a result, one of the deletion mutants showed that the 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 as shown in Figure 1 (see –). Therefore, a synthetic double-stranded oligonucleotide, 5'-d [CAGCCATGGGGTTCC], was added to the 5' end of this plasmid.
were ligated using ligase. After cutting this with Ncol, Ncol of E. coli expression vector pKK233-2
After binding to the site and filling-in the 3'-end, it was circularized with ligase. This expression vector was transformed into Escherichia coli NM522, an IacI ^Q strain. It was confirmed that this expression plasmid produced glucose isomerase within the bacterial cells upon addition of IPTG by immunoblotting of the disrupted bacterial cell supernatant and activity measurement. This bacterium has been deposited with the National Institute of Microbial Technology, and its deposit number is FERM P-9717. [Effects of the Invention] The present invention provides a gene encoding glucose isomerase derived from Streptomyces griseofuscus S-41, 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. You can make a big contribution.

[Brief explanation of drawings]

FIG. 1 shows the base sequence of the glucose isomerase gene.

Claims (1)

[Claims] 1. A 5.7 kb compound corresponding to glucose isomerase, present on the chromosome of Streptomyces griseofuscus S-41, and excised with the restriction enzyme BamH1.
Genes included in the fragment fraction. 2. The gene according to claim 1, wherein the gene has the base sequence shown in FIG. 3 Corresponding to glucose isomerase, the chromosome of Streptomyces griseophuscus S-41
A recombinant having DNA containing the promoter region, the complete translation region of the enzyme, the enzyme gene, and a part of the xylulokinase gene expressed in polycistronic cells in the 5.7 kb-BamH1 fragment fraction. 4. The recombinant according to claim 3, wherein the DNA is a prokaryotic or eukaryotic vector. 5. The recombinant according to claim 3, which has the gene and the base sequence shown in FIG. 6 Corresponding to glucose isomerase, obtained from Streptomyces griseofuscus S-41,
Complete digestion of BamH1 by sucrose density gradient centrifugation
The DNA fragment obtained in the 5.7kb-DNA fragment fraction and excised with the restriction enzyme Sphl contains most of the structural gene of the enzyme, including the promoter region.
A microorganism that has a vector into which DNA has been integrated. 7 Microorganisms such as Escherichia coli and Bacillus
7. The microorganism according to claim 6, which is S. subtilis or S. cerevisiae.