JP2989638B2 - Acremonium chrysogenum promoter - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to A. chrysogenum (A. chrysogenum).
genum), a novel DNA with promoter activity
It concerns compounds.

(Conventional technology and problems to be solved) A filamentous fungus, Acremonium chrysogenum, is widely used as a bacterium producing cephalosporin C which is a raw material of various cephalosporin antibiotics that are practically used as pharmaceuticals. And is one of the industrially important microorganisms. Heretofore, the only technique that has been used to improve the cephalosporin C-producing ability of the strain is the classical mutation technique. However, in recent years, with the improvement of recombinant DNA technology, transformation systems (for example, Queener et al., M.
icrobiology 1985.American Society for Microbiolog
y, (1985) pp 468-472, Skatrud et al., Current Genetics.
(1987) 12: 337-348], opening the way for genetic engineering approaches to improve breeding of superior strains.
[Eg Skaturd et al., BIO / TECHNOLOGY (1989) 7,477].
In general, in order to successfully carry out gene manipulation in a certain microorganism, in addition to the development of the above-described transformation system, it is necessary to develop an expression vector having a structure such as a promoter and a terminator that efficiently functions in the microorganism. . At present, the development of such expression vectors in Acremonium chrysogenum has been delayed compared to other biological systems, and the isolation of a promoter that is an important component of the expression vector is also a few examples (for example, However, it is hard to say that only the existence of JP-A-63-301790, JP-A-61-254192 and JP-A-64-80295] is sufficient. Therefore, isolation of a novel promoter that functions efficiently in Acremonium chrysogenum has been desired by those skilled in the art. Generally, it is considered that an efficient promoter exists in the 5 'upstream region of a gene encoding a protein (hereinafter abbreviated as a high expression gene) which is expressed in a host cell in a large amount. Focusing on this point, in Saccharomyces cerevisiae, which is the most advanced fungal expression vector, the genes of various glycolytic enzymes and the major cytoskeletal proteins are expressed as highly expressed genes. One of them, such as the actin gene, has been isolated, and its promoter has been used as a component of a useful expression vector. [For example, Kingsm
an et al., Gene (1983) 24, 1-14, Ling-Pai Ting et al. BBR,
C. (1986) 136.1063-1070] (Means for Solving the Problems) In view of the above points, the present inventors have proposed that phosphoglycerate kinase (hereinafter referred to as PGK and PGK), which is a glycolytic control gene, is obtained from Acremonium chrysogenum. Abbreviated) as well as the actin gene were isolated. The inventors have found that the regions containing the promoters of these genes can be used for the development of expression vectors using acremonium as a host, and have completed the present invention.

That is, the present invention has a promoter activity of the phosphoglycerate kinase gene of Acremonium chrysogenum, a DNA fragment containing at least a part of the base sequence represented by the following formula 1, A DNA fragment having the promoter activity of the actin gene of Acremonium chrysogenum and containing at least a part of the base sequence represented by the following formula 2: The DNA fragment of the present invention was isolated together with a protein coding region separated by introns and a 3 'untranslated region following the protein coding region. A part of this protein coding region is used as a component of an expression vector when a desired gene is expressed as a fusion protein. In addition, it is generally considered that a signal for terminating transcription efficiently, including a terminator, is present in the 3 'untranslated region,
This is also used as a component of the expression vector. Therefore, these regions are also included in the present invention. The specific nucleotide sequence containing the coding region and the 3 'untranslated region of the PGK gene and actin gene derived from Acremonium chrysogenum and the 3' untranslated region are shown in FIGS. 4 and 5 together with the sequence of the promoter-containing fragment of the present invention. Was. The present invention further provides a series of expression vectors for Acremonium chrysogenum, wherein the promoter-containing fragment terminator-containing fragment is incorporated into a plasmid in various ways.

The promoter-containing DNA fragment of the present invention can be generally constructed by the following steps.

(1) Chromosomal DNA from Acremonium chrysogenum
And cut with an appropriate restriction enzyme (for example, Mbo I or the like).

(2) The chromosomal DNA library of Acremonium chrysogenum is constructed by incorporating the DNA fragment obtained in (1) into an appropriate phage vector (eg, EMBL3, EMBL4, etc.).

(3) A DNA fragment containing the PGK gene and at least a part of the actin gene coding region derived from the other species whose structure has been clarified is obtained.

(4) The DNA obtained in (3) is labeled with ³² P, the chromosome gene DNA library obtained in (2) is subjected to plaque hybridization, and plaques showing complementarity with the DNA probe are selectively separated. I do.

(5) DNA is extracted from the selected phage, Southern hybridization is carried out using the same probe as above, the target gene is located on a restriction enzyme fragment of an appropriate size, and it is subcloned into a plasmid vector.

(6) The nucleotide sequence of the DNA fragment subcloned in (5) is determined, and by comparing with the corresponding gene structure derived from other species, it is confirmed that the obtained gene is the target gene. Determine the code region. The promoter of the present invention is usually present in a DNA fragment located upstream of the coding region 5 '.

(7) The promoter is present on the DNA fragment of the present invention by ligating the DNA fragment containing the 5 ′ upstream region and a drug resistance marker gene derived from bacteria in an arrangement suitable for expression and introducing it into Acremonium chrysogenum. Make sure you have General procedures required for handling E. coli, phage, and DAN in the above steps are those routinely performed by those skilled in the art, and are described, for example, in the experimental operation manual (T. Maniatis et al., Molecular Clonig A
Laboratory Manual, Cold Spring Harbor Laboratory 1
982, 1989). All of the enzymes, reagents and general-purpose vector DNA of Escherichia coli used are commercially available products.Unless otherwise specified, it is possible to completely achieve their purposes according to the use conditions specified in the products. it can.

In the above (1), strains such as Acremonium chrysogenum ATCC 11550 and Acremonium chrysogenum IS-5 can be used as a DNA extraction source.

The Acremonium chrysogenum IS-5 strain was deposited on February 5, 1990 with the Microorganisms and Industrial Technology Research Institute of the Ministry of International Trade and Industry of Japan under the deposit number of Microtechnical Laboratory No. 11232. This strain was transferred to the International Depositary on November 5, 1990 based on the Budapest Treaty on the International Recognition of Deposit of Microorganisms, as No. 3153, FERMBP-3153.

Extraction of total DNA from the bacterium can be performed, for example, by the method described in Johnstone et al. [Johnstone et al., EMBO Journal (1985) 41307-131].
1] or Minuth et al. [Minuth et al., Curre
nt Genetics (1982) 5: 227-231]. Examples of the PGK genes derived from other species in the above (3) and (6) include, for example, the PGK gene of Saccharomyces cerevisiae [Hitzemau et al., The Jourral of Biological Chemis.
try (1980) 25,12073-12080], [Hitzeman et al. Nucleic
Acids Research (1982) 10,7791-7808], and the PGK gene of Aspergillus Nidulans (Clements et al. Geue (1986) 44 97-105). Examples of the actin gene include, for example, the actin gene of Saccharomyces cerevisiae (Gallwitz et al.,
Nucleic Acids Research (1980) 8.1043- 1055] [Gall
Witz et al., Proc. Natl. Acad. Sci. USA (1980) 77.2546-254.
9], human β-actin gene [NAKAJIMA et al., Proc.
Acad Sci. USA (1985) 82 6133-6137]. These genes can be obtained by the method described in the above-mentioned literature, or by synthesizing a DNA oligomer corresponding to a known base sequence and hybridizing using the oligomer. The synthesis of the DNA oligomer can be performed using a commercially available DNA synthesizer according to the operation procedure. The method for determining the DNA base sequence in (6) is also the known dideoxy method [Sanger et a
l., Proc. Nati. Acad. Sci. USA (1977) 73,5463].

In the above (7), as a drug resistance marker gene derived from a bacterium, a neomycin phosphotransferase gene [for example, Beck et al., Gene (1982) 19327-33]
6], hygromycin B phosphotransferase gene [for example, Gritz et al., Gene (1983) 25 179-18].
8] etc. are used. Transformation of Acremonium chrysogenum using these drug resistance marker genes can also be performed according to a known method [for example, Queene
r et al., Microbiology 1985.American Society for Microb
iology, (1985) pp468-472]. When a promoter fragment derived from Acremonium chrysogenum is ligated to the above marker gene in an arrangement suitable for expression, it is known that the transformation efficiency of Acremonium chrysogenum by the marker gene is significantly increased. [Skatrud
Current Genetics (1987) 12: 37-348] Therefore, to determine whether or not the DNA fragment of the present invention contains a functional promoter, construct a plasmid in which the fragment and the above marker gene are ligated in a suitable arrangement for expression. It can be confirmed by examining the transformation frequency of Acremonium chrysogenum with the plasmid.

(Examples) The abbreviations or abbreviations described in the examples are as follows.

CM medium: 20 g sucrose / 0.5 g potassium dihydrogen phosphate / 0.5 g dipotassium hydrogen phosphate / 0.5 g potassium chloride / 0.5 g magnesium sulfate (7 hydrate) /0.01 g iron (II) sulfate (7 hydrate) / Sodium nitrate 3g / Yeast extract 4g / Peptone 10
g dissolved in water 1.

 CM solid medium: CM medium containing 1.5% agar.

GAG medium: glycerol 40g / asparagine 4g / calcium chloride 0.1g / sodium chloride 0.1g / trace metal solution [magnesium sulfate (7 hydrate) 4g / iron (II) sulfate (7 hydrate) 0.4g /
Manganese sulphate (tetrahydrate) 0.16g / Zinc sulphate (heptahydrate) 0.4g
/ Anhydrous copper sulfate 0.04g dissolved in water 1] 25ml / 0.1M
Medium containing 30 ml of phosphate buffer (pH 7.0) in water 1, 6 × SSC: 0.9 M sodium chloride / 90 mM sodium citrate, 20 × SSPE: sodium chloride 210 g / sodium dihydrogen phosphate (dihydrate) 31.2 g / 0.5M EDTA (EDTA: ethylenediaminetetraacetic acid) 40 ml dissolved in water 1, 50 × Denhartz: Ficoll 5 g / polyvinylpyrrolidone 5 g / bovine serum albumin 5 g dissolved in water 500 ml.

P-buffer: 0.6 M potassium chloride / 0.01 M, magnesium chloride / 0.025 M calcium chloride PEG solution: 25% polyethylene glycol (molecular weight about 400
0) /0.01M Tris (pH8.0) /0.05 calcium chloride / 0.6M potassium chloride.

Example 1 [Isolation of Acremonium chrysogenum phosphoglycerate kinase (PGK) gene] (1) Preparation of a gene library of Acremonium chrysogenum Acremonium chrysogenum IS-5 strain (Microtechnical Laboratories No. 3153) , FERM BP-3153) using the method of Johnston et al. For Aspergillus nidulans [Reference: Embo Journal (EMBO)
J.), 4 , 1307-1131, 1985,]. Then, about 60 μg of the total DNA was partially digested with a restriction enzyme MboI, and then treated with alkaline phosphatase. On the other hand, 10 μg of Lambda Vector EMBL 3 (Promega Biotex)
Digestion was complete with BamHI and EcoRI and the short EcoRI-BamHI linker moiety was removed by isopropanol precipitation.

Next, about 1 μg of the above-mentioned partially digested DNA fragment and about 2 μg of a vector having a BamHI terminal were ligated using T4 ligase, and were encapsulated in lambda phage particles. The thus-obtained recombinant phage suspension was appropriately diluted, and E. coli NM539 (Promega Biotech)
And the number of plaques that appeared was counted. As a result, this suspension was found to contain 3 × 10 ⁵ recombinant phages. This phage solution was used as an Acremonium chrysogenum gene library and stored at 4 ° C.
The detailed methods and conditions for the preparation of the donor DNA and the vector described above, and the binding thereof, etc.
ischauf) et al. [Journal of Molecular Biology (J. Mol. Biol), 17
0, encapsulation of 827-842 1983] Also within the lambda particles of DNA, using the Promega Biotech packaging extract was performed according to the protocol attached thereto.

(2) Preparation of Probe pYPGK1 (this plasmid having a 2.9 Kb HindIII fragment containing the entire PGK gene derived from Saccharomyces cerevisiae) was prepared by digesting HindIII digested Saccharomyces total DNA with PBR327.
(ATCC37516), a nucleotide sequence of the Saccharomyces PGK gene reported by Hitzman et al. [Reference:
Nucleic Acids Research
s.), 10 7791-7808, 1982]: 5'-CAGATCATCAAGAAGTAATTATCT-3 '
Obtained by screening using ) 20μ
g was digested with Hind III and EcoR I, and subjected to 1% agarose gel electrophoresis. And Maniatis (T.Maniatis)
Et al. Molecular Cloning A Laboratory
Manual, Cold Spring Harbor Laboratory Press, 1982 (T. Maniatis et al., Molecular Clo
ning A Laboratory Manual, Cold Spring Harbor Labora
tory 1982, hereafter abbreviated this manuscript as an experiment of Manuatis. ) 2.9 according to the method described on pages 164-165.
The Kb fragment was recovered from the gel and purified. Then, about 200 ng of this fragment was used using a nick translation kit manufactured by Takara Shuzo Co., Ltd., and according to the protocol attached to it.
[Α- ³² P] deoxycytidine triphosphate (dCTP) 50 μCi
Labeled with. Then, after heating the reaction solution at 70 ° C. for 10 minutes,
Nick columns manufactured by Pharmacia (Pharmacia, Nick-c
olumn ^TU ) to obtain a probe having a radioactivity of about 10 ⁷ cpm. (Hereinafter, this probe is referred to as YP-probe.

(3) Screening by hybridization Phage solution (gene library) obtained in (1)
Was infected with NM539, and a total of 2
× 10 ⁴ plaques were formed. These plaques were prepared by the method of Benton WD et al. [Literature: Science (S
196 , 180-182, 1977]], and the DNA is fixed to a nitrocellulose filter, subjected to alkali denaturation and neutralization treatment, immobilized on the DNA, and hybridized with the YP-probe obtained in the above (2). Was. Hybridization was performed with 30% formamide, 5 × Denhardz, 5 × SS
Performed at 42 ° C. for 16 hours using a solution containing YP-probe at a final concentration of 5 × 10 ⁵ cpm / ml with PE, 0.1% SDS. Subsequently, the filter is washed twice in a 6 × SSC solution containing 0.1% SDS at room temperature for 10 minutes each, and further washed with 0.1% SDS.
The plate was washed in a × SSC solution at 42 ° C. for 30 minutes. Autoradiography was then performed at -80 ° C for 16 hours using an intensifier screen. As a result, seven positive spots were found. Phages were extracted from the agar region corresponding to the four positive spots, and plaque hybridization was performed again according to the above-described steps.
One purified positive phage clone was obtained. These clones were named λ-PGK1,2,3,4, respectively.

(4) Subcloning of PGK gene and restriction of its position From the four phage clones obtained in (3), a method described in Grossberger [Reference: Nucleic Acids. Research, 15 , 673]
DNA was extracted according to 7]. This lambda DNA is then
After cutting with HI and performing agarose gel electrophoresis,
Southern hybridization using a P-probe [Methods: Literature: Southern, Journal of Molecular Biology (J. Mol. Bio
l.), 98 , 503-517, 1975]. In addition, hybridization, filter washing | cleaning, etc. were performed like what was described in (3). As a result, it was found that only the approximately 5.5 Kb BamHI fragment present in all the clones hybridized with the probe. This fragment was recovered and purified from agarose gel using Geneclean (GENE CLEAN ^™ , Funakoshi) according to the attached protocol. On the other hand, pUC18 (Takara Shuzo Co., Ltd.) used as a vector was digested with BamHI and treated with alkaline phosphatase. Then, both were ligated with T4-ligase, and E.co.
li Introduced to JM105. Ampicillin (Amp), 100μg / ml,
Six white colonies growing on an L-broth agar medium containing 0.004% of 5-bromo-4-chloro-3-indolyl-β-galactoside (X-Gal) were selected, and a simple method (Maniatis The plasmid DNA was extracted by BamHI digestion analysis, and it was found that the target fragment was inserted into the plasmids in 5 out of 6 clones. Furthermore, analysis by Southern hybridization was performed in the same manner as described above, and it was confirmed that this insert was the target fragment.
One of these plasmids was named pGK5.

The plasmid pGK5 was digested with various restriction enzymes and analyzed by agarose gel electrophoresis. As a result, the restriction map of the 5.5 Kb insert shown in FIG. 1 was obtained. Next, Southern hybridization was performed using a YP-probe to limit the PGK coding region present in this fragment. As a result, it was estimated that at least a part of the PGK coding region was present in the Pst I-Stu I fragment of about 0.7 Kb.

(5) Nucleotide sequence of Acremonium chrysogenum PGK gene First, a 0.7 kb Pst I-Stu I fragment estimated to contain a part of the coding region of the PGK gene was ligated to the S13 of M13mp18 and M13mp19.
Each fragment was subcloned between maI and PstI, and a part of each sequence was determined based on the method of Sanger et al. (Sanger, F, Science Science 214 , 1981, etc.). By comparing with a known PGK gene, the direction of the gene and which part of the PGK-protein this region encodes were estimated. Sequencing was performed upstream and downstream from the region, and finally a 3306 bp base sequence covering the entire region indicated by the underline in FIG. 1 was determined.

Specific experimental procedures for nucleotide sequence determination were performed using Takara's sequencing kit according to the attached protocol. FIG. 3 shows the determined total nucleotide sequence and predicted translation products. Genetic information processing software (SD
Computer analysis of this sequence using C Software Development Co., Ltd.) revealed the following, and through the above-described steps, obtained confirmation that the isolated gene was a true PGK gene. 1) The PGK gene of Acremonium chrysogenum is composed of 418 amino acids and has a molecular weight of 44,3.
Encodes a 00 dalton protein. 2) The code area is
It is separated by two 145,64 bp introns. PGK of Aspergillus nidulans, whose intron is different in size but located at the same filamentous fungus
It was identical to the gene. 3) The primary structure of the amino acid predicted from the nucleotide sequence is very similar to that of PGK derived from human, Saccharomyces cerevisiae and Aspergillus nidulans, showing 68, 70 and 75% homology, respectively. Was. Bal II-Kpn whose structure was determined as described above
The fragment I (region (A) of FIG. 1) covers a region extending 1251 bp 5 ′ upstream from the start codon (ATG) of the PGK gene, and the PGK promoter of the present invention is considered to be present on the fragment. Can be Examples 2 and 3 below strongly support that this estimation is correct.

According to the steps shown in FIG. 5 [Construction of pPGKM5] Example 2, the neomycin phosphotransferase gene from a bacterial (hereinafter abbreviated as Km ^R gene.) The derived Acremonium chrysogenum
Plasmid pPG for expression under the control of the PGK promoter
KM5 was built. The respective steps will be described below.

(1) Preparation of pGKBL The pramid pPGK-5 obtained in Example 1- (4) was digested with BglII, and a 3.6 kb fragment containing the PGK gene was separated and purified.
The fragment was prepared in Example 1- (4), and BamHI of pUC18.
PGKBL was obtained by insertion into the site. At the same time, a plasmid in which the above fragment was inserted in the opposite direction to pGKBL was also obtained and named pGKBL '.

(2) Preparation of pGKCS Plasmid pGKBL obtained in (1) above was transformed with Mlu I and Xho I
And a 4.8 kb fragment was separated and purified. The fragment was ligated to a synthetic linker represented by the following formula to obtain pGKCS. formula: Perform the same operation using pGKBL 'instead of pGKBL.
pGKCS 'was also constructed. In pGKCS and pGKCS ', a fragment containing a PGK promoter and terminator derived from Acremonium chrysogenum contains a unique restriction enzyme site (Bgl II, Xho
It is a plasmid having a structure connected in a suitable arrangement for expression via I), and is a useful starting material when constructing a vector for expressing various foreign genes in Acremonium chrysogenum. The above linker is a DNA synthesizer model 3 from Applied Biosystems.
Using 80-A, it was synthesized as two single strands in the usual manner.

(3) Preparation plasmid pEX002 of pPGKM5 was cleaved with BamH I and Bgl II, to separate and purify a fragment of 1.5kb including Km ^R gene. The fragment was Bgl I
PGK digested with I and treated with alkaline phosphatase
By binding to CS in the direction shown in FIG. 5, pPGKM5 was obtained. The plasmid pEX002 used above was lac UV5
Promoter and Km ^R derived from transposon 5 (Tu5)
An expression vector for Escherichia coli having a gene, the construction method of which is described in JP-A-63-74488.

In Example 2 above, separation of the restriction enzyme digested fragment
Preparation of DNA fragments from agarose gel is performed by Gene Clean (GEN), all performed by 1% agarose gel electrophoresis.
E.CLEAN ^™ , manufactured by Funakoshi Co., Ltd.) according to the attached protocol. Basic operations such as ligation of plasmid and DNA fragment, transformation of Escherichia coli, and preparation and analysis of a plasmid obtained as a result of subcloning were all carried out in accordance with the method described in the manual of Maniatis.

Example 3 [Transformation of Acremonium chrysogenum using pPGKM5] (1) Preparation of protoplasts: A mycelium of Acremonium chrysogenum IS-5 (about 1 cm ² ) grown on a CM solid medium at 30 ° C. for 5 days The corresponding mycelium) was inoculated into 50 ml of a CM medium, and cultured at 30 ° C. for 3 days on a rotary shaker (250 rpm). Further, 1 ml of the bacterial solution was inoculated into 50 ml of GAG medium and cultured at 30 ° C. for 20 hours on a rotary shaker (250 rpm). 50 ml of the obtained culture solution was centrifuged at 3500 rpm for 10 minutes to precipitate mycelium, washed with 0.9% NaCl solution, and diluted with McIlvaine buffer solution containing 0.1M dithiothreitol (0.1M McIlvaine). Citric acid, 0.2M sodium phosphate, pH7.3) 2
The suspension was suspended in 0 ml and gently shaken at 30 ° C. for 1 hour. Then, the mycelium was precipitated by centrifugation at 3200 rpm for 10 minutes, washed with P-buffer, and then 10 mg of Novozyme (Novo).
suspended in 10 ml of P-buffer containing
Shake gently at ℃ for 1 hour. The reaction below at 800 rpm
The supernatant obtained by centrifugation for 30 seconds is filtered with filter paper (TOYO FILTER PAPER
Filtration using 5A) dispersed mycelia and protoplasts. Then, the filtrate was centrifuged at 3000 rpm for 5 minutes to precipitate protoplasts, washed once with P-buffer, and suspended in P-buffer so that the protoplast had a concentration of 3 × 10 ⁸ / ml. .

(2) Transformation of protoplasts with pPGKM5 To 0.1 ml of the protoplast suspension obtained in the above (1), 5 μg (10 μ) of plasmid pPGKM5 was added, and then 0.05 ml.
Was added and mixed lightly. After allowing to stand on ice for 25 minutes, 1 ml of the same PEG solution was added, and the mixture was allowed to stand at room temperature for another 30 minutes. 0.2 ml each of the thus obtained transformed protoplast suspension was used as a protoplast regeneration medium [Reference (Iso
gai et al .: BRM medium described in Agric. Biol. Chem. 1987, 51, 2321-2329)] on a plate containing 25 ml.
After culturing at 20 ° C for 20 hours, 5 ml of the same BRM medium containing 3 mg of G418 and kept at 50 ° C was overlaid. Then, G418-resistant transformants were selected by culturing at 28 ° C. for 10 to 20 days. When the above experiment was performed several times, pPGKM5.5μ
50 to 150 G418 resistant strains appeared per g. On the other hand, when the same experiment was performed using the control plasmid pEX002, only 0 to 2 G418-resistant strains appeared. The above results indicate that pPGK having the sequence shown in the above formula 1
This strongly suggests that the promoter of Acremonium chrysogenum PGK gene is contained in the Xba I-Bgl II fragment on M5. Note in the chromosome of the above-resistant strains, that plasmid used to introduce is inserted, Pst I fragment of pEX002 containing most of the Km ^R gene coding region (about 900 bp) as a probe, Southern hybridization experiment I was assured.

Example 4 [Cloning of Actin Gene] (1) Screening of Actin Gene-Containing Clones by Hybridization Probe of Hinf I 400 bp fragment (Wako Pure Chemical Industries) containing the third exon of human β-actin gene labeled with ³² P (Hereinafter abbreviated as ACT probe), and
The Acremonium chrysogenum gene library prepared in (1) was screened under the same conditions as in Example 1- (3), and four phages hybridizing with the probe were obtained.

(2) Subcloning of actin gene and restriction of its position DNA was extracted from one of the phages obtained in (1) and named λACT5. Then, λACT5 was changed to Xho I, Sa
After digestion with lI and agarose gel electrophoresis, Southern hybridization was performed using the ACT probe. The resulting 5.4 Kb Xho I fragment, 1.
It was found that two Sal I fragments having a size of 3 Kb and 1.5 Kb hybridized with the probe. Therefore, these three fragments (Xho I-5.4K fragment, Sal I-1.5K fragment, Sa
The lI-1.3K fragment) was subcloned into the SalI site of pUC18 to obtain pACT5X, pACT5SS, and pACT5SL.
Then, a partial restriction map of these plasmids was prepared, and by overlapping them, a partial restriction map of a DNA fragment of about 6 Kb which seems to contain the actin gene was prepared as shown in FIG.

The Southern hybridization was performed under the same conditions as in Example 1- (4) except that the ACT probe was used.

(3) Determination and decoding of base sequence From the results of (2) above, at least a part of the actin coding region (a part corresponding to the third exon of human β-actin) in the 0.7 kb Sma I-Xho I fragment. Was strongly suggested, so the nucleotide sequence of this portion was determined first. By comparing with a known actin gene, the direction of the gene, the presence or absence of an intron, and which part of the actin protein this region encodes were estimated. Subsequently, sequencing was performed upstream and downstream from the region, and finally, a 3748 bp base sequence covering the entire region indicated by the underline (B) in FIG. 2 was determined. FIG. 4 shows the determined total nucleotide sequence and predicted translation products. As a result of comparative analysis of the amino acid sequence of the nucleic acid sequence and the amino acid sequence of the translation product with that of known actin, the following was found, and it was confirmed through the above-mentioned steps that the isolated gene was a true actin gene.

1) The actin gene of Acremonium chrysogenum is
It encodes a protein consisting of 375 amino acids and having a molecular weight of 41800 daltons. This residue number is identical to all other actins except for vertebrate skeletal muscle actin (α-type).

2) The amino acid sequence predicted from the nucleotide sequence was very similar to that of known actin, and showed 92% and 90% homology to actin of Saccharomyces cerevisiae and human γ-type actin, respectively. The NsiI-SalI-fragment isolated and structure determined as described above (FIG. 2)
Covers a region extending 1293 bp 5 ′ upstream from the start codon of the actin gene, and the actin promoter of interest in the present invention is considered to be present on the fragment. The following Examples 5 and 6 strongly supported that the above estimation was correct.

According to the steps shown in Figure 6 [Construction of pACTHY83] Example 5, bacterial hygromycin B phosphotransferase gene (hereinafter HYB ^R
Abbreviated as gene. ) Was constructed under the control of an actin promoter derived from Acremonium chrysogenum to construct a plasmid pACTHY83. The respective steps will be described below.

(1) Preparation of pACTΔNP Plasmid pACT5X obtained in Example 4- (2) was
A 5.3 Kb fragment was prepared by simultaneous digestion with stI. Then, the fragment was religated (self-cyclized) using T4 ligase to obtain pACTΔNP.

(2) Preparation of pACTB1, pACTB2, and pACTB3 pACTΔNP obtained in (1) was first digested with NcoI, and the resulting sticky ends were digested with DNA polymerase Klenow fragment (hereinafter DNA po
l.) and four types of deoxynucleotide triphosphates (deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, thymidine triphosphate, hereinafter abbreviated as 4dNTPS). Convert to end. The 5 'end is then phosphorylated and consists of the following sequence Was ligated to the end with T4 ligase and then digested with BamHI. The digest was subjected to agarose gel electrophoresis and 4 Kb of D
The NA fragment was separated and purified. The fragment was self-closed by T4 ligase to obtain pACTB1. Also, two linkers having different sequences and different numbers of bases The same operation was performed using Takara Shuzo Co., Ltd. to obtain pACTB2 and pACTB3, respectively.

(3) Preparation of pACTCS1, pACTCS2, pACTCS3 The plasmid pACTSS obtained in Example 4- (2) is digested with BamHI, blunt-ended using DNA pol. And 4dNTPS, and then self-closing with T4 ligase. This yielded a plasmid, pACTSSΔBam, which lost the BamHI site. Subsequently, the plasmid was digested with Sca I and EcoR I, and a 0.9 Kb fragment considered to contain a terminator of the actin gene was separated and purified. Then, the fragment was inserted between SmaI and EcoRI of pACTB1 to obtain pACTCS1. The 0.9 kb fragment was inserted between SmaI and EcoRI of pACTB2 and pACTB3 to obtain pACTCS2 and pACTCS3, respectively. In these three plasmids, a fragment containing an actin promoter and a terminator derived from Acremonium chrysogenum is placed in a position suitable for expression via a unique restriction enzyme cleavage site BamHI (located immediately downstream of the actin start codon ATG). It is a plasmid having a linked structure and is a useful starting material when constructing vectors for expressing various target genes as fusion proteins in Acremonium chrysogenum. By using any of these three plasmids, the desired gene can be ligated in the same reading frame as the actin gene.

(4) pACTHY83 Preparation plasmid pLG83 [Pasteur Institute, Julian Davis (Julian Davies) obtained from exchanges] was digested with BamH I, was separated and purified fragments of 1.3Kb containing HYB ^R gene.
The fragment was digested with BamHI and ligated with pACTCS1 treated with alkaline phosphatase in the direction shown in FIG. 6 to obtain pACTHY83. Note that the pLG83 is a vector for yeast having HYB ^R gene, its properties are known literature [Gritz et al., Gene (1983) 25.179~188] are described. In Example 5, the basic operations such as separation and purification of a restriction enzyme digested fragment, ligation of a plasmid and the fragment, transformation of Escherichia coli, and preparation and analysis of a plasmid obtained as a result of subcloning are all the same as those in Example 2. I went to.

Example 6 Transformation of Acremonium chrysogenum with pACTHT83 Using pACTHY83 and pLG83,
A transformation experiment of Chrysogenum IS-5 strain was performed. pACTHY
When 5 μg of 83 was used, 40 to 100 transformants (hygromycosis B-resistant strain) were obtained, whereas pLG83 was used.
When no transformant was used, no transformant appeared. This means that Hind I on pACTHY83 having the sequence shown in Formula 2 above
It strongly suggests that the Acremonium chrysogenum actin gene promoter is contained in the II-BamHI fragment (FIG. 6). In the above experiment, protoplast preparation of Acremonium chrysogenum IS-5 strain was performed in the same manner as in Example 3- (1), and protoplast transformation was performed except that 4.5 mg hygromycin B was used instead of G418. All were performed in the same manner as in Example 3- (2).

(Effect of the Invention) By using the promoter of the present invention, various genes can be efficiently expressed in Acremonium chrysogenum.

Also, various genes involved in cephalosporin fermentation,
By efficiently expressing in Acremonium chrysogenum under the control of the promoter of the present invention, the fermentation yield of cephalosporin can be expected to be improved. Further, by using the promoter of the present invention as described in detail in Examples, it becomes possible to construct an efficient transformation vector for Acremonium chrysogenum.

[Brief description of the drawings]

FIG. 1 shows a restriction map of a DNA fragment containing the Acremonium chrysogenum PGK gene. FIG. 2 shows a restriction map of a DNA fragment containing the Acremonium chrysogenum actin gene. 1 and 2 Indicates the exon of each gene. In each gene, the 5 'end of the first exon and the 3' end of the final exon are unknown. FIG. 3 is in FIG. Shows the chlorine sequence of the region (A) shown by. Also expected PG
The amino acid sequence of the K protein is shown below the base sequence.
FIG. 4 is in FIG. Indicates the base sequence of the region (B) indicated by. The amino acid sequence of the predicted actin protein is shown below the base sequence. FIG. 5 shows the process of preparing plasmid pPGKM5. Sixth
The figure shows the process of preparing plasmid pACTHY83. The abbreviations or abbreviations newly used in the figures are as follows. B: BamH I / Bg: Bgl II / B ・ B: Binding site between BamH I and Bgl II
/ P: Pvu II / Ps: Pst I / M: Mlu I / K: Kpn I / S: Sma I / Sa / Sal
I / St: Stu I / Sc: Sca I / X: Xho I / Xb: Xba I / N: Nsi I / Nc: Nc
o I / E: EcoR I / RV: EcoR V / H: Hind III / Bs: BssH II / Ps ・ N
s: binding site between Pst I and Nsi I / 2 μori: origin of replication of yeast 2 μ plasmid / CYCP: promoter of yeast iso-1-cytochrome C gene / CYCT: terminator of yeast iso-1-cytochrome C gene / PGKP : Acremonium chrysogenum PGK gene promoter / PGKT: Acremonium chrysogenum PGK gene terminator / ACTP: Acremonium chrysogenum actin gene promoter / ACTT: Acremonium chrysogenum actin gene terminator

Claims (2)

(57) [Claims] (1) Acremonium chrysogenum (A. chryso)
genum) having a promoter active portion of a phosphoglycerate kinase gene and having at least a part of the following nucleotide sequence: 2. Acremonium chrysogenum (A.chryso)
genum) has a promoter active portion of the actin gene and has at least a part of the base sequence shown below.
fragment;