JPH0458891A - Promoter of acremonium chrysogenum - Google Patents

Abstract

NEW MATERIAL:A DNA fragment containing at least a part of the base sequence of formula and having a promoter active part of a phosphoglycerate kinase gene of Acremonium chrysogenum. USE:Construction of manifestation vector using Acremonium as a host. PREPARATION:Chromosome DNA of Acremonium chrysogenum is incised with restriction enzyme MboI, etc., and the fragment is integrated into a phage vector EMBL3, etc., to construct a chromosome DNA library of Acremonium chrysogenum. A DNA fragment containing at least a part of region coding pGK gene originated from other kind of life, an actin gene and GAPD gene is labeled with <32>P, a plaque exhibiting complementarity to the fragment is separated and DNA is extracted therefrom. The DNA is subjected to Southern hybridization using the above probe and subcloned to a plasmid vector.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to Acremonium chrysogenum (A.

chrysogenum) and relates to a novel DNA compound having promoter activity.

(Conventional technology and issues to be solved) Acremonium chrysogenum, a filamentous fungus, is widely used as a producing bacterium for cephalosporin C, which is a raw material for various cephalosporin antibiotics that are used as pharmaceuticals. It is one of the industrially important microorganisms.

Conventionally, the only technique used to improve the cephalosporin C production ability of the strain was the classical mutation method. However, in recent years, with the improvement of recombinant DNA technology, transformation systems [for example, Queener
et al., Microbiology 1985. Amer
ican 5ociety forMicrobiol
ogy, (1985) pp468-472.5k
atrud et al., Current Genetics (
1987) 12: 337-348], opening the way to applying genetic engineering approaches to breeding and improving superior bacterial strains. [For example, 5katrud et al., BIO/TE
CHNOLOGY (1989) 7.477]. In general, in order to carry out genetic manipulation successfully in a given microorganism, in addition to the development of the above-mentioned transformation system, it is necessary to develop an expression vector equipped with structures such as promoters and terminators that function efficiently in the microorganism. ing. Currently, the development of such expression vectors for Acremonium chrysogenum is delayed compared to other biological systems, and there are only a few examples regarding the isolation of the promoter, which is an important component [e.g. , Tokukai Sho (63-301790),
Tokukai Akira (61-254192) Tokukai Akira (64-8029
5)'] is still far from being sufficient. Therefore, it has been desired by those skilled in the art to isolate a new promoter that functions efficiently in Acremonium chrysogenum.

It is generally believed that efficient promoters exist in the 5' upstream region of genes encoding proteins that are abundantly expressed in host cells (hereinafter referred to as highly expressed genes). Focusing on this point, we focused on Satucharomyces cerevisiae (S, c
erevisiae), the genes for various glycolytic-controlling enzymes and the actin gene, which is one of the major cytoskeletal proteins, have been isolated as highly expressed genes, and their promoters can be used as components of useful expression vectors. has been done. [For example, Kingsman et al., Gen
e (1983) 24. 1-14. Ling-Pai
Ting et al. B, B, R, C, (1986)
136. 1063-1070) (Means for Solving the Problems) In view of the above points, the present inventors have developed phosphoglycerate kinase (hereinafter abbreviated as PGK), which is a glycolytic-controlling gene, from Acremonium chrysogenum. The gene for cellaldehyde-3-phosphate dehydrogenase (hereinafter abbreviated as GAPD) and the gene for actin were isolated. Then, they learned that the regions containing the promoters of these genes could be used to develop expression vectors using Acremonium as a host, leading to the completion of the present invention.

That is, the present invention has the promoter activity of the phosphoglysade kinase gene of Acremonium chrysogenum;
D containing at least a part of the base sequence shown in the following formula 1
NA fragment, Formula 1: %Formula% ■ A DNA fragment having the promoter activity of the Acremonium chrysogenum actin gene and containing at least a part of the base sequence shown in the following Formula 2, Formula 2: %Formula% CGGAGTTGCGTAATCGGCTGCTTCT
ATTTCAGATGGTGCGAGGGAGTACT
CCTACTCACGATCTTGAATCACAGG
AGGTCCCCATCAAAGCCACATGCCG
ACGTCGTTTACGAGACACGGTACAT
GGTACATCCGAAGACGGGACAGCAG
GAAGCACCTAAAGACGCTTCCCTC
GACATGGAAACACCCCATTGGGCCA
GGCGGCAAGGAGCAGGAGCAGGAGC
AGGCAGTTGCTTTCGATGATGCTCG
ATCTCGCGCCGAACCGTGATTAGGT
ACTGATGCCATCGGTGCCGGCCAGG
CTGGCACCGGCCTGCCTTGATGCGA
GATGCCTACTCGTACTATGCCTACA
GGTATGGGCTTTCCGCGTGTCGTCA
GCTTGCGACCGCGCGGCTGCTGACG
ACCCAAGGCAAGCTGGTAACATGGC
GGCACGAAATTTCTCTCTGCCTGCT
CGTCCTCTTGGTGTGGAGGGGTACG
AGTGCAGGTATGATGGGACGGCAGA
GGAGTGACGGAGGCTGTGCGGTTGG
CACGAGTACTGTACGAGTACTCGTA
CTGTAGGTGCAGCGACTGTGGTGGT
ACTGCTAGGTGGAATTGGGTCCAG
AGGCATGCAGCTCCCAGCCACCGTC
GTTAACCAAATCAGTTAAAGCAGCAA
CGCAAACCCGCCCCCGTTTTTCTGCC
AGAAAATTTGGGCGGTGTCGTCGTGCCC
CAGTCGTTGTTGCCCGCCCTTGTCT
GGTCGCCTACAAGGGCTGCACCACAG
GTAACAACAGCCCGCCCCAGGTCCT
TGTAGGTGCCCAGTGAGTGCCCGGT
GCCCACAAGTTTCTCGTAGGCATCC
ACTAGGCGGACTTGGAAGCCCATCA
GTGATGCTTCCCTCCTTTTCCCCCTC
CACATCTCACTCACGTCACGCAAGC
CAACCCTCTCTCTCCCCCCGTCTCCAT
TCCATCTTCTTTCTCTCCACGACCCT
TAAGAGTCCCTCCTGCTCACGTCGA
CCATCCTTCGCTCCCAGCCCACGA
CATCTGCATCGTCTGGGCTTCTTGA
CACTCTGTCATTTTCTTCCTTATAAAA
ACCTCTTTACCGCTCTTCCCGTAAT
CCGACGCC ■ A DNA fragment having promoter activity of the glyceraldehyde-3-phosphate dehydrogenase gene of Acremonium chrysogenum and containing at least a part of the base sequence shown in the following formula 3.

Formula 3: % Formula % The DNA fragment of the present invention was also isolated with a protein coding region separated by an intron and a <3' untranslated region following it. A part of this protein coding region is used as a component of an expression vector when expressing a desired gene as a fusion protein. Furthermore, it is generally believed that the 3' untranslated region contains signals, including terminators, for efficient transcription termination.
This is also used as a component of an expression vector.

Therefore, these areas are also included in the present invention. Figure 3.4 shows the specific nucleotide sequences containing the intron-intersected coding regions and 3' untranslated regions of the PGK gene, actin gene, and GAPD gene derived from Acremonium chrysogenum, together with the sequence of the promoter-containing fragment of the present invention.
．． 5. Furthermore, the present invention also provides a series of expression vectors for Acremonium chrysogenum, in which the promoter-containing fragment and terminator-containing fragment are integrated into plasmids in various ways.

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

(1) Chromosome D from Acremonium chrysogenum
Extract NA and use appropriate restriction enzyme (e.g. Mbo 1 etc.)
Cut with.

(2) Incorporate the DNA fragment obtained in (1) into a suitable phage vector (e.g. EMBL3, EMBL4, etc.) to obtain the chromosomal DNA of Acremonium chrysogenum.
Build A library.

(3) PGK genes, actin genes, and GAPDs from other species that have already been isolated and whose structures have been clarified
A DNA fragment containing at least a portion of the gene coding region is obtained.

(4) Label the DNA obtained in (3) with 32p and (
Plaque hybridization is performed with the chromosomal gene NA library obtained in 2), and plaques showing complementarity with the DNA probe are selected and separated.

(5) Extract DNA from the selected phage, perform Southern hybridization using the same probe, position the gene of interest on a restriction enzyme fragment of an appropriate size, and subclon it into a plasmid vector.

(6) By determining the base sequence of the DNA fragment subcloned in (5) and comparing it with the corresponding gene structure derived from other species, it is possible to confirm that the obtained gene is the desired gene and its exact Determine the code region. The promoter of the present invention is usually 5′ of the coding region.
It is present within the DNA fragment located upstream.

(7) By linking the DNA fragment containing the above 5' upstream region and a bacterial drug resistance marker gene in a suitable arrangement for expression and introducing it into Acremonium chrysogenum, a promoter is present on the DNA fragment of the present invention. Make sure there is. The general operations required for handling E. coli, phage, and DNA in the above steps are those commonly performed by those skilled in the art, and include, for example, Maniatis (Maniatis).
(T, Maniati) et al.
s et al, Molecular Cloni
g A Laboratory Manual, C
o1d Spring Harbor Labo
r-atory 1982.1989),
Easy to implement. The enzymes, reagents, and E. coli general-purpose vector DNA used are all commercially available products, and unless otherwise specified, the purpose can be fully achieved if the usage conditions specified by the product are followed. can.

In the above (1), strains such as Acremonium chrysogenum ATCC 11550 and Acremonium chrysogenum 15-5 can be used as the DNA extraction source.

Acremonium chrysogenum strain 15-5 has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry on February 5, 1990, under the deposit number No. 11232 of the Institute of Microbiology.

In addition, total DNA extraction from the bacterium can be performed using, for example, John's
Johnstone et at, E
MBO Journal (1985) 41307-131
1) method or the method of Minuth et al.
Current Genetic
s (1982) 5:227-231).

The PGK genes derived from other species in (3) and (6) above include, for example, Satucharomyces cerevisiae.
(Saccharomyces cerevisiae
) PGK gene [Hitzemau et al., The Jo
urral of Biological Chemistry
try (1980) 25.12073-120803
, CI (Itzeman et al. Nucleic Ac1
ds Research (1982) 10.7791-
78083.

Aspergillus nidulans (Aspergillus nidulans)
1us Nidulans) PGK gene [C1<em>
ents et al. Geue (1986) 4497-105
Examples include E. In addition, as an actin gene, for example, the actin gene C of Satucharomyces cerevisiae
Ga11w1tz et al., Nucleic Acids Re
5earch (1980) 8.1043-1055]
CGa11w1tz et al., Proc, Natl, Ac
ad, Sci, USA (1980) 77°25
46-2549), human β-actin gene [
NAKA-JIMA et al., Proc. Natl.
Acad Sci, (JSA (1985) 826
133-6137: ], etc. Also, G.A.P.
The D gene is a cDN encoding human GAPD.
A [(e.g., Hanauer et al. EMBOJ, (
19B4)3. 2627-2633:].

GAPD gene (H
Holland et al. J, Biol, Chem, (198
0)25525962605), the GAPD gene of Aspergillus nidulans [Punt et al.
988) 6949-573, etc. These genes can be obtained by methods described in the above-mentioned documents, or by synthesizing DNA oligomers corresponding to known base sequences and hybridization using the oligomers. The DNA oligomer can be synthesized using a commercially available DNA synthesizer according to its operating procedures. The method for determining the DNA base sequence in (6) is also the well-known dideoxy method [Sanger et al.
al,, Proc.

Nati, Acad, Sci, [JSA (1977
)73.5463) is used.

In (7) above, the bacterial-derived drug resistance marker gene includes the neomycin phosphotransferase gene [for example, Beck et al., Gene (
1982) 19327-336), hygromycin B phosphotransferase gene [e.g.
Zetal, Gene (1983) 25179-
188) etc. are used.

Transformation of Acremonium chrysogenum using these drug resistance marker genes can also be carried out according to known methods [for example, Queener et al., Micr.
biology 1985. American 5
ociety for Microbiology,
(1985) pp468-472]. It is known that when a promoter fragment derived from Acremonium chrysogenum is linked to the above marker gene in a configuration suitable for expression, it significantly increases the efficiency of transformation of Acremonium chrysogenum by the marker gene. C
3katrud et al., Current Genetics (
1987) 12:37-348:] Therefore, the present invention DN
Whether or not the A fragment contains a functional promoter can be determined by constructing a plasmid in which the fragment and the above marker gene are linked in an arrangement suitable for expression, and examining the frequency of transformation of Acremonium chrysogenum with the plasmid. It can be confirmed.

(Example) The abbreviations and symbols described in the examples are as follows.

CM medium: sucrose 20g/potassium dihydrogen phosphate 0.5
g/dipotassium hydrogen phosphate 0. ．． , 5 g / potassium chloride 0.5 g / magnesium sulfate (heptahydrate) 0.5 g /
Iron sulfate (IF) (heptahydrate) 0.01g/sodium nitrate 3g/yeast extract 4g/peptone 10
g dissolved in water 11.

CM solid medium: CM medium containing 1.5% agar.

GAG medium: 40 g glycerol/4 g asparagine/
Calcium chloride 0.1 g / Sodium chloride 0.1 g / Trace metal solution [Magnesium sulfate (7 hydrate) 4 g / Iron (II) sulfate (7 hydrate) 0.4 g / Manganese sulfate (tetrahydrate) 0.16 g / Zinc sulfate (heptahydrate) 0.4 g/anhydrous copper sulfate 0.04 g in water II! dissolved in] 25d1
0.1 M phosphate buffer y - (pH 7,0) 307
Medium containing rLl in water 11, 6XSSC: 0.9
M sodium chloride/90mM sodium citrate, 20
X 5SPE: Sodium chloride 210 g / Sodium dihydrogen phosphate (dihydrate) 31.2 g 10.5 M
EDTA (EDTA is ethylenediaminetetraacetic acid) 40
- dissolved in 11 ml of water, 50 x Denharz: 5 g of Ficoll/5 g of polyvinylpyrrolidone/5 g of bovine serum albumin dissolved in 5007 nl of water.

P-buffer: 0.6M potassium chloride10. O
IM, magnesium chloride 10.025M calcium chloride PEG solution: 25% polyethylene glycol (molecular weight 4000) 10.01M Tris (pH 8,0) 10
．． 05 Calcium chloride 10.6M Potassium chloride.

Example 1 [Isolation of Acremonium chrysogenum phosphoglycerate kinase (PGK) gene] (1) Creation of Acremonium chrysogenum gene library Johnston (1,L,
Johnstone et al. used the method for Aspergillus nidulans [Reference: Embojournal (EM
BOJ, ), 4.1307-1311.1985. )
Extracted according to.

Then, 60 μg of this total DNA was partially digested with the restriction enzyme MboI, and then treated with alkaline phosphatase. On the other hand, 10 Mg of lambda vector EMBL 3 (Promega Biotex) was completely digested with BamHI and EcoRI, and short EcoR
A portion of the I-BamHI linker was removed.

Next, about 1 μg of the partially digested DNA fragment and BamHI
Approximately 2 μg of the vector having the terminal end was ligated using T4 ligase and encapsulated into lambda phage particles. The recombinant phage suspension obtained in this way was appropriately diluted and added to E. coli (E. coli) NM539 (
Promegabiotic) and the number of plaques that appeared was counted. As a result, this suspension was found to contain 3 x 105 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 vector and the binding of the two were those described by Frischauf et al. [Journal of Molecular Biology (J, Mo1. Biol),
170.827-8421983] DNA was encapsulated into lambda particles using Promega Biotic's packaging extract according to the attached protocol.

(2) Preparation of probe pYPGK 1 (P derived from Satucharomyces cerevisiae)
This plasmid, which has a 2.9 Kb HindIII fragment containing the entire GK gene, was prepared by converting the HindIII digest of Satucharomyces total DNA into PBR327 (ATCC 375
16') into the HindIII site of the Satucharomyces PGK gene [Reference: Nucleic Acid Research (Nucleic A
c1ds Res, ), 1077917808, 1
Synthetic oligonucleotide designed based on 982):
5'-CAGATCATCAAGAAGTAATTAT
It was obtained by screening using CT-3'. ) 20 μg was digested with HindIII and EcoRI, and then subjected to 1% red gel electrophoresis. And Maniatis (T.) et al.
Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Publishing, 19
1982 (T., Maniatis et al., Mo.
1ecular Cloning A Lab.

ratory Manual, Co1d Sprin
g Harbor Laboratory 1982, hereinafter this belt will be abbreviated as Manuatis actual equipment. ) p16
2.9 Kb according to the method described in 4-165.
The fragment was recovered from the gel and purified. Approximately 200 ng of this fragment was then added to [α-32P]deoxycytidine triphosphate (dCTP) using a nick translation kit manufactured by Takara Shuzo Co., Ltd., according to the attached protocol.
) Labeled with 50 μCi. Then, the reaction solution was heated at 70°C for 1
After heating for 0 minutes, a Nick column manufactured by Pharmacia (P
harmacia, Nick-columnTIJ
) to obtain a probe with radioactivity of approximately 1107 cp. (Hereinafter, this probe will be referred to as the YP-probe.) (3) A portion of the phage solution (gene library) obtained in hybridization screening (1) was infected with NM 539, and spread on four plates. A total of 2×10′ plaques were formed. These plaques were collected using the method of Benton (W, D.) et al. [Reference: Science, 196.18
0-182.1977°], transferred to a nitrocellulose filter, denatured with alkali and neutralized to fix the DNA, and then hybridized with the YP-probe obtained in (2) above. Hybridization was carried out in 30% formamide, 5x Denharz,
5XSSPE.

0°1% SDS, and final concentration 5X10'cpm/7nl
1 at 42°C using a solution containing YP-probe at
I went for 6 hours. The filter was then soaked with 0.1% SDS.
Washed twice for 10 min each in 6X SSC solution containing
Washed at 2°C for 30 minutes. Autoradiography was then performed at -80°C for 16 hours using an intensifier screen. As a result, 7 positive spots were found. Among them, phages were extracted from the agar regions corresponding to the four positive spots, and plaque hybridization was performed again according to the above steps to obtain four purified positive phage clones. These clones were named λPGKI and 2.3.4, respectively.

(4) From the four phage clones obtained in (3) subcloning the PGK gene and limiting its position, the method described by Grossberger [Reference: Nucleic Acid Research (Nucleic Ac1d)
s, Re5search).

DNA was extracted according to [15, 6737]. Next, this lambda DNA was cut with BamHI and subjected to agarose gel electrophoresis, followed by Southern hybridization using a YP-probe.
Southern), Journal of Molecular Biology (J, Mo1. Biol,), 9
8,503-517゜1975]. Note that hybridization, filter washing, etc. were performed in the same manner as described in (3). As a result, it was found that only the approximately 5.5 Kb BamHI fragment present in all clones hybridized with the probe. This fragment is called SCENE-CLEAN (GENE-CLEAN).
Funakoshi Co., Ltd.) was used to recover and purify the product from agarose gel according to the attached protocol. On the other hand, pLlc18 (Takara Shuzo Co., Ltd.) used as a vector was cut with BamH and treated with alkaline phosphatase. Then, the two were ligated using T4-ligase, and E. col.
It was introduced into iJM 105. Ampicillin (Amp),
100μg/-25-bromo-4-chloro-3-indolyl-β-galactoside (X-Gal), 0.00
Six white colonies grown on L-broth agar medium containing 4% were selected, and plasmid DNA was extracted using a simple method (method described in Maniatis' experimental book, pages 368 to 369).
was extracted and analyzed by BamHI digestion.6
It was found that the target fragment was inserted into the plasmid of 5 clones. Furthermore, analysis by Southern hybridization was performed in the same manner as above, and it was confirmed that this insert was the desired fragment. One of these plasmids was named pGK5.

As a result of cutting the plasmid pGK5 with various restriction enzymes and analyzing it by agarose gel electrophoresis, a restriction enzyme cleavage map of the 5.5 Kb insert shown in FIG. 1 was obtained. Next, in order to limit the PGK coding region present in this fragment, Southern hybridization was performed using a YP-probe. As a result, approximately 0.7Kb
It was estimated that at least a part of the PGK coding region exists within the Pstl-3tul fragment of .

(5) Base sequence of Acremonium chrysogenum PGK gene First, a 0.7 Kb Pstl-5tul fragment, which was estimated to contain a part of the coding region of the PGK gene, was inserted into M13m.
SmaI-PstI of p18 and M13mp19
In between, a portion of each sequence was subcloned using the method of Sanger et al. (Sanger, F. Science 5
science 214.1981, etc.). By comparing this with known PGK genes, we estimated the direction of the gene and which part of the PGK protein this region encodes. Sequencing is performed upstream and downstream from the region, ultimately covering the entire region underlined in FIG. 1, 3306
The base sequence of bp was determined.

The specific experimental procedure for base sequencing was carried out using Takara's Sea Fencing Kit according to its attached protocol. The entire determined base sequence and predicted translation products are shown in FIG. Genetic information processing software (SD
As a result of computer analysis of this sequence using C Software Development Co., Ltd., the following was found, and through the above-mentioned steps, confirmation was obtained that the isolated gene was the true PGK gene. 1) The PGK gene of Acremonium chrysogenum encodes a protein consisting of 418 amino acids and having a molecular weight of 44,300 daltons. 2
) The coding region is separated by two introns consisting of 145.64 bp. Although the size of the intron was different, its location was the same as that of the PGK gene of Aspergillus nidulans, a filamentous fungus. 3)
The primary structure of amino acids predicted from the base sequence is human,
Satucharomyces cerevisiae.

It was very similar to that of PGK from Aspergillus nidulans, showing 68.70.75% homology, respectively. In addition, Bgff whose structure was determined as above
The in■-Kpnl fragment [Fig. 1 (A) region] is 1251b+ 5' upstream from the start codon (ATG) of the PGK gene.
), which is the object of the present invention.
The K promoter is believed to be present on the fragment.

Examples 2 and 3 shown below strongly support that this assumption is correct.

Example 2 Construction of CpPGKM 5] A plasmid for expressing the bacterial neomycin phosphotransferase gene (hereinafter abbreviated as KmR gene) under the control of the PGK promoter derived from Acremonium chrysogenum according to the steps shown in FIG. pPGKM5 was constructed. Each step will be explained below.

(1) Preparation of pGKBL Plamid pPGK-5 obtained in Example 1-(4) was transformed into Bgl
A 3.6 Kb fragment containing the PGK gene was isolated and purified. The fragment was prepared in Example 1 (4), pU
By inserting into the BamHI site of c18, pGKBL
I got it.

A plasmid in which the above fragment was inserted in the opposite direction to pGKBL was also obtained at the same time, and this was named pGKBL'.

(2) Preparation of pGKC3 The plasmid pGKBL obtained in (1) above was cut with Mlu I and Xho I, and a 4.8 kb fragment was isolated and purified.

pGKC3 was obtained by ligating this fragment with a synthetic linker represented by the following formula. Formula: %Formula% pGKC3' was also constructed by performing the same procedure as above using pGKBL' instead of pGKBL. pcKcs and p
GKC3' is PG derived from Acremonium chrysogenum
This is a plasmid with a structure in which a fragment containing a K promoter and a terminator is connected via Unique restriction enzyme sites (BglII, XhoI) in an arrangement suitable for expression. It is a useful starting material when constructing vectors for expression. The above linker is a DNA synthesizer model 380 manufactured by Applied Biosystems.
-A was synthesized as two single strands using a conventional method.

(3) Construction of pPGKM5 Plasmid pEXOO2 is cut with BamHI and BgAII, and a 1.5 kb fragment containing the KmR gene is separated and purified.

This fragment was cut with BgffII, treated with alkaline phosphatase, and ligated to pGKC8 in the direction shown in FIG. 7 to obtain pPGKM5. The plasmid pEXOO2 used above is an expression vector for Escherichia coli having the 1acU■5 promoter and the KmR gene derived from transposon 5 (Ta2), and its construction method is described in JP-A No. 63-74488. .

In Example 2, the restriction enzyme-digested fragments were all separated by 1% agarose gel electrophoresis, and the DNA fragments were prepared from the agarose gel using Sheen Clean (G
The test was carried out using ENE-CLEANTM (manufactured by Funakoshi) according to the attached protocol. Also,
Basic operations such as ligation of plasmids and DNA fragments, transformation of E. coli, and preparation and analysis of plasmids obtained as a result of subcloning were all performed in accordance with the methods described in the actual equipment of Maniac Nice.

Example 3 [Transformation of Acremonium chrysogenum using pPGKM5] (1) Preparation of protoplasts
Acremonium chrysogenum l5 grown for C5 days
-5 mycelia (mycelia corresponding to about 1 crj) were added to C
M medium 501d was inoculated, and a rotary shaker (250r,
p, m) and cultured at 30°C for 3 days. Furthermore, the bacterial liquid is 1m7! was inoculated into 50-0 GAG medium and cultured on a rotary shaker (25 Or, p, m) at 30°C for 20 hours. Obtained culture solution 5077!7! 350Or, p
, m for 10 minutes to precipitate mycelia, and then centrifuge at 0.0 m.
9% NaC! ! McIlvain containing 0.01M dithiothreitol was washed with a solution of
ne) Suspended in buffer solution (0.1M citric acid, 0.2M sodium phosphate, pH 7.3) 20- and gently shaken at 30°C for 1 hour.

The mycelium was then pelleted by centrifugation at 3200 r, p, m for 10 minutes, washed with P-buffer, and then treated with Novozyme (
Novo) at a concentration of 1 On+g/m/
-Buffer 1077!7! and gently shaken at 30°C for 1 hour. The following reaction was carried out at 80 Or, p, m for 3
The supernatant obtained by centrifugation for 0 seconds was filtered using filter paper (TOYOFILTE).
Mycelia and protoplasts were dispersed by filtration using RPAPER5A). Then, the filtrate was heated to 300O
After centrifugation at r, p, m for 5 minutes to precipitate protoplasts, they were washed once with P-buffer and suspended in P-buffer to a concentration of 3 x 101l/7nl of protoplasts.

(2) Protoplast transformation with pPGKM5 After adding 5 μg (10 μl) of plasmid pPGKM5 to 0.1 μl of the protoplast suspension obtained in (1) above,
0.057 nl of PEG solution was added and mixed briefly.

After standing on water for 25 minutes, the same PEG solution was added thereto, and the mixture was left standing at room temperature for an additional 30 minutes. The thus obtained transformed protoplast suspension was added to a protoplast regeneration medium [Literature (lsogai et al.: Ag
ric, Bio1. Chem, 1987.51.2
321-2329) was spread on a plate containing 25d and cultured at 15°C for 20 hours, the same BRM medium 5- containing 3mg of G418 and kept at 50°C was overlaid. . and 10 at 28℃
By culturing for ~20 days, a transformed strain that became G418 resistant was selected. After conducting the above experiment several times, it was found that 50 to 150 04 cells per 5.5 μg of pPGKM.
18 resistant strains have emerged. On the other hand, when the same experiment was conducted using the control plasmid pEXOO2,
Only 0-2 0418-resistant strains appeared. The above results strongly suggest that the promoter of the Acremonium chrysogenum PGK gene is contained within the Xbal-Bg11I fragment on pPGKMS, which has the sequence shown in formula 1 above. The insertion of the plasmid used for introduction into the chromosome of the above-mentioned resistant strain was confirmed by a Southern hybridization experiment using the Pstl fragment (approximately 900 bp) of pEXOO2, which contains most of the KmR gene coding region, as a probe. It was done.

Example 4 [Actin gene cloning] (1) Screening for actin gene-containing clones by hybridization A Hinf I 40Qbp fragment (Wako Pure Chemical Industries, Ltd.) containing the third exon of the human β-actin gene labeled with 32P was probed ( The Acremonium chrysogenum gene library prepared in Example 1-(1) was screened under the same conditions as Example 1-(3), and the four probes that hybridized with the probe were screened under the same conditions as Example 1-(3). obtained phages.

(2) DNA was extracted from one of the phages obtained by subcloning the actin gene and limiting its position (1), and named it λACT5. Next, λACT5 was digested with XhoI and 5affI, and agarose gel electrophoresis was performed, followed by Southern hybridization using the above ACT probe.

As a result, it was found that a 5.4 Kb Xho I fragment and two types of 5aβ I fragments having sizes of 1.3 Kb and 1.5 Kb hybridized with the probe. Therefore, these three types of fragments (Xho I-5, 4 fragment, Sad
Fragment at I-1,5, Sa I! pACT5X was obtained by subcloning the fragments I-1 and 3) into the sa and gi sites of pLlc18, respectively.

pACT5SS and pACT5SL were obtained. Next, partial restriction enzyme maps of these plasmids were prepared, and by overlapping them, a partial restriction enzyme map of a DNA fragment of approximately 6 Kb thought to contain the actin gene was prepared as shown in FIG. 2.

The above Southern hybridization was performed under the same conditions as in Example 1-(4) except for the use of the ACT probe.

(3) Base sequence determination and decoding From the results of (2) above, 0.7kb Small-Xho
Within the L fragment, at least a portion of the actin coding region (
Since it was strongly suggested that a portion corresponding to the third exon of human β-actin exists, the nucleotide sequence of this portion was first determined. By comparing this with known actin genes, we estimated the direction of the gene, the presence or absence of introns, and which part of the actin protein this region codes for. Next, the sequence was determined upstream and downstream from the region, and the final result is shown in Figure 2 underlined (B).
A 3748 bp base sequence covering the entire region shown was determined. The entire determined base sequence and predicted translation products are shown in FIG. As a result of comparative analysis of the nucleic acid sequence and the amino acid sequence of the translation product with those of known actin,
The following was found, and through the above steps, it was confirmed that the isolated gene was a true actin gene.

1) The actin gene of Acremonium chrysogenum encodes a protein consisting of 375 amino acids and a molecular weight of 41,800 daltons. This number of residues is the same as in all other actins, except for skeletal muscle actin (α-type) in mammals.

2) The amino acid sequence predicted from the base sequence was extremely similar to that of known actin, and showed 92% and 90% homology with Satucharomyces cerevisiae actin and human γ-type actin, respectively. The N5iI-3aβI-fragment (Fig. 2), which was isolated and whose structure was determined as described above, covers a region extending 1293 bp 5' upstream from the start codon of the actin gene, and is the target of the present invention, the actin promoter. is believed to be present on the fragment. Examples 5 and 6 shown below strongly support that the above estimation is correct.

Example 5 (Construction of pAcTt(Y83)) According to the steps shown in FIG.
A plasmid pACTHY83 was constructed to express the phosphotransferase gene (hereinafter abbreviated as HYBR gene) under the control of the actin promoter derived from Acremonium chrysogenum. Each step will be explained below.

(1) Preparation of plasmid pACT5X obtained in Example 4-(2) into pACT8NP
A 5.3 Kb fragment was prepared by simultaneous digestion with il and Pstl.

The fragments were then religated (self-circling) using T4 ligase to obtain pACTΔNP.

(2) pAcTBl, pACTB2. , pACTB
The pACTΔNP obtained in 3. Preparation (1) was first incubated with Neo I
The resulting sticky ends were digested with DNA polymerase Klenow fragment (hereinafter abbreviated as DNApoC) and four types of deoxynucleotide triphosphates (deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, and thymidine). Triphosphate (hereinafter abbreviated as 4dNTPS) is used to convert the ends to blunt ends. The 5' end is then phosphorylated, and T is added to the end of BamHI, which consists of the following sequence.
After ligation with 4 ligase, it was digested with BamHI. The digested product was subjected to agarose gel electrophoresis, and a 4Kb DN
The A fragment was isolated and purified. The fragment was then self-circularly closed using T4 ligase to obtain pACTB1. Furthermore, the same operation as above was performed with a different sequence and number of bases from the above linker, and pACTB2. pACTB3 was obtained.

(3) pAcTcsl, pACTC32, pACTC
33 Preparation Example 4-Plasmid pACT obtained in (2)
SS was digested with BamHl and DNApoA, and 4
After the ends were blunted using dNTPS, the plasmid pACTSSΔBam, which had lost the Bam81 site, was obtained by self-circulating with T4 ligase. The plasmid was then treated with 5caI and Ec.

After digestion with R1, a 0.9 Kb fragment thought to contain the terminator of the actin gene was isolated and purified.

The fragment was then transformed into Sma I-EcoR of pAcTBl.
pACTC81 was obtained by inserting between I and pACTC81. In addition, the 0.9 Kb fragment from the above was inserted into pACTB2. pACTB3
By inserting between SmaI-EcoRI of
pACTC32 and pACTC33 were obtained, respectively.

These three plasmids are suitable for expression using a fragment containing the actin promoter and terminator derived from Acremonium chrysogenum via the unique restriction enzyme cleavage site Bam) II (located immediately downstream of the actin initiation codon ATG). It is a plasmid with a connected structure and is a useful starting material when constructing vectors for expressing various genes of interest as fusion proteins in Acremonium chrysogenum. By using any of these three types of plasmids, it becomes possible to link a desired gene in the same reading frame as the actin gene.

(4) Preparation of pACTHY83 Plasmid pLG83 (obtained from Professor Julian Davies, Bastour Institute) was cut with BamH, and 1.
A 3 Kb fragment was isolated and purified. The fragment was digested with BamH and treated with alkaline phosphatase to create pAcTcs.
By binding with l in the direction shown in Figure 8, pACT
HY83 was obtained. The above pLG83 is a vector for yeast having the HYB'' gene, and its properties are described in the known literature [Gritz et al., Gene (1983) 25
．． 179-188]. Example 5
All basic operations such as separation and purification of the restriction enzyme-digested fragment, ligation of the plasmid and the fragment, transformation of E. coli, and preparation and analysis of the plasmid obtained as a result of subcloning were performed in the same manner as in Example 2.

Example 6 Transformation of Acremonium chrysogenum with pACTHT83 Using the aforementioned pACTHY83 and pLG83, a transformation experiment of Acremonium chrysogenum strain 15-5 was carried out. When 5 μg of pACTHY83 was used, 40-1
In contrast, when pLG83 was used, no transformants appeared at all when pLG83 was used. This means that the old ndII[-Bam
This strongly suggests that the Acremonium chrysogenum actin gene promoter is contained within the HI■ fragment (Fig. 8). In the above experiment, protoplast preparation of Acremonium chrysogenum 15-5 strain was carried out in the same manner as in Example 3-(1), except that 4.5 mg hygromycin B was used instead of G418 for protoplast transformation. , were carried out in the same manner as in Example 3-(2).

Example 7 [Cloning of GAPD gene] (1)
Screening of GAPD gene-containing clones by hybridization Hin derived from human GAPD-cDNA labeled with 32p
cIIACCI about 1. IKb fragment [human GAPD-c
The DNA clone was an oligonucleotide 5' CCTGGCCA synthesized based on the base sequence of human GAPD cDNA already reported by Hanauer et al.
A human lung-derived λgtllc-DNA library (C1
ontech Laboratories, Inc.
) was obtained by screening according to conventional methods. ] as a probe (hereinafter abbreviated as GAP-probe), the Acresonium chrysogenum gene library prepared in Example 1-(1) was screened under the same conditions as Example 1-(3), and the probe We obtained four phage clones that hybridized with.

(2) Subcloning of GAPD gene and restriction of its position DNA was extracted from the four types of phages obtained in (1) above, and λGAPI, λGAP2. λGA
P3. It was named λGAP4. These four types of λ DNA were then simultaneously digested with BamHl and Pstl, subjected to agarose gel electrophoresis, and then Southern hybridization was performed using the above GAP probe. As a result, approximately 3.7 Kb exists in all the above four types of λ-DNA.
It was revealed that only the old Pstl-Bam fragment of Pstl-Bam hybridized with the probe. Therefore, the DNA fragment was subcloned between Pstl and Bam of pUc18 to obtain pGAPl.

The plasmid was then digested with various restriction enzymes and analyzed by agarose gel electrophoresis.
A partial restriction enzyme map of the A fragment was prepared as shown in FIG. Furthermore, in order to limit the GAPD coding region present in this fragment, Southern hybridization was performed using a GAP probe, and an approximately 2.7 Kb KpnI-
It was estimated that the entire GAPD coding region was present in the Xhol fragment.

(3) Determination and deciphering of base sequence The base sequence of 2659 bp between Kpn I and Xho 1 was determined according to a conventional method. The entire base sequence and predicted translation products are shown in FIG. Comparative analysis of the nucleic acid sequence and the amino acid sequence of the translation product with those of known GAPD revealed the following, and through the above steps, it was confirmed that the isolated gene was a true GAPD gene.

(1) The GAPD gene derived from Acremonium chrysogenum consists of 337 amino acids and has a molecular weight of 36,200.
Codes for the Dalton protein.

(2) The coding region is separated by two introns of 217 bp and 65 bp, and their positions are completely identical to the positions of the 5th intron and 6th intron, respectively, present in the Aspergillus 2dulans gene. I was doing it. It was also estimated that an intron exists within the 5' untranslated region, similar to the GAPD gene of Aspergillus nidulans.

(3) The amino acid sequence of the translation product predicted from the base sequence is very similar to known GAPD, and includes, for example, human, Saccharomyces cerevisiae, Aspergillus
GAPD derived from Nidulans and 66% and 6, respectively.
4% and 77% homology. Furthermore, 20 residues thought to be involved in the formation of the active site of GAPD were completely conserved in Acremonium chrysogenum GAPD. The PstJ-Bam old fragment (approximately 3.7 Kb) on pGAPl isolated as described above contains a region extending approximately 1.7 Kb 5 upstream from the start codon of the GAPD gene, and is not suitable for the purpose of the present invention. It is thought that the GAPD promoter exists on this fragment. The examples shown below strongly support that this assumption is correct.

Example 8 Plasmid pEG-APHY for expressing the HYB" gene under the control of the GAPD promoter derived from Acremonium chrysogenum according to the steps shown in FIG.
83 was constructed. Each step will be explained below.

(1) Preparation of pGAPΔ series First, pGAPI obtained in Example 7 was digested with EcoRV and then treated with nuclease Ba731 to generate a 100 to 200 bp deletion from the EcoRV site to the inside of the DNA. Then, the end digested with Baff31 was
After repairing the ends to blunt ends using NApol and 4 dNTPS, BglI was added using T4 ligase.
After rinsing with I and digesting the reaction product with BgffiII, agarose gel electrophoresis was performed to separate and purify the DNA fragments, self-circulation was performed with T4 ligase, and E. coli was transformed with the reaction solution. Eight transformed (ampicillin resistant) colonies obtained in this way were randomly selected, the plasmid possessed by each was extracted, and each pGAPΔ1
It was named ~Δ8. Then, these eight plasmids were
Each was simultaneously digested with Bgj2II and 5acl, and the resulting fragments of 200 to 400 bp were separated and purified. The fragments were subcloned between BamHISacI of M13mp18 and their nucleotide sequences were determined. As a result, in each plasmid, from the EcoRV site to the GAP
The deletion point on the 5' side of the D gene was revealed as shown in Figure 1O.

(2) pEGAP3. pEGAP5 and pEGAP8
Preparation of pGAPΔ3 obtained in 1 above was combined with BgA'II and Ps
A fragment of approximately 1.6 Kb was prepared by digestion with tl. On the other hand, pGKC3 obtained from the height layer side 2-(2) is mixed with BgffII and P
stl digestion, and a 3.6 Kb fragment was isolated and purified.

These two fragments are then ligated with T4 ligase to create p
Obtained EGAP3. Also, instead of pGAPΔ3, pGA
PΔ5. By performing the same operation using pGAPΔ8, pEGAP5. pEGAP 8 was produced respectively. These three plasmids are the GAPD promoter derived from Acremonium chrysogenum, the PGK
The fragment containing the terminator is the Unique restriction enzyme cleavage site B.
g (! II (several ~ from GAPD start codon ATG
It is located in the T stream of several tens of bp. ), it is a plasmid with a structure that is connected in a configuration suitable for expression, and it is a useful starting material when constructing vectors to express various target genes as fusion proteins in Acremonium chrysogenum. . By using any of these three types of plasmids, it becomes possible to link a desired gene in the same reading frame as the GAPD gene.

(3) Preparation of pEGAP83 Example 5-1 containing the HYBR gene prepared in (4)
, 3 Kb BamHI fragment was added to the B of pEGAP3 above.
pEGAP83 was obtained by inserting into the gj711 site in the direction shown in FIG. In Example 8, all basic operations such as separation and purification of restriction enzyme-digested fragments, ligation of plasmids and the fragments, transformation of E. coli, and preparation and analysis of plasmids obtained as a result of subcloning are the same as in Example 2. I did the same.

Example 9 Transformation of Acremonium chrysogenum with pEGAP83 Using pEGAP83 obtained in Example 8 and pLG83 as a control, Acremonium chrysogenum was transformed with pEGAP83 and pLG83 as a control.
A transformation experiment was conducted on strain 5-5. As a result, pEGA
When 5 μg of P83 was used, 20 to 80 transformants were obtained. On the other hand, when pLG83 was used, no transformants appeared at all, as in the previous case. This means that Bgl on pEGAPa containing the sequence shown in claim 3.
This strongly suggests that the II-Pstl fragment (approximately 1.6 Kb) contains the promoter of the Acremonium chrysogenum GAPD gene.

(Effects of the Invention) By using the promoter of the present invention, it becomes possible to efficiently express various genes in Acremonium chrysogenum.

Furthermore, by efficiently expressing various genes involved in cephalosporin fermentation in Acremonium chrysogenum under the control of the promoter of the present invention, it is expected that the fermentation yield of cephalosporins will be improved. Furthermore, as detailed in the Examples, by using the promoter of the present invention, it becomes possible to construct an efficient transformation vector for Acremonium chrysogenum.

[Brief explanation of drawings]

FIG. 1 shows a restriction enzyme map of a DNA fragment containing the Acremonium chrysogenum PG' gene. Figure 2 shows DNA containing the Acremonium chrysogenum actin gene.
A restriction enzyme map of the fragment is shown. Figure 3 shows Acremonium.
A restriction enzyme map of a DNA fragment containing the Chrysogenum GAPD gene is shown. ■ in Figures 1, 2 and 3 indicate exons of each gene. In each gene, the 5' end of the first exon,
and the 3' end of the final exon is unknown. FIG. 4 shows the chlorine arrangement in the region (A) indicated by ← in FIG. Furthermore, the predicted amino acid sequence of the PGK protein is shown below the base sequence. Figure 5 is indicated by H in Figure 2 (B
) shows the base sequence of the region. The predicted amino acid sequence of actin protein is also shown below the base sequence. FIG. 6 shows the base sequence of the region (C) indicated by ← in FIG. Furthermore, the predicted amino acid sequence of GAPD protein is shown below the base sequence. FIG. 7 shows the production process of plasmid pPGKM5. FIG. 8 shows the process of creating plasmid pACTHY83. FIG. 9 shows the production process of pEGAP83. Figure 10 shows Eco in the pGAPΔ series.
The deletion point from the RV site to the 5' side of the GAPD gene (8g
nn linker) indicates the attachment point. The new abbreviations and symbols used in the figures are as follows. B:BamHI/Bg:BgA II/B -B:B
Binding site between amHI and Bgj211/P: PvuI
[/Ps:Pstl/M:MIuI/ni:Kpnl/S
:SmaI/Sa:Sa,j I/St:5tuI/S
c:5caI/X:Xhol/Xb:Xbal/NN5
iI/Nc:Ncol/E:EcoRI/R¥:Ec
oRV/H:1(ind[I/Bs:Bs5HII/P
s-Ns: Pstl and N5iI binding site/2uor
i: Origin of replication of yeast 2μ plasmid/CYCP: Promoter of yeast iso-1-cytochrome C gene/CY
CT: Terminator of yeast iso I-cytoflore C gene/PGKP: Promoter of Acremonium chrysogenum PGK gene/PGKT: Terminator of Acremonium chrysogenum PGK gene/AC
TP: Acremonium chrysogenum actin gene promoter/ACTT: Acremonium chrysogenum actin gene terminator/GAPDP:
Acremonium chrysogenum GAPD gene promoter/L: Bgffi U l1nker (GG
AAGATCTTCC) insertion site/■~■: pGAP
Δ1 to Δ8

Claims (1)

[Claims] 1) Acremonium chrysogenum (A. chryso
A DNA fragment containing the promoter active portion of the phosphoglycerate kinase gene of ge-num) and having at least part of the base sequence shown in formula 1 below.Formula 1: [There is a gene sequence. ] 2) Acremonium chrysogenum (A. chryso
A DNA fragment containing the promoter active portion of the actin gene (Ge-num) and having at least part of the base sequence shown in Formula 2 below.Formula 2: [There is a gene sequence. ] 3) Acremonium chrysogenum (A. chryso
DNA fragment Formula 3: [There is a gene sequence. ]