JPH0767649A - Gene of homoserin o-acetyltransferase - Google Patents

Abstract

PURPOSE:To obtain a new DNA fragment useful in improvement of cephalosporin C-producing ability of Acremonium chrysogenum. CONSTITUTION:A DNA fragment containing a homoserin O-acetyltransferase gene derived from Acremonium chrysogenum and coding e.g. a protein having 1864 bp length and having an amino acid sequence a part of which is expressed by the formula. This gene is obtained by extracting a chromosomal DNA from Acremonium chrysogenum IS-5 strain (FERM P-11232), etc., partially decomposing the DNA with MboI, etc., integrating the DNA fragment into a vector to build a chromosomal DNA library and labelling DCPC-ATF gene and screening the gene, extracting the DNA from the selected clone and treating the DNA according to ordinary method.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a DNA fragment derived from Acremonium chrysogenum and containing a homoserine o-acetyltransferase gene which is one of methionine biosynthetic enzymes.

[0002]

2. Description of the Related Art Cephalosporin C, which is a starting material for clinically important cephalosporin antibiotics, is produced by a fermentation method using a filamentous fungus, Acremonium chrysogenum. Conventionally, the technique used to improve the cephalosporin C production ability of the bacterium was mutagenesis followed by random screening of high-producing strains. However, in recent years, development of a transformation system using the bacterium as a host (see, for example, Queener et al., Microbio).
logic 1985. American Society
for Microbiology, (1985) 4
68-472) and cephalosporin C biosynthetic enzyme genes have been cloned one after another, opening the way for genetically engineered approaches to breeding good strains (eg, Skatrud et al., BIO / TECHNOLOGY.
(1989) 7, 477). Cephalosporin C is 5 types of enzymes in Acremonium chrysogenum starting from 3 kinds of amino acids (L-α-aminoadipic acid, which is an intermediate of biosynthesis of L-cysteine, L-valine, and L-lysine). It is known that it is biosynthesized through a 6-step reaction by (for example, Martin et al., T
reds in Biotechnology (19
85) 3: 39-44). To date, genes encoding four of these biosynthetic enzymes have been cloned from Acremonium chrysogenum (see, for example, Samon et al., Nature (1985) 3
18, 191, Samsung et al., BIO / TECHNO
LOGY (1987) 5, 1207, Gutierre
s et al., Journal of Bacteria
y (1991) 173, 2354-2365, EP04.
50758), which is being applied to molecular breeding. On the other hand, it has been conventionally known that Acremonium chrysogenum exhibits higher productivity when methionine is used as a source of sulfur existing in the cephalosporin C skeleton than inorganic sulfur such as sulfuric acid. (For example, Treichl
er et al. (1979) Genetics o
f industrial microorganis
ms. Proceedings of the Thi
rd International Symposium
m on Genetics of Industry
al Microorganisms. America
n Society for Microbiology
y, Washington, D.Y. C. , 97-10
4). Furthermore, it has been reported that addition of methionine to the medium not only causes an effect as a good source of sulfur (cysteine) but also activates the cephalosporin C biosynthetic enzyme group (for example, Sawada et al. a
l. FEMS Microbiol Lett (198
0) 9, 281-284). Therefore, it is considered that the genes involved in methionine biosynthesis provide a useful means for molecular breeding of cephalosporin C-producing bacteria, like the above-mentioned cephalosporin C biosynthesis genes (for example, improvement in productivity, methionine It is considered that low-cost inorganic sulfur can be effectively used for creating strains that can be used similarly to methionine). S. cerevisiae (hereinafter simply referred to as yeast), N. Similar to crassa and the like, methionine is also normally used in Acremonium chrysogenum in two pathways (one is the pathway for producing homocysteine by sulfurization of o-acetylhomoserine with hydrogen sulfide, and the other is It is known to be biosynthesized by the methylation reaction of homocysteine supplied by a so-called sulfur transfer pathway from cysteine to homocysteine via cystathionine. By the way, homoserine o-acetyltransferase, which is one of methionine biosynthetic enzymes, is acetyl-C.
A substance that transfers the acetyl group of oA to homoserine and is essential to both of the above two types of homocysteine supply pathways o-
It produces acetylhomoserine. Therefore, this enzyme plays a central role in methionine biosynthesis and is considered to be an effective target for strain improvement by the above-mentioned molecular breeding. However, the gene of this enzyme has not been isolated and identified from Acremonium chrysogenum.

[0003]

DISCLOSURE OF THE INVENTION The present invention improves homologues of Acremonium chrysogenum by isolating the gene for homoserine o-acetyltransferase, which is derived from Acremonium chrysogenum and is one of the enzymes involved in methionine biosynthesis. In addition, the purpose is to use this.

[0004]

The present inventors isolated a DNA fragment containing a homoserine o-acetyltransferase gene from an Acremonium chrysogenum gene library, and partially isolated its nucleotide sequence (the entire coding region and Part of the non-coding region) determined. Furthermore, they have found that the corresponding c-DNA restores the methionine auxotrophy of homoserine o-acetyltransferase-deficient yeast and completed the present invention. That is, the present invention provides (1) a DNA fragment containing a homoserine o-acetyltransferase gene derived from Acremonium chrysogenum. (2) D according to (1), which contains a gene encoding a protein having the amino acid sequence shown in the sequence listing (SEQ ID NO: 1)
NA fragment. (3) The DNA fragment according to (1) or (2) defined by the restriction enzyme map shown in FIG. (4) The nucleotide sequence between BamHI and BalI in FIG. 1 is shown in the sequence listing (SEQ ID NO: 2) (1), (2)
Alternatively, it relates to the DNA fragment described in (3). In addition, the DNA fragment of the present invention is a suitable vector (for example, p
ACTHY83, pPGK5 (JP-A-4-58891)
It can be introduced into Acremonium chrysogenum by ligation to Acremonium chrysogenum transformation vector). As a result, it is possible to create a bacterium in which the enzyme activity is enhanced or suppressed. Therefore,
The present invention also includes Acremonium chrysogenum transformed with a recombinant DNA that carries all or part of the DNA fragment of the present invention. In the course of this study, a cDNA encoding a protein having the amino acid sequence shown in the sequence listing (SEQ ID NO: 1) was obtained by using a part of the DNA fragment of the present invention as a probe. One of the nucleotide sequences is shown in the sequence listing (SEQ ID NO: 1). These cd
A NA fragment also functions as a promoter fragment in Acremonium chrysogenum (for example, JP-A-4-58891).
It is considered that it can be used for the same purpose as the chromosomal DNA fragment by ligating with Therefore, a group of cDNA compounds derived from RNA transcribed from a chromosomal DNA fragment, such as the DNA shown in the sequence listing (SEQ ID NO: 1), is also included in the present invention.

The DNA fragment of the present invention can be constructed by the following steps. (1) Chromosomal DNA from Acremonium chrysogenum
Is extracted and partially digested with an appropriate restriction enzyme (eg, MboI etc.). (2) The DNA fragment obtained in (1) is incorporated into an appropriate vector to obtain an Acremonium chrysogenum chromosome DN.
Build A library. (3) The deacetylcephalosporin C acetyltransferase gene from Acremonium chrysogenum that has been isolated and has a clear structure (hereinafter DCPC)
-Abbreviated as ATF gene) or a DNA fragment containing at least a part of the coding region of the homoserine o-acetyltransferase gene derived from another species of organism. (4) Label the DNA obtained in (3) and hybridize with the library obtained in (2),
A clone showing homology with the probe is selected and separated. (5) Extract DNA from the clone selected in (4),
Southern hybridization is carried out using the above probe to locate the gene of interest on a restriction enzyme fragment of an appropriate size and subclone it into a plasmid vector. (6) The nucleotide sequence of the thus obtained DNA fragment is determined, and homoserine o- is obtained by comparing with a corresponding gene structure derived from another species organism and the amino acid sequence translated therefrom.
The presence of the acetyltransferase gene is confirmed and the coding region is located.

In the above steps, general operations necessary for handling DNA, Escherichia coli as a recombinant host, etc. are commonly performed by those skilled in the art, for example, Maniat.
is et al.'s experimental manual (T. Maniatis et a
l. , Molecular Cloning A Lab
oratory Manual, Cold Sprin
g Harbor Laboratory 1982, 1
989) and can be easily implemented. In addition, general operations necessary for handling yeast as a recombinant host include, for example,
Sherman et al.'S experiment book (F. Sherman et al.
al. , Laboratory Course Ma
natural for Methods in Yeast
Genetics. Cold Spring Harb
orLaboratory, Cold Spring
It can be easily carried out according to Harbor, NY 1986). Commercially available products can be used for all the enzymes and reagents used, and unless otherwise specified, these objects can be completely achieved according to the use conditions specified for the product. In the above (1), the DNA extraction source is Acremonium chrysogenum ATCC1155.
0, strains such as Acremonium chrysogenum IS-5 can be used. In addition, Acremonium chrysogenum IS
The -5 strain has been deposited on February 5, 1990, at the Institute for Microbial Technology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, with the deposit number of Micromachine Research Institute No. 11232. Also, the total DNA from the bacterium
Extraction can be performed, for example, by Johnston et al. (Johnst
one et al. , EMBO Journal (1
985) 4 1307-1311), or
Minuth et al., Cur.
rent Genetics (1982) 5: 227-.
It can be performed according to the method of 231). Above (2)
Examples of the vector include, for example, EMBL3 and EM.
Lambda phage vector such as BL4 (STRATAGENE) or pHC79 (Bethesda)
Research Laboratories <BRL>
Cosmid vector) can be used. Examples of the DCPC-ATF gene in the above (3) include, for example, a gene derived from Acremonium chrysogenum IS-5 (Matsuda et al., Biochem. B).
iophys. Res. Commun. (1992) 1
86, 40-46) or its cDNA (Matsu
da et al. , Biochem. Biophy
s. Commun. (1992) 182,995-10
11). In addition, homoserine derived from other species
Examples of the acetyltransferase gene include the MET2 gene derived from yeast (S. cerevisiae) (Langin et al., Gene (1986).
49, 283-293). These genes are synthesized by the method described in the above literature or by synthesizing a DNA oligomer corresponding to the already revealed nucleotide sequence,
It can be easily obtained by screening a DNA library or a cDNA library using the oligomer as a probe. In addition, chromosomal DNA by PCR using two kinds of primers corresponding to both ends of the gene, or m-R by RTPCR method.
It is also possible to clone directly from NA. (6)
As the amino acid sequence of the known homoserine o-acetyltransferase in the above, a sequence derived from the above yeast or a sequence derived from Ascobolus immersus (Goyon et al., Gene (1988) 6
3,279-308) and the like can be used. Further, as another method for confirming the presence of the homoserine o-acetyltransferase gene, a complementation test using the gene-deficient yeast described in Examples described later, or destruction of the gene in Acremonium chrysogenum ( For example, Matsuda et al., Biochem.
Biophys. Commun. (1992) 186.
40-46).

Hereinafter, the present invention will be described in detail with reference to Examples.
The present invention is not limited to the examples.
The abbreviations and abbreviations used in Examples and Reference Examples are as follows. Maniatis Experiment Book: (T. Maniatis et
al. , Molecular Cloning A
Laboratory Manual, Cold Sp
ring Harbor Laboratory 19
82) N-3 seed medium: Corn steep liquor 40 g / beet 20 g / ammonium acetate 2 g / sitose 40 g
Dissolved in 1 liter of water. Main medium: beet 30 g / defatted soybean 40 g / corn steep liquor 10 g / ammonium acetate 5 g / ammonium sulfate 7 g / calcium sulfate 8 g / calcium carbonate 15 g / sitose 60 g / methyl oleate 41.5 in 1 liter of water. CM medium: sucrose 20 g / potassium dihydrogen phosphate 0.5
g / dipotassium hydrogen phosphate 0.5 g / potassium chloride 0.
5 g / magnesium sulfate (heptahydrate) 0.5 g / iron (II) sulfate (heptahydrate) 0.01 g / sodium nitrate 3 g /
4 g of yeast extract / 10 g of peptone dissolved in 1 liter of water. CM solid medium: CM medium containing 1.5% agar SC-Trp medium: yeast nitrogen base lacking amino acids (BACTOYEAST NITROGEN
BASE w / o AMINO ACIDS: DIFC
O) 6.7 g / glucose 20 g / adenine sulfate 0.0
2 g / methionine 0.02 g / lysine hydrochloride 0.03 g
/ Phenylalanine 0.05g / Agar (BACTO-A
GAR: DIFCO) 20 g is dissolved in 1 liter of water,
Prepared solid medium. SC-Trp-Met medium: A medium obtained by removing methionine from the above SC-Trp medium. 6 × SSC: 0.9 M sodium chloride / 90 mM sodium citrate. 20 × SSPE: Sodium chloride 210 g / sodium dihydrogen phosphate (dihydrate) 31.2 g / 0.5 M EDT
A 40 ml dissolved in 1 liter of water. 50 × Denhartz: Ficoll 5 g / polyvinylpyrrolidone 5 g / bovine serum albumin 5 g dissolved in water 0.5 liter.

[0008]

【Example】

[0009]

Example 1 Isolation of the homoserine o-acetyltransferase gene a. Preparation of Acremonium chrysogenum gene library The total DNA of the Acremonium chrysogenum IS-5 strain (Microtechnology Research Institute No. 11232) was converted into Johnston (IL.
Johnston et al. Used the method used for Aspergillus nidulans (Reference: EMBO J. (198).
5) Extraction was carried out according to 4,1307-1311. Then 60 μg of this total DNA was partially digested with MboI and then treated with alkaline phosphatase. On the other hand, 10 μg of lambda vector EMBL3 (Promega Biotech) was completely digested with BamHI and EcoRI, and the short EcoRI-BamHI linker portion was removed by isopropanol precipitation. Then 1 μg of the partially digested DNA fragment
And 2 μg of a vector having a BamHI end were ligated with T4 ligase and encapsulated in lambda phage particles. The recombinant phage suspension thus obtained was diluted appropriately and E. coli NM539
(Promega Biotech) was infected and the number of plaques that appeared was counted. As a result, this suspension is about 3 x 10
^It was found to contain ⁵ recombinant phages. This phage solution was used as an Acremonium chrysogenum gene library and stored at 4 ° C. In addition, the above-mentioned donor DN
Detailed methods and conditions for preparation of A and vector, and ligation of both are described in Frischau.
f) and others were adopted (Reference: Journal
Of Molecular Biology (J. Mol. Bio
l. ) (1983) 170, 827-842). Also D
Encapsulation of NA into lambda particles was performed using a packaging extract from Promega Biotech, Inc. according to the protocol attached thereto. b. Preparation of Probe The method for constructing this plasmid containing the plasmid pCCS1 [DCPC-ATF-encoding cDNA insert is described in EP-0450758. ] 10 μg was digested with EcoRI and then subjected to 1% agarose gel electrophoresis. 1.2 including the DCPC-ATF code area
A 5 Kb DNA fragment was gene-cleaned (GENE.CL
EAN ^™ , sold by Funakoshi Co., Ltd.) was used to collect and purify from the agarose gel according to the attached protocol. Then, 50 ng of the fragment was added to a multiprime DNA labeling system (Multiprime DNA label).
ing system: Amersham) and labeled with (α- ³² P) deoxycytidine triphosphate 50 μCi according to the protocol attached thereto. Then, the reaction solution was heated at 70 ° C. for 10 minutes, and then nick column (Pharmacia, Nick-col) manufactured by Pharmacia.
(umn ^TU ) to obtain a probe having a radioactivity of about 10 ⁷ cpm. Hereinafter, this probe is referred to as an ATF-probe. c. Screening by hybridization A part of the phage solution (DNA library) obtained in a was infected with NM539, and a total of 4 x 1 was spread on 4 plates.
0 ³ plaques were formed. These plaques were subjected to the method of Benton WD et al. (Reference: Sc
Ience (1977) 196, 180-182), transferred to a nitrocellulose filter, alkali-denatured and neutralized to immobilize the DNA, and then hybridized with the ATF-probe obtained in the above b. Hybridization is performed with 30% formamide,
It was carried out at 37 ° C. for 15 hours using a solution containing 5 × Denhartz, 5 × SSPE, 0.1% SDS, and SGLY-probe at a final concentration of 4 × 10 ⁵ cpm / ml. Then, the filter was washed twice in a 2 × SSC solution containing 0.1% SDS at room temperature for 10 minutes each time, and further washed with 0.1%.
Was washed in a 0.2 × SSC solution containing SDS at 42 ° C. for 30 minutes. Then, autoradiography was performed at -80 ° C for 18 hours using an intensifier screen. As a result, 3 dark positive spots and 2 thin spots were found. Of these, phages were extracted from the agar regions corresponding to the two thin positive spots, and plaque hybridization was performed again according to the above steps to obtain two purified positive phage clones.
These clones were designated as λ-GMET1 and 2, respectively. The dark positive spot is derived from the clone carrying the DCPC-ATF gene. d. Subcloning of MET2 gene and limitation of its position From the two kinds of phage clones obtained in c, the method described in Grossberger (Reference: Nuc
leic Acids. Research (1987)
DNA was extracted according to 15, 6737). Next, this lambda DNA was digested with BamHI, subjected to agarose gel electrophoresis, and then subjected to Southern hybridization using an ATF-probe (for the method, refer to: Southern, (J. Mol. Biol.)).
(1975) 98, 503-517).

Hybridization, filter washing and the like were performed in the same manner as described in c. As a result, it was revealed that only about 8 Kb of BamHI existing in both clones hybridized with the probe. Therefore, this fragment was recovered from the agarose gel and purified in the same manner as in the above b. Next, this fragment was subcloned into the BamHI site of pUC18 (Takara Shuzo Co., Ltd.), which is a general-purpose E. coli vector, and the resulting plasmid was named pAME.
It was named TB8. Then, the above-mentioned two kinds of lambda DNAs were digested with SalI and subjected to Southern hybridization as described above. As a result, it was found that the approximately 2.7 Kb fragment present in both clones strongly hybridized with the probe. Therefore, this fragment was also subcloned into the SalI site of pUC18, and the resulting plasmid was designated as pAMETS3.
I named it. In addition, subcloning (ligation reaction between DNA fragments, transformation of Escherichia coli with a hybrid plasmid generated by the reaction, preparation of a plasmid from the obtained transformant, analysis thereof, etc.) is performed by the method described in Maniatis's experimental manual. It was carried out according to. See also pAMETB
8, pAMETS3 was used to carry out Southern hybridization as above, and it was confirmed that the inserts carried by both plasmids were the desired fragments. Next, both plasmids were cleaved with various restriction enzymes and analyzed by agarose gel electrophoresis to prepare a partial restriction enzyme map of each insert. Further, these were overlapped to complete a map of about 10 Kb region presumed to contain the homoserine o-acetyltransferase 2 gene derived from Acremonium chrysogenum. (First
Figure) e. Nucleotide sequence of homoserine o-acetyltransferase gene First, 0.8 Kb of BamHI-SalI estimated to contain at least a part of the coding region of homoserine o-acetyltransferase gene by analysis in c.
The entire base sequence of the fragment (the region where the above two types of inserts overlap) is determined by the method of Sanger et al. [Pro. N
atl. Acad. Sci. USA, 74 (1977)
5463]. Then, by comparing with a known homoserine o-acetyltransferase gene, the direction of the gene and which part of the homoserine o-acetyltransferase protein was encoded by this region were estimated. Then, sequencing was carried out upstream from the region, and finally, a 2588 bp nucleotide sequence covering the entire region underlined in FIG. 1 was determined. The specific experimental procedure for determining the nucleotide sequence was performed using a sequencing kit of Takara (Takara Shuzo) according to the attached protocol. The determined entire base sequence and the expected translation product are shown in the sequence listing (SEQ ID NO: 2).

[0011]

Example 2 Isolation of homoserine o-acetyltransferase cDNA a. Acremonium chrysogenum poly A ⁺ RNA extraction Acremonium chrysogenum IS-5 mycelium grown on CM solid medium at 30 ° C. for 3 days was treated with N3 seed medium 5
0 ml was inoculated, and 1 ml of the obtained culture solution, which was cultured at 25 ° C. for 3 days, was transferred to a 500 ml flask containing 30 ml of main medium,
Culturing was carried out at 0 ° C for 3 days. The mycelium collected from 10 ml of the bacterial solution by suction filtration was frozen in liquid nitrogen and ground with a mortar and pestle. From the bacterial crushed powder thus obtained, TOTAL RNA ISOLATION K
Using IT (Invitrogen), 800 μg of total RNA was extracted according to its attached protocol. Then, 27 μg of poly A ⁺ RNA was obtained from this total RNA by performing an oligo (dT) cellulose chromatography operation according to Maniatis's protocol. b. Preparation of cDNA library Using the cDNA synthesis system (Amersham), according to the protocol, 1 from the poly A ⁺ RNA obtained in the above a.
Single-stranded DNA and then double-stranded cDNA were synthesized. Then, a cDNA library of 2 × 10 ⁵ Acremonium chrysogenum was prepared using the cDNA cloning system λgt10 (codeRPN1257 from Amersham) according to the protocol. c. Preparation of probe 1.5 Kb EcoRV fragment derived from pAMETB8 prepared in Example 1d (EcoRV-EcoRV in FIG. 1)
Was labeled with (α- ³² P) deoxycytidine triphosphate in the same manner as in Example 1b and used in the hybridization experiment described below. After that, this probe was
Called T-Probe d. Screening by hybridization A part of the phage solution obtained in b was infected with NM539 to form a total of 3 × 10 ⁴ plaques on 8 plates. These plaques were transferred to a nitrocellulose filter in the same manner as in Example 1c to immobilize DNA, and then hybridized with the MET probe obtained in the above c. Hybridization is performed with 50% formamide, 5 x Denhardt's, 5 x SSPE, 0.1% S
M at DS and final concentration of 4 × 10 ⁵ cpm / ml
It was performed at 42 ° C. for 15 hours using a solution containing the ET probe. The filter was then washed twice in a 2 × SSC solution containing 0.1% SDS for 10 minutes each at room temperature,
5 in 0.2xSSC solution with additional 0.1% SDS
It was washed at 5 ° C for 30 minutes. Autoradiography was then performed for 18 hours at -80 ° C using an intensifier screen. As a result, 3 positive spots were found. Phages were extracted from the agar region corresponding to the spots, and plaque hybridization was performed again according to the above steps to obtain three purified positive phage clones. And these clones were designated as λ-CMET1, 2, 3 respectively. e. Structural analysis of cDNA From the three phage clones obtained in d above, Example 1d
As a result of preparing and analyzing λDNA in the same manner as
It was revealed that MET2 contained the longest approximately 1.9 Kb cDNA insert. Therefore, this fragment was subcloned into pUC18 (the obtained plasmid was designated as pUMET2) to prepare a partial restriction enzyme map, and then cDNA was prepared using the same experimental method as in Example 1e.
The entire base sequence of the A insert was determined. The 1864 bp nucleotide sequence thus obtained is shown in the sequence listing (SEQ ID NO: 1). 1 from ATG starting at the 202nd position in this DNA sequence
There was an open reading frame (ORF) capable of encoding 500 amino acids up to the TAG ending at 704. The O
Sequence listing of the amino acid sequence translated from RF (SEQ ID NO: 1)
It is shown in the lower row of the nucleotide sequence. As expected, this sequence showed high similarity to that of homoserine o-acetyltransferase from other species. Also, by comparing with the nucleotide sequence of the homoserine o-acetyltransferase gene determined in Example 1e, it was revealed that the gene of the present invention has two introns.

[0012]

Example 3 Complementation test of homoserine o-acetyltransferase-deficient yeast a. Construction of pYMET2 A plasmid for expressing homoserine o-acetyltransferase cDNA derived from Acremonium chrysogenum in baker's yeast according to the steps shown in FIG. pY
ACMET2 was constructed. The process will be described below.

First, the plasmid pUME obtained in Example 2e
T2 was digested with SacII located upstream of the start codon, the cut end was blunted using a blunting kit (Takara Shuzo), and then BglII linker [d (p
CAGATCTG): Takara Shuzo] was connected. The reaction was then co-digested with BglII and BamHI,
Approximately 1.75K containing homoserine o-acetyltransferase cDNA when subjected to agarose gel electrophoresis
The fragment of b was recovered from the gel and purified. The fragment and BamH
PYAC cleaved with I and treated with alkaline phosphatase
T3 [Matsuda et al. , Bioche
m. Biophys. Res. Commun. (199
2) 182,995-1001] with T4 ligase to obtain a yeast actin promoter on pYACT3.
And homoserine o-acetyltransferase cD
Plasmid pYACMET2 in which NA was ligated in the forward direction
Got In addition, a plasmid pYACRMET2 in which the above promoter and cDNA were linked in the opposite direction was also obtained. In the above steps, separation and purification of DNA fragments produced by restriction enzyme digestion, ligation reaction between DNA fragments, transformation of Escherichia coli with plasmids produced by the reaction, preparation of plasmids from the obtained transformants and analysis thereof, etc. The basic operation of was carried out according to the method described in Example 1 and Maniatis's experimental manual. b. Introduction into MET2 mutant yeast The plasmids pYACMET2 and pYA obtained in the above a.
The CRMET2 is a lithium acetate method [Ito et a
l. J. Bacteriol (1983) 153, 1
63-168], has a mutation in the MET2 gene,
Yeast strain STX known to show methionine requirement
66-4A (a ade2 gal2 lys4 me
t2 pha2 prt3 trp1 rad18). Thus, the colonies appearing in the SC-Trp medium were subcultured in the SC-Trp-Met medium and tested for methionine auxotrophy, and it was found that all the transformants with pYACMET2 lost the auxotrophy. On the other hand, all the pYACRMET2 transformants showed methionine auxotrophy. From the above results, it was confirmed that the DNA fragment obtained by the present invention encodes a protein having the same function as the yeast MET2 gene product, that is, homoserine o-acetyltransferase.

[0014]

The DNA fragment of the present invention is ligated to a vector,
Introduction into Acremonium chrysogenum makes it possible to regulate intracellular levels of homoserine o-acetyltransferase, which plays a central role in methionine metabolism. As a result, the creation of a strain that is advantageous for cephalosporin fermentation can be expected. Further, the present invention DN
A can also be used as an auxotrophic marker gene when transforming Acremonium chrysogenum.

[0015]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1864 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Acremonium chrysogenum (Acremonium c)
hrysogenum) Strain name: IS5 Sequence characteristics Characteristic symbol: CDS Location: 202..1704 Method by which the characteristics were determined: E sequence TTTTTTTTTT TTGTGTATAG ATTTCCTGTT GTAATTGTTC AATCTCACAT AGCAGCACTCACCCCCTGCGCCGCCCACACACATCCACCCACATCCACCCACATTCATCCACCCACATCCACCCACATTCATGCCCCCATTATCTTGTC CAAGCTGCTCCACCCACATCACCCCCCATTATCTTGTC CAAG 180 GCCGAGTTTC AATCGCCAGT G ATG GCC TCC CAA ACG CCT TCC AAC GGC ACT 231 Met Ala Ser Gln Thr Pro Ser Asn Gly Thr 1 5 10 GCG GGC ACC GCA GCT CAG AGA TAC GAA AGA ACA CAA TCA CAG CCT GAG 279 Ala Gly Thr Ala Ala Gln Arg Tyr Glu Arg Thr Gln Ser Gln Pro Glu 15 20 25 AAC CCG TTC GCG GCT CTC ATC CCG CAC CAG CAG ATC GCC ATC ATC CCG 327 Asn Pro Phe Ala Ala Leu Ile Pro His Gln Gln Ile Ala Ile Ile Pro 30 35 40 GAA TTC ACC CTC GAA TCC GGC GTG ACC CTC TAC AAT GTC CCC GTA GCC 375 Glu Phe Thr Leu Glu Ser Gly Val Thr Leu Tyr Asn Val Pro Val Ala 45 50 55 TAC ACG ACG AGG GGC AAG TTG TCC CCC GAG CGG GAC AAT GCT ATG GTC 423 Tyr Thr Thr Arg Gly Lys Leu Ser Pro Glu Arg Asp As n Ala Met Val 60 65 70 ATC TGC CAT GCC CTC ACT GGT AGC TCT GAC GTG GGC GAC TGG TGG GGT 471 Ile Cys His Ala Leu Thr Gly Ser Ser Asp Val Gly Asp Trp Trp Gly 75 80 85 90 CCC CTT CTT GGT GGT TCG GGC AAG GCT TTC GAC GAG ACG CGC TTC TTT 519 Pro Leu Leu Gly Gly Ser Gly Lys Ala Phe Asp Glu Thr Arg Phe Phe 95 100 105 ATC GTG TGC ATG AAC AGC CTG GGA AGC CCC TAC GGC ACC GCG AGC CCG 567 Ile Val Cys Met Asn Ser Leu Gly Ser Pro Tyr Gly Thr Ala Ser Pro 110 115 120 GTG ACG GCC AAG GAT GGC GAC CCG TCC AAG GGT CGG TAC GGA CCC GAG 615 Val Thr Ala Lys Asp Gly Asp Pro Ser Lys Gly Arg Tyr Gly Pro Glu 125 130 135 TTT CCC CTG ACG ACG ATT AGG GAT GAC GTC AGG CTT CAT AAG TTG TTG 663 Phe Pro Leu Thr Thr Ile Arg Asp Asp Val Arg Leu His Lys Leu Leu 140 145 150 CTC GAC GAC CTA GGC GTG AAG CGG GTT GCC TCC GTC GTC GGC GGG TCC 711 Leu Asp Asp Leu Gly Val Lys Arg Val Ala Ser Val Val Gly Gly Ser 155 160 165 170 CTG GGT GGC ATG TTT GTA CTG GAG TGG GCG TAC CTG GGC AAG GAC TAC 759 Leu Gly Gly Met Phe Val Leu Glu Trp Ala Ty r Leu Gly Lys Asp Tyr 175 180 185 ATC CGG AGC ATC GTC CCC ATC GCC ACG TCC AGC CGG CAC AGC GCG TGG 807 Ile Arg Ser Ile Val Pro Ile Ala Thr Ser Ser Arg His Ser Ala Trp 190 195 200 TGT ATC AGC TGG GGC GAG GCA CAG CGC CAG AGC ATC TAC GCG GAC CCC 855 Cys Ile Ser Trp Gly Glu Ala Gln Arg Gln Ser Ile Tyr Ala Asp Pro 205 210 215 AAG TAC GAC GAT GGC TAC TAC TCC TTC GAC GAC CCG CCG TCC ACC GGC 903 Lys Tyr Asp Asp Gly Tyr Tyr Ser Phe Asp Asp Pro Pro Ser Thr Gly 220 225 230 CTA GGA GCG GCG CGC ATG TCG GCG CTG CTC ACC TAC CGC AGT CGC AAC 951 Leu Gly Ala Ala Arg Met Ser Ala Leu Leu Thr Tyr Arg Ser Arg Asn 235 240 245 250 TCG TTC GAG TCC CGG TTC GGC CGC AAT ATC CCG GAC CCG GCC AAG GTG 999 Ser Phe Glu Ser Arg Phe Gly Arg Asn Ile Pro Asp Pro Ala Lys Val 255 260 265 CAG ACA ATC AGC GGT CGA CCG CCG CCC TCA ACG CCG TCT GAG GCG CAT 1047 Gln Thr Ile Ser Gly Arg Pro Pro Pro Ser Thr Pro Ser Glu Ala His 270 275 280 TTT CAC ATC CAC AAC GAC GGG CAC AAC GTC AAG CGG ACG ACA CTC TCC 1095 Phe His Ile His Asn Asp Gly His Asn Val Lys Arg Thr Thr Leu Ser 285 290 295 CCG AGG GAC AGC CAG CGG AGT CTC GGG TCG GCT GGC GCC ACG GAA GCA 1143 Pro Arg Asp Ser Gln Arg Ser Leu Gly Ser Ala Gly Ala Thr Glu Ala 300 305 310 GCC ATC GAG AGA CAG CAA CAG CAG GAG GGC CAG TCG GCA GAC CCC CAG 1191 Ala Ile Glu Arg Gln Gln Gln Gln Glu Gly Gln Ser Ala Asp Pro Gln 315 320 325 330 TTC AGC GGA CCG CCC AAG AAC TCG AGC CTG ACA GGC GGC GAC TCG CTG 1239 Phe Ser Gly Pro Pro Lys Asn Ser Ser Leu Thr Gly Gly Asp Ser Leu 335 340 345 CCG ACG TCG ACG TAC TTC TCG GCG CAG TCC TAC CTG CGG TAC CAG GGC 1287 Pro Thr Ser Thr Tyr Phe Ser Ala Gln Ser Tyr Leu Arg Tyr Gln Gly 350 355 360 ACC AAG TTC GTC TCC CGC TTC GAC GGC AAC TGC TAC ATC GCC ATG ACG 1335 Thr Lys Phe Val Ser Arg Phe Asp Gly Asn Cys Tyr Ile Ala Met Thr 365 370 375 CGC AAG CTG GAC ACG CAC GAC GTA TCC CGC AAC CGC GCG CCG ACC GTG 1383 Arg Lys Leu Asp Thr His Asp Val Ser Arg Asn Arg Ala Pro Thr Val 380 385 390 GCG GAG GCG CTG GCC CAG ATC GAG CAG CCG ACG CTG GTG CTG GGC ATC 1431 Ala Glu A la Leu Ala Gln Ile Glu Gln Pro Thr Leu Val Leu Gly Ile 395 400 405 410 GAG AGC GAC GGG CTC TTC ACC TTT GCC GAG CAG GAG GAG ATT GCG CGG 1479 Glu Ser Asp Gly Leu Phe Thr Phe Ala Glu Gln Glu Glu Ile Ala Arg 415 420 425 CAC GTC AAG GAT GCG CGG CTG GAG CGC ATC GAC AGC CCC GAG GGC CAC 1527 His Val Lys Asp Ala Arg Leu Glu Arg Ile Asp Ser Pro Glu Gly His 430 435 440 GAC GCC TTC CTG CTC CAG TTT GAG CAG GTG AAC CGG TAC GTG CTC AAC 1575 Asp Ala Phe Leu Leu Gln Phe Glu Gln Val Asn Arg Tyr Val Leu Asn 445 450 455 TTT TTC AGG GAG GTG CTG CCG GAT ATC ATG GGC AAG GAA GGC GTG GCG 1623 Phe Phe Arg Glu Val Leu Pro Asp Ile Met Gly Lys Glu Gly Val Ala 460 465 470 CCC GAG GTA GCG TCA GTG GGG GAG ATT GTC GAG TCG AGC ACG TTT GGG 1671 Pro Glu Val Ala Ser Val Gly Glu Ile Val Glu Ser Ser Thr Phe Gly 475 480 485 490 GAG GCC GAG GTG GAG GAC ATA ACG GCT TGG TGAGGCTGTT GGGCGCTCAT 1721 Glu Ala Glu Val Glu Asp Ile Thr Ala Trp 495 500 TACTAGTACC AGGGTATTTT GCATACCTGT CAATGCTGCC ACGAGGCTCT AGTGTGGATG 1781 GCGGTGGTGG TGG TGGTGGT AGTGGTAGTG GTGGTGTTGA ACCAATAGAA TGGGTGTGCT 1841 GAGCCAAAAA AAAAAAAAAA AAA 1864 SEQ ID NO: 2 Sequence length: 2588 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: Genomic DNA Origin organism name: Acremonium chrysogenum (Acremonium c
hrysogenum) Strain name: IS5 Sequence features Characteristic symbol: intron Location: 608..723 1115..1183 Method for determining features: E sequence TGGCCAAGCC TCGCGTGCTA TGGACCAGGT CGTCGGGATGAGGTGGTGAT TGGGGGGGGG 60 TTGTTGGCGA AGAGGATCGC GCCTCGGA GGCGACGATG TTTGGAAAGG TACGTACCGA CGTGCCTACT 180 GAGACCCACA TTTGATATGT CTGACTGTTT GGTTGCTGAC TCATCTCATG AAGAGAAGGC 240 CGAAAATCGC TGGGTGGGTG AGCACGCCAT ATAAAGTGGG CCAAGATCCC AGGTTCATCA 300 TCGTCAACCA CCGCCAGTGG GTGAATAGAG ACACGATTCA TTCGAAACTA CTTTTTTTTT 360 TTTGTGTATA GATTTCCTGT TGTAATTGTT CAATCTCACA TAGCAGCACT CCTGCCTCGC 420 CGCGTCTCGC CCGCCATCCT CTTATCTTGT CCAAGCAGCA GTCCCGCGGC TCCCCGTACA 480 TCGCATACGC CAATTCTCAC CCACAAAAGT CTGTATCACC AACACTCCCC GGCCGAGTTT 540 CAATCGCCAG TG ATG GCC TCC CAA ACG CCT TCC AAC GGC ACT GCG GGC ACC 591 Met Ala Ser Gln Thr Pro Ser Asn Gly Thr Ala Gly Thr 1 5 10 GCA GCT CAG AGA TAC G GTCAGTACAC CCCCATCTAT CACGCCTCCT ATCACCCTCA 647 Ala Ala Gln Arg Tyr G 15 CCCTCGCGTC CCCCCCGATA TCGGAATGTC CCTCCGTCGA GAAAGTACAA AGGACCGATG 707 GCTGAATACC TGCAAG AA AGA ACA CAA TCA CAG CCT GAG AAC CCG TTC GCG lu Arg Thr Gln Ser Gln Pro Glu Asn Pro Phe Ala 758 20 25 30 GCT CTC CACC CAC GCA CAC ATC TTC ACC CTC 806 Ala Leu Ile Pro His Gln Gln Ile Ala Ile Ile Pro Glu Phe Thr Leu 35 40 45 GAA TCC GGC GTG ACC CTC TAC AAT GTC CCC GTA GCC TAC ACG ACG AGG 854 Glu Ser Gly Val Thr Leu Tyr Asn Val Pro Val Ala Tyr Thr Thr Arg 50 55 60 GGC AAG TTG TCC CCC GAG CGG GAC AAT GCT ATG GTC ATC TGC CAT GCC 902 Gly Lys Leu Ser Pro Glu Arg Asp Asn Ala Met Val Ile Cys His Ala 65 70 75 CTC ACT GGT AGC TCT GAC GTG GGC GAC TGG TGG GGT CCC CTT CTT GGT 950 Leu Thr Gly Ser Ser Asp Val Gly Asp Trp Trp Gly Pro Leu Leu Gly 80 85 90 GGT TCG GGC AAG GCT TTC GAC GAG ACG CGC TTC TTT ATC GTG TGC ATG 998 Gly Ser Gly Lys Ala Phe Asp Glu Thr Arg Phe Phe Ile Val Cys Met 95 100 105 110 AAC AGC CTG GGA AGC CCC TAC GGC ACC GCG AGC CCG GTG ACG GCC AAG 1046 Asn Ser Leu Gly Ser Pro Tyr Gly Thr Ala Ser Pro Val Thr Ala Lys 115 120 125 GAT GGC GAC CCG TCC AAG GGT CGG TAC GGA CCC GAG TTT CCC CTG ACG 1094 Asp Gly Asp Pro Ser Lys Gly Arg Tyr Gly Pro Glu Phe Pro Leu Thr 130 135 140 ACG ATT AGG GAT GAC GTC AG GTAGGTTTGG CCTCGCTTTT GGGGATCGCA 1144 Thr Ile Arg Asp Asp Val Ar 145 GTGGGGATGG TGATGCGAGT GCTGACGGGG CACAAATAG G CTT CAT AAG TTG TTG 1199 g Leu His Lys Leu Leu 150 CTC GAC GAC CCC CGT GGC CCC GCC CTA GGC GGG TCC 1247 Leu Asp Asp Leu Gly Val Lys Arg Val Ala Ser Val Val Gly Gly Ser 155 160 165 170 CTG GGT GGC ATG TTT GTA CTG GAG TGG GCG TAC CTG GGC AAG GAC TAC 1295 Leu Gly Gly Met Phe Val Leu Glu Trp Ala Tyr Leu Gly Lys Asp Tyr 175 180 185 ATC CGG AGC ATC GTC CCC ATC GCC ACG TCC AGC CGG CAC AGC GCG TGG 1343 Ile Arg Ser Ile Val Pro Ile Ala Thr Ser Ser Arg His Ser Ala Trp 190 195 200 TGT ATC AGC TGG GGC GAG GCA CAG CGC CAG AGC ATC TAC GCG GAC CCC 1391 Cys Ile Ser Trp Gly Glu Ala Gln Arg Gln Ser Ile Tyr Ala Asp Pro 205 210 215 AAG TAC GAC GAT GGC TAC TAC TCC TTC GAC GAC CCG CCG TCC ACC GGC 1439 Lys Tyr Asp Asp Gly Tyr Tyr Ser Phe Asp Asp Pro Pro Ser Thr Gly 220 225 230 CTA GGA GCG GCG CGC ATG TCG GCG CTG CTC ACC TAC CGC AGT CGC AAC 1487 Leu Gly Ala Ala Arg Met Ser Ala Leu Leu Thr Tyr Arg Ser Arg Asn 235 240 245 250 TCG TTC GAG TCC CGG TTC GGC CGC AAT ATC CCG GAC CCG GCC AAG GTG 1535 Ser Phe Glu Ser Arg Phe Gly Arg Asn Ile Pro Asp Pro Ala Lys Val 255 260 265 CAG ACA ATC AGC GGT CGA CCG CCG CCC TCA ACG CCG TCT GAG GCG CAT 1587 Gln Thr Ile Ser Gly Arg Pro Pro Pro Ser Thr Pro Ser Glu Ala His 270 275 280 TTT CAC ATC CAC AAC GAC GGG CAC AAC GTC AAG CGG ACG ACA CTC TCC 1631 Phe His Ile His Asn Asp Gly His Asn Val Lys Arg Thr Thr Leu Ser 285 290 295 CCG AGG GAC AGC CAG CGG AGT CTC GGG TCG GCT GGC GCC ACG GAA GCA 1679 Pro Arg Asp Ser Gln Arg Ser Leu Gly Ser Ala Gly Ala Thr Glu Ala 300 305 310 GCC ATC GAG AGA CAG CAA CAG CAG GAG GGC CAG TCG GCA GAC CCC CAG 1727 Ala Ile Glu Arg Gln Gln Gln Gln Glu Gly Gln Ser Ala Asp Pro Gln 315 320 325 330 TTC AGC GGA CCG CCC AAG AAC TCG AGC CTG ACA GGC GGC GAC TCG CTG 1775 Phe Ser Gly Pro Pro Lys Asn Ser Ser Leu Thr Gly Gly Asp Ser Leu 335 340 345 CCG ACG TCG ACG TAC TTC TCG GCG CAG TCC TAC CTG CGG TAC CAG GGC 1823 Pro Thr Ser Thr Tyr Phe Ser Ala Gln Ser Tyr Leu Arg Tyr Gln Gly 350 355 360 ACC AAG TTC GTC TCC CGC TTC GAC GGC AAC TGC TAC ATC GCC ATG ACG 1871 Thr Lys Phe Val Ser Arg Phe Asp Gly Asn Cys Tyr Ile Ala Met Thr 365 370 375 CGC AAG CTG GAC ACG CAC GAC GTA TCC CGC AAC CGC GCG CCG ACC GTG 1919 Arg Lys Leu Asp Thr His Asp Val Ser Arg Asn Arg Ala Pro Thr Val 380 385 390 GCG GAG GCG CTG GCC CAG ATC GAG CAG CCG ACG CTG GTG CTG GGC ATC 1967 Ala Glu Ala Leu Ala Gln Ile Glu Gln Pro Thr Leu Val Leu Gly Ile 395 400 405 410 GAG AGC GAC GGG CTC TTC ACC TTT GCC GAG CAG GAG GAG ATT GCG CGG 2015 Glu Ser Asp Gly Leu Phe Thr Phe Ala Glu Gln Glu Glu Ile Ala Arg 415 420 425 CAC GTC AAG GAT GCG CGG CTG GAG CGC ATC GAC AGC CCC GAG GGC CAC 2063 His Val Lys Asp Ala Arg Leu Glu Arg Ile Asp Ser Pro Glu Gly H is 430 435 440 GAC GCC TTC CTG CTC CAG TTT GAG CAG GTG AAC CGG TAC GTG CTC AAC 2111 Asp Ala Phe Leu Leu Gln Phe Glu Gln Val Asn Arg Tyr Val Leu Asn 445 450 455 TTT TTC AGG GAG GTG CTG CCG GAT ATC ATG GGC AAG GAA GGC GTG GCG 2159 Phe Phe Arg Glu Val Leu Pro Asp Ile Met Gly Lys Glu Gly Val Ala 460 465 470 CCC GAG GTA GCG TCA GTG GGG GAG ATT GTC GAG TCG AGC ACG TTT GGG 2207 Pro Glu Val Ala Ser Val Gly Glu Ile Val Glu Ser Ser Thr Phe Gly 475 480 485 490 GAG GCC GAG GTG GAG GAC ATA ACG GCT TGG TGAGGCTGTT GGGCGCTCAT 2257 Glu Ala Glu Val Glu Asp Ile Thr Ala Trp TG500 TACTAGCTAGTG ACGACTGTAGGAGTATTAGCATTGAGGA TGGGTGTGCT 2377 GAGCCACATG TGCCGTCAAC TTGCGCCCTT TCCATTCTAG GATGCCTATG AATGGGGTTG 2437 AAGAACATAA ATAATGCGAG GTGGACTGGC GGGGACTGGC GGTTGGCCCA TGAACCATGC 2497 TCAAACGTTC 2497 TCAAACCTTC 2497 TCAAACCTTC 2497 TCAAACCTTC 2497 TCAAACGTTC

[Brief description of drawings]

FIG. 1 shows a partial restriction enzyme map of the DNA fragment of the present invention.
Consecutive restriction enzyme recognition sites are numbered from left to right in the figure for convenience. In the figure, ── indicates the region where the base sequence was determined, and → indicates the direction of the homoserine o-acetyltransferase gene. The BalI recognition site was found in the process of determining the base sequence and may exist at positions other than those shown in the figure. Also, between SalI and BamHI,
The existence of at least two SalI sites has been clarified, but the exact position thereof has not been determined, and therefore it is not shown in the figure.

FIG. 2: Plasmids pYACMET2 and pYACR
The manufacturing process of MET2 is shown. The abbreviations and abbreviations used in FIG. 2 are as follows. MET: Homoserine o-acetyltransferase gene, YA
P: Yeast-derived actin promoter, YTT:
Yeast-derived Trp5 (tryptophan synthase gene terminator, trp1: yeast Trp1 (phosphoribosylanthranilic acid isomerase) gene, 2μ: yeast 2
Origin of replication of μ plasmid

Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display area C12R 1: 645) C12R 1: 645)

Claims (4)

[Claims] 1. A DNA fragment containing a homoserine o-acetyltransferase gene derived from Acremonium chrysogenum. 2. The DN according to claim 1, which encodes a protein having an amino acid sequence shown in the sequence listing (SEQ ID NO: 1).
A fragment. 3. The DNA fragment according to claim 1, which is defined by the restriction enzyme map shown in FIG. 4. BamHI-BalI in FIG.
The DNA fragment according to claim 1, 2 or 3, wherein the base sequence between them is shown in the sequence listing (SEQ ID NO: 2).