JPH08336391A - O-acetylhomoserine sulfhydrylase gene - Google Patents

Abstract

PURPOSE: To obtain the subject gene useful for improving the cephalosporin C productivity of Acremonium chrysogenum bacteria. CONSTITUTION: First, o-acetylhomoserine sulfhydrylase is purified, part of the amino acid sequence thereof is determined, a partial CDNA fragment coding the enzyme is isolated, and a DNA fragment containing an o-acetylhomoserine (abbreviated as OAH) sulfhydrylase gene is isolated from an Acremonium chrysogenum gene library using the above CDNA fragment as a probe. The OAH sulfhydrylase gene extracted from Acremonium chrysogenum IS-5 (FERM BP-3153) includes a gene coding an amino acid sequence of the formula. When a plasmid pTOAHI containing this gene is transduced into an Acremonium chrysogenum IS-5 strain, a transformant potentiated in the ability to synthesize OAH sulfhydrylase is obtained.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a DNA fragment containing an O-acetylhomoserine sulfhydrylase (hereinafter abbreviated as OAH sulfhydrylase) gene derived from Acremonium chrysogenum and the D fragment thereof.
The present invention relates to Acremonium chrysogenum transformed with a recombinant DNA incorporating an NA fragment to enhance the enzyme activity.

[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 [eg, Queener et al., Microbiology 1985. A]
American Society for Microbiology, (1985) 468-472)
And the cloning of the cephalosporin C biosynthetic enzyme gene have been carried out one after another, opening the way to apply a genetic engineering approach to breeding of excellent strains [eg, Skatrud et al., B.
IO / TECHNOLOGY (1989) 7, 477].

Cephalosporin C is acremonium
In Chrysogenum, three amino acids (L-cysteine, L-valine, L-lysine which is a biosynthetic intermediate of L-lysine)
It is known that α-aminoadipic acid) is used as a starting material and biosynthesized through a 6-step reaction with 5 kinds of enzymes [eg, Martin et al., Trends in Biotechnolog].
y (1985) 3: 39-44]. To date, most of the genes involved in these steps have been cloned from Acremonium chrysogenum [eg Samon et al., Nature.
(1985) 318,191, Samson et al., BIO / TECHNOLOGY (1987).
5,1207, Gutierres et al., Journal of Bacteriology (199
1) 173,2354-2365, Japanese Patent Application No. 5-077367,
EP0450758], is being applied to molecular breeding.

On the other hand, the above-mentioned Tori cephalosporin C is synthesized using three kinds of amino acids which are the primary metabolites of the producing bacterium as a raw material. Can be a strategy. Among these amino acids, cysteine has been comparatively extensively studied in the context of its biosynthesis and cephalosporin C fermentation. Similar to S. cerevisiae (hereinafter simply abbreviated as yeast) and N. crassa (Pan mold), cysteine is also considered to be biosynthesized by two pathways in Acremonium chrysogenum [eg Martin] Et al, Microbiological Reviews (1980) 44,
230-251].

One is a pathway for producing cysteine by sulfurization of O-acetylserine with hydrogen sulfide, and the other is the production of cysteine from homocysteine produced by demethylation of methionine or sulfurization of O-acetylhomoserine. It is a route to cysteine via. The analysis of various mutant strains has revealed that the latter, that is, the reverse sulfur transfer pathway via cystathionine, plays an important role in the fermentative production of cephalosporin C in Acremonium chrysogenum. [For example, Treichler et al. (1979) Genetics of i
ndustrial microorganisms. Proceedings of the Third
International Symposium on Geneticsof Industrial
Microorganisms. American Society for Microbiology,
Washington, DC, 97-104].

Therefore, genes of enzymes involved in the reverse sulfur transfer pathway (cystathionine γ-lyase, cystathionine β-synthase, OAH sulfhydrylase, etc.) produce cephalosporin C in the same manner as the above-mentioned cephalosporin C biosynthesis gene. It is thought to provide a useful tool for molecular breeding of fungi. Recently, the present inventors have cloned the cystathionine γ-lyase and cystathionine β-synthase genes from Acremonium chrysogenum, and succeeded in creating an Acremonium chrysogenum strain in which the respective enzyme activities were enhanced by using them. (Specification of Japanese Patent Application No. 5-101882, Japanese Patent Application No. 5-21311)
No. 6).

On the other hand, OAH sulfhydrylase, which is an enzyme that catalyzes the reaction of producing homocysteine, which is a substrate of cystathionine β-synthase, from OAH and hydrogen sulfide, also analyzes the enzyme-deficient strain to ferment cephalosporin C. , Particularly, it has been pointed out that it plays an important role in fermentation production in a medium mainly composed of an inorganic sulfur source [eg,
Treichler et al. (1979) Genetics of industrial micr
oorganisms. Proceedings of the Third International
Symposium on Genetics of Industrial Microorganism
s. American Society for Microbiology, Washington,
DC, 97-104]. However, the OAH sulfhydrylase gene derived from this bacterium has not yet been isolated and identified.

[0009]

The present invention is derived from Acremonium chrysogenum and is one of the enzymes involved in the biosynthetic pathways of cysteine and methionine, OAH.
The purpose of this study is to isolate the sulfhydrylase gene and use it to improve Acremonium chrysogenum.

[0010]

The present inventors succeeded in purifying OAH sulfhydrylase from Acremonium chrysogenum, determined a part of the amino acid sequence of the enzyme, and obtained the amino acid information. Based on this, a partial cDNA fragment encoding the enzyme was isolated. Next, using the cDNA fragment as a probe, a DNA fragment containing the OAH sulfhydrylase gene was isolated from the Acremonium chrysogenum gene library, and its nucleotide sequence was partially (entire coding region and part of non-coding region). Decided. Furthermore, the OAH sulfhydrylase activity of Acremonium chrysogenum into which a recombinant DNA in which a part of the DNA fragment is incorporated into a vector is introduced is a parent strain (non-introduced strain).
I found that it was rising compared to. We have
The present invention has been completed based on these findings. That is, the present invention provides (1) O derived from Acremonium chrysogenum
DNA fragment containing AH sulfhydrylase gene, (2)
The OAH sulfhydrylase gene contains a gene encoding the amino acid sequence shown in the sequence listing (SEQ ID NO: 1).
The NA fragment, (3) OAH sulfhydrylase gene, is included between the restriction enzyme sites of PstI-SmaI of about 2.8 Kb defined by the restriction enzyme map shown in FIG.
A DNA fragment having at least PstI, NheI, and NruI restriction enzyme sites, (4) PstI in FIG.
A DNA fragment whose base sequence between -SmaI is the base sequence shown in the sequence listing (SEQ ID NO: 2), (5) (1) (2) (3) or
(4) Recombinant DNA in which the whole or a part of the described DNA fragment is incorporated into a vector, (6) (5) Recombinant DNA
Acremonium chrysogenum transformed with.

The present inventors also screened a cDNA library derived from Acremonium chrysogenum using the above probe, and isolated a cDNA containing the entire coding region of OAH sulfhydrylase. The cDN
The nucleotide sequence of A is shown in the sequence listing (SEQ ID NO: 1) together with the amino acid sequence encoded thereby. These cDNA
By ligating the compound with a promoter fragment that functions in Acremonium chrysogenum (for example, Japanese Patent Laid-Open No. 58891/1992), the same manner as the DNA fragment of the present invention,
It can be used to improve Acremonium chrysogenum. Therefore, the RN transcribed from the DNA fragment of the present invention
A group of cDNA compounds derived from A is also included in the present invention.

The DNA fragment and recombinant of the present invention can be constructed by the following steps. 1: Determination of partial amino acid sequence of OAH sulfhydrylase. (1) Purify OAH sulfhydrylase from Acremonium chrysogenum. (2) The purified enzyme obtained in (1) is subjected to limited decomposition with a proteolytic enzyme such as Pseudomonas endoprotease Asp-N and fractionated by reverse phase chromatography or the like. (3) The partial amino acid sequence of the degraded peptide fragment obtained in (2) is determined. By the above operation, the sequence listing SEQ ID NOs: 3, 4, 5
Also, it was revealed that the three types of sequences shown below were contained in OAH sulfhydrylase. 1. Asp-Asn-Ile-Val-Ser-Thr-Ser-Asn-Leu-Tyr-Gly-Gly
-Thr-Tyr-Asn 2.Asp-Lys-Gly-Val-Pro-Leu-Ile-Val-Asp-Asn-Thr-Phe
-Gly-Ala-Gly-Gly-Tyr-Phe-Ile-Arg-Pro-Val-Asp-His-G
ly 3.Asp-Ala-Lys-Thr-Leu-Ala-Ile-His-Pro-Trp-Ser-Thr
-Thr-His-Glu The sequences of these peptide fragments show high homology to the amino acid sequence of yeast-derived OAH sulfhydrylase whose structure has already been clarified. The sequence is 102 of yeast OAH sulfhydrylase.
It was revealed that they correspond to the -116, 175-199, and 384-398th amino acid sequences, respectively. The method for purifying OAH sulfhydrylase in (1) above will be described in detail in Examples. The operations such as limited decomposition and chromatography in the above (2) are well known experimental manuals (for example, "Protein structural analysis for gene cloning", Hisashi Hirano, 1
In 993, it can be carried out according to the method described in Tokyo Kagaku Dojin). In addition to Asp-N, trypsin, lysyl endopeptidase, etc. can be used as the proteolytic enzyme. The amino acid sequence of (3) above can be obtained, for example, from Shimadzu Corporation or Applied B
It can be determined using a gas phase sequencer manufactured by iosystems. 2. Cloning of OAH sulfhydrylase gene (1) Chromosomal DNA is extracted from Acremonium chrysogenum and partially digested with an appropriate restriction enzyme (eg, MboI). (2) The DNA fragment obtained in (1) is incorporated into an appropriate vector to construct a chromosomal DNA library of Acremonium chrysogenum. (3) The DNA probe prepared based on the partial amino acid sequence information determined in 1 above is labeled, hybridized with the library obtained in (2), and clones showing homology with the probe are selected and separated. To do. (4) DNA is extracted from the clone selected in (3), Southern hybridization is performed using the same probe as above, the target gene is located on a restriction enzyme fragment of appropriate size, and it is subcloned into a plasmid vector. . (5) The nucleotide sequence of the DNA fragment thus obtained is determined,
The code area is positioned. (6) D containing the entire coding region of OAH sulfhydrylase
NA fragment (fragment that is presumed to include regions such as promoter and terminator of the gene) is acremonium
A recombinant DNA is prepared by incorporating it into a chrysogenum transformation vector. (7) Acremonium chrysogenum is transformed with the above recombinant DNA. (8) The OAH sulfhydrylase activity of the transformant obtained in (7) is measured to confirm the effect of introducing the DNA fragment of the present invention.

In the above steps, the general procedures necessary for handling DNA and Escherichia coli as a recombinant host are usually carried out by those skilled in the art. .Maniatis et al., Molecular Cloning
A Laboratory Manual, ColdSpring Harbor Laboratory
1982, 1989) and can be easily implemented. In addition, commercially available products can be used for all of 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, Acremonium chrysogenum IS-5 [Ministry of International Trade and Industry, Institute of Industrial Technology, Institute for Microbial Technology;
No. 232, Micro Engineering Research Article 3153 (FERM BP-3
153) as a deposit can be used. In addition, extraction of total DNA from the bacterium can be performed, for example, by Johnstone et al. [John
stone et al., EMBO Journal (1985) 4 1307-1311], or Minuth et al. [Minuth et al., Current Gene
tics (1982) 5: 227-231]. Examples of the vector in (2) above include E
Lambda phage vectors such as MBL3 and EMBL4 (STRATAGENE) or pHC79 (Bethesda Researc)
h Laboratories: BRL) or the like can be used. The DNA probe of (3) above is purified O
Polymerase chain reaction using mixed primers designed based on the partial sequence of AH sulfhydrylase (P
CR). For example, a double-stranded cDNA synthesized from Acremonium chrysogenum RNA according to a conventional method is used as a template, and O of SEQ ID NO: 6 in the Sequence Listing is used.
PCR is carried out using the AH1 primer and the OAH2 primer of SEQ ID NO: 7 in the sequence listing, and the amplified 830 bp partial cDNA fragment can be used as a probe.

Further, labeling of the probe is carried out by labeling with a radioisotope or digoxigenin which has been widely used in the past.
Any of non-radioactive compounds such as biotin can be used. Examples of the Acremonium chrysogenum transformation vector in (7) above include pPGKM5 and pPGKM5.
ACTHY83 or the like (Japanese Patent Application Laid-Open No. 4-58891) can be used. In addition, Acremonium in (7)
For example, transformation of chrysogenum can be performed by the method of Queener et al. [Queener et al: Microbiology 1985, American Society].
for Microbiology (1985) p468-472].

OAH in 1- (1) and 2- (8) above
The sulfhydrylase activity can be measured, for example, by the method described by Yamagata et al. [S. Yamagata et.al. Methods in ENZYM
OLOGY 143 (1987) 465-469]. Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to the Examples. The abbreviations and abbreviations described in the examples are as follows.

Maniatis' Experiment Book: (T. Maniatis et a
l., Molecular Cloning A Laboratory Manual, Cold Spr
ing Harbor Laboratory 1982) 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 ml contained 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 / yeast extract 4 g / peptone 10
g dissolved in 1 liter of water. CM solid medium: CM medium containing 1.5% agar.

GAG medium: 40 g of glycerol / 4 g of asparagine / 0.1 g of calcium chloride / 0.1 g of sodium chloride / trace metal solution “magnesium sulfate (heptahydrate)”
4 g / iron (II) sulfate (heptahydrate) 0.4 g / manganese sulfate (tetrahydrate) 0.16 g / zinc sulfate (heptahydrate) 0.4 g
/0.04 g of anhydrous copper sulfate dissolved in 1 liter of water "25 ml / 0.1M phosphate buffer (p
H7.0) A medium containing 30 ml in 1 liter of water.

TEDPG-buffer: 50 mM Tris (pH 7.8) / 1 mM EDTA / 0.1 mM DTT /
0.2 mM pyridoxal phosphate / 30% glycerol. P-buffer: 0.6M potassium chloride / 0.01M magnesium chloride / 0.025M calcium chloride. AspN-buffer: a mixture of 8M urea / 50 mM sodium phosphate (pH 8.0) and 45 mM DTT in a ratio of 10: 1.

PEG solution: 25% polyethylene glycol (-4000) /0.01 M Tris (pH 8.0) /
0.05M calcium chloride / 0.6M potassium chloride. 6
X 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.

In the following examples, unless otherwise specified, the DNA fragments generated by restriction enzyme digestion were separated and purified from an agarose gel by Gene Clean (GENE CLE).
AN ^{TM (} Funakoshi Co., Ltd.) according to the attached protocol to perform basic operations in subcloning, plasmid construction, etc. (ligation reaction between DNA fragments, transformation of Escherichia coli with hybrid plasmid generated by the reaction,
Preparation of a plasmid from the obtained transformant and its analysis, etc.) were all carried out according to the above Maniatis experiment manual.

[0023]

EXAMPLES Next, examples of the present invention will be described.
It does not limit the present invention in any way.

[0024]

Example 1 a. Purification of OAH sulfhydrylase. Acremonium chrysogenum IS-5 (FERM B
The P-3153) strain was cultured in an N-3 seed medium at 28 ° C. for 70 hours and then centrifuged (5000 g, 5 minutes) to obtain a bacterial cell having a wet weight of about 230 g. The cells were washed with TEDPG-buffer, and the collected cells were suspended again in 400 ml of TEDPG-buffer. This suspension was sonicated while cooling with ice water (200 W, 2 minutes),
The supernatant after centrifugation at 100,000 g for 1 hour was recovered as a crude enzyme solution. Crude enzyme solution has total protein of 2295m
g, total activity 34.9 U.

900 ml of the crude enzyme solution thus obtained was adsorbed on a Q-Sepharose column (5 × 11 cm) which had been equilibrated with TEDPG-buffer in advance.
After washing with 330 ml of the same buffer, TED
Elution was performed with a linear concentration gradient (total volume 600 ml) of a salt concentration of 0 to 200 mM in PG-buffer. The flow rate was 0.5 ml / min.

As a result, 110 ml of an OAH sulfhydrylase active fraction (total protein: 176 mg, total activity: 25.3 U) was recovered. Then, a part of this active fraction was concentrated to 7 ml using Centriprep and adsorbed on monoQ HR5 / 5 (manufactured by Pharmacia) equilibrated with TEDPG-buffer in advance. After washing with 20 ml of the same buffer, elution was carried out with a linear concentration gradient of a salt concentration of 0 to 200 mM in TEDPG-buffer, and 0.5 ml of the main active fraction (total protein 0.5 mg, total activity 1.38 U). ) Got. Then, a 5 M sodium chloride solution was added to this active fraction so that the final concentration was 0.3 M, and the fraction was concentrated to 0.1 ml using Centricon.

This concentrated solution was preliminarily equilibrated with TEDPG-buffer containing 0.3 M sodium chloride, Protein.
It was filled in Pak300 (manufactured by Waters) and gel filtration was performed at a flow rate of 0.3 ml / min. As a result, OAH sulfhydrylase was found to have a 1.3 ml fraction (total protein 0.08 mg, having an activity peak near 180 kd).
It was recovered as a total activity of 0.4 U). This active fraction was desalted and concentrated using Centriprep and Ultrafree (cut 10,000, manufactured by Millipore) to give 0.07 ml.
It was recovered as an aqueous solution of (total protein 0.07 mg).

A part of the purified sample thus obtained (3
(Equivalent to μg) was subjected to boiling reduction treatment in the presence of β-mercaptoethanol, and then subjected to SDS-polyacrylamide gel electrophoresis, whereupon a single band was detected at a position of a molecular weight of about 49 kd. In the above process, the OAH sulfhydrylase activity was measured as follows. 10mMO
-Acetylhomoserine, 8 mM sodium sulfide, 0.0
A total volume of 250 microliters solution consisting of 75 mM pyridoxal phosphate, 100 mM potassium phosphate (pH 7.2) and enzyme solution (10 to 50 microliters) was prepared and reacted at 30 ° C. for 5 minutes. Subsequently, OAH sulfhydrylase activity was determined by quantifying homocysteine in the reaction solution. Homocysteine is quantified by Kred
ich et al. (NMKredich and MABecker Methods i
n Enzymology Vol.17.PartB.p459-470).

That is, 0.2 ml of reaction solution and 1 mM
Mix 1 ml of nitrous acid (mixture of 0.1 M nitrous acid and 0.4 N sulfuric acid 1:99), leave at room temperature for 6 minutes, and
Add 1 milliliter of ammonium sulfamate,
Mix and leave at room temperature for 2 minutes. Then, 2% mercury chloride dissolved in 0.4N hydrochloric acid, 6.88% sulfanilamide dissolved in 0.4N hydrochloric acid and 0.2% N-1-naphthylethylenediamine dihydrochloride dissolved in 0.4N hydrochloric acid were added to the reaction solution. It was determined by adding 1.6 ml of a solution prepared by mixing salt at a ratio of 1: 4: 2 and measuring the absorbance at 540 nm. b. Determination of partial amino acid sequence 15 microliters of purified enzyme (15μ
(corresponding to g) is freeze-dried and then AspN-buffer 2
It was dissolved in 7.5 microliters and incubated at 50 ° C. for 15 minutes. After cooling to 20 ° C, 2.5 microliters of 100 mM iodoacetamide was added to the reaction solution, and the mixture was incubated at 20 ° C for 15 minutes. Subsequently, 170 microliters of sterilized water and 75 ng of Endoproteinase Asp-N (Boehringer) were added to the reaction solution and reacted at 37 ° C. for 17 hours to fragment the enzyme protein.

The reaction solution thus obtained was mixed with μRPCC2 /
It was adsorbed on a C18SC2.1 / 10 (Pharmacia) column and eluted with a linear concentration gradient of 0 to 100% acetonitrile containing 0.01% TFA to fragment the decomposed peptide fragment. The N-terminal amino acid sequences of three of the peptide fragments that were subdivided and recovered were determined by a gas phase sequencer (PSQ-1 system manufactured by Shimadzu Corporation). The determined amino acid sequence is shown in the sequence listing and below. 1. Asp-Asn-Ile-Val-Ser-Thr-Ser-Asn-Leu-Tyr-Gly-Gly
-Thr-Tyr-Asn 2.Asp-Lys-Gly-Val-Pro-Leu-Ile-Val-Asp-Asn-Thr-Phe
-Gly-Ala-Gly-Gly-Tyr-Phe-Ile-Arg-Pro-Val-Asp-His-G
ly 3.Asp-Ala-Lys-Thr-Leu-Ala-Ile-His-Pro-Trp-Ser-Thr
-Thr-His-Glu The sequence of these peptide fragments shows homology with the amino acid sequence of yeast-derived OAH sulfhydrylase whose structure has already been clarified. Is the yeast OAH sulfhydrylase 102-1
16, 175-199, corresponding to the 384-398th amino acid sequence, that is, these three peptides are peptide 1 from the N-terminal to the C-terminal of OAH sulfhydrylase derived from Acremonium chrysogenum,
It became clear that they were arranged in the order of a few.

[0031]

Example 2 Cloning of OAH Sulfhydrylase 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 inoculated into 50 ml of N-3 seed medium and cultured at 25 ° C for 3 days. did.
1 ml of the obtained culture solution was transferred to a 500 ml flask containing 30 ml of the main medium,
The cells were cultured at 25 ° C for 3 days. The mycelium collected from 10 ml of the bacterial solution by suction filtration was frozen in liquid nitrogen,
Ground with a mortar and pestle. Using the TOTAL RNA ISOLATION KIT (Invitrogen) from the crushed bacterial powder thus obtained, the total R was measured according to the attached protocol.
800 μg NA was extracted. The total RNA was then subjected to oligo (dT) cellulose chromatography according to Maniatis's protocol to obtain poly A ⁺ RN.
A 27 μg was obtained. b. Preparation of cDNA library Using the cDNA synthesis system (Boehringer), and following the a. Poly A ⁺ R obtained in
NA (4 μg) to single-stranded DNA, then double-stranded cDNA
A was synthesized. Next, an EcoRI adapter (Takara Shuzo) was attached to the end of the cDNA by the action of T4 ligase, and then a Chroma Spin 400 column (CHROMA SPIN400 col) was attached.
umn: Clontech), free adapters and short cDNA fragments of 500 bp or less were removed.

CD having the EcoRI end thus obtained
The NA fragment was transferred to the EcoRI arm of λZipLox (BRL
, And then infected with E. coli SURE strain to obtain a cDNA library derived from Acremonium chrysogenum IS-5 strain consisting of about 2 × 10 ⁶ clones. In the above step, a linker primer having a sequence described in Nojima et al. In the method for constructing a Biomanual Series 2 gene library (Yodosha) was used as a primer for the synthesis of single-stranded cDNA in place of oligo dT. . In addition, GIGAPACK II Gold (ST
RATAGENE Co., Ltd.) was used and the protocol attached thereto was used. c. Isolation of OAH Sulfhydrylase Partial cDNA Two types of primers synthesized based on partial amino acid sequence information of OAH sulfhydrylase derived from Acremonium chrysogenum revealed in Example 1 (OAH1: No. 8 of the above peptide fragment 1) To the 14th sequence, OAH2: antisense primer corresponding to the 6th to 12th sequences of the above peptide fragment 3) and the double-stranded cDNA obtained in b as the template DNA.
By performing PCR using A, a cDNA fragment encoding a part of OAH sulfhydrylase was obtained. The details of the experiment are described below. The above primer OAH
The sequences of 1 and OAH2 are shown in SEQ ID NOs: 6 and 7 of the sequence listing, respectively.

PCR was performed using a 10-fold concentration of reaction buffer (TaKa
Buffer attached to Ra Taq: Takara Shuzo Co., Ltd.) 5 microliters, 2.5 mM dNTP mixed solution 5 microliters,
2.5 microliters of each of the above primers OAH1 and OAH2 (each having a concentration of 2 μg / milliliter),
Taq DNA polymerase (TaKaRa Taq .: Takara Shuzo) 0.
5 microliters, above b. Double-stranded cDNA2 obtained in
Prepare a reaction system consisting of 32.5 microliters of sterile deionized water and microliters (equivalent to about 20 ng),
Incubation at 94 ° C for 1 minute, 50 ° C for 2 minutes, 72 ° C for 2 minutes perkin Elmer Cetus D
NA Thermal Cycler (Perkin / Elmer / Cetus /
30 cycles were carried out using DNA. Thermal. Cycler). When the obtained reaction solution was analyzed by agarose gel electrophoresis, a band of about 900 bp was detected.

Then, the fragment was extracted from the gel and pC was obtained using the TA cloning system (Invitrogen).
After cloning into RII, its nucleotide sequence was determined according to the method of Sanger et al. [Proc. Nat. Acad. Sci. USA 74 5463 (1977)] by Applied Biosyste.
ms) 373 DNA Sequencer. As a result of sequence analysis, a single open reading frame was found that could correctly encode all the partial amino acid sequences of the three peptide fragments determined in Example 1.

As a result of the above, cD encoding a part of OAH sulfhydrylase of Acremonium chrysogenum
It was judged that the NA fragment could be isolated. And the part cd
The NA fragment (about 500 ng) was labeled with digoxigenin-labeled deoxyuridine triphosphate (DIG-dUTP), and
It was called an AH probe and was subjected to the following hybridization. The labeling with DIG-dUTP is performed by the non-radio system DNA labeling system (DNA Labeling Kit: Boe
hringer) and according to the protocol attached thereto. d. Isolation of OAH Sulfhydrylase cDNA Above b. Escherichia coli SURE strain (STRATAGENE) was infected with a part of the cDNA library constructed in 1. to form about 100,000 plaques on L-broth agar medium (10 plates). Then, the plaques were transferred onto High Bond N (Amersham) according to the attached protocol of Amersham, subjected to alkali denaturation and neutralization treatment, and UV-irradiated D
NA was fixed. The filter thus obtained was used as described in c. It was hybridized with the OAH probe obtained in.

Hybridization is 50%
Formamide, 5 × SSC, 0.02% SDS, 2% Blocking Reagent (Boehringer), 0.
Performed at 42 ° C. for 16 hours using a solution containing 1% N-lauroyl sarcosine and a DIG-dUTP-labeled OAH probe (added after heat denaturation treatment at 100 ° C. for 5 minutes) at a final concentration of 10 ng / ml. It was

Then, the filter was washed twice in 2 × SSC solution containing 0.1% SDS at room temperature for 10 minutes each time, and further in 0.1 × SSC solution containing 0.1% SDS at 68 ° C. It was washed twice for 15 minutes each. Then, a detection kit using an anti-digoxigenin antibody labeled with alkaline phosphatase and a luminescent substrate AMPPD (DIG Lumine
scent Detection Kit: Boehringer) was used to detect the clone hybridized with the OAH probe according to the attached protocol. As a result, 20 hybridization positive plaques were found. So, after purifying 10 of these positive plaques,
The phage contained in each plaque was transformed into E. coli DH10.
B (ZIP) strain (BRL) was infected with recombinant λDN
Conversion from A to recombinant plasmid DNA and recovery were performed.

The thus obtained 10 kinds of plasmids were designated as E
When cleaved with coRI and analyzed by agarose gel electrophoresis, it was revealed that the plasmid named pCOAH1 had the longest cDNA insert (about 1.6 Kb). Therefore, the cD of pCOAH1
The entire base sequence of the NA insert is shown in the above c. It was determined by the same method as.

1538 bp DNA thus determined
The nucleotide sequence is shown in the sequence listing (SEQ ID NO: 1). In this DNA sequence, there was an open reading frame (ORF) capable of encoding 433 amino acids from ATG starting at the 79th position to TCC ending at the 1377th position. The amino acid sequence translated from the ORF is shown below the base sequence in the sequence listing (SEQ ID NO: 1). As a result of computer search, this amino acid sequence showed about 60% homology with that of budding yeast OAH-OAS sulfhydrylase.

[0040]

Example 3 Isolation of the OAH Sulfhydrylase Gene a. Preparation of Acremonium chrysogenum gene library The total DNA of the Acremonium chrysogenum IS-5 strain was analyzed by Aspergillus
Method used for Nidulans [Reference: EMBOJ. (1985)
4, 1307-1311]. And this whole DNA
60 μg was partially digested with MboI and then treated with alkaline phosphatase. On the other hand, the lambda vector EMBL3
(Promega Biotech) 10 μg of BamHI and E
It was digested to completion with coRI and the short EcoRI-BamHI linker moiety was removed by isopropanol precipitation. Then, 1 μg of the partially digested DNA fragment and BamHI
2 μg of a vector having an end was ligated with T4-ligase and encapsulated in lambda phage particles.

The thus-obtained recombinant phage suspension was appropriately diluted, and Escherichia coli NM5 was obtained.
39 (Promega Biotech) was infected and the number of plaques that appeared was counted. As a result, this suspension is 3 x 1
It was found to contain 0 ⁵ recombinant phages. This phage solution was used as an Acremonium chrysogenum gene library and stored at 4 ° C.

Detailed methods and conditions for the preparation of the above-mentioned donor DNA and vector, and their binding and the like were those described by Frischauf et al. [Reference: Journal of Molecular Biology (Journal of Molecular Biology). J. Mol. Biol) (1983) 170 p827-842]. The encapsulation of the DNA in the lambda particles was performed using the packaging extract of Promega Biotech, Inc. according to the protocol attached thereto. b. Screening by hybridization a. Above. Phage solution (DNA library)
A part of the cells was infected with NM539 to form a total of about 6000 plaques on three plates. These plaques were treated in Example 2d. After transferring to a High Bond N filter in the same manner as above, alkali denaturation and neutralization treatments were carried out, and DNA was fixed by UV-irradiation. The plaques showing a positive signal were screened by hybridizing with the OAH probe obtained in the above.

The operations such as hybridization, filter washing, and detection of hybridization signal were all carried out in the above-mentioned Example 2d. I went the same way. As a result, 6 positive spots were found. Therefore, phages were extracted from the agar regions corresponding to these positive spots, and plaque hybridization was performed again according to the above steps to obtain 6 purified positive phage clones. c. Subcloning of OAH Sulfhydrylase Gene and Limitation of Its Position Above b. The method described in Grossberger from 6 types of phage clones obtained in [Reference: Nucleic Acid
S. Research (1987) 15,6737]. This λDNA is then digested with HindIII,
After performing agarose gel electrophoresis, Southern hybridization was carried out using an OAH probe [For the method, refer to Southern, (J. Mol. Biol.) (1975) 98, 503-5.
17] Incidentally, the operations such as hybridization, washing of the filter (High Bond N), detection of signals and the like are carried out according to the above b. The procedure was the same as that described in. As a result, it was revealed that the HindIII fragment of about 4.7 Kb, which is commonly present in 5 of the 6 λ clones, hybridizes with the probe.

Acremonium chrysogenum IS
-5 strain-derived chromosomal DNA was digested with HindIII,
When the Southern analysis described above was performed, it was about 4.7 in this case as well.
Hybridization signals were observed only for the Kb fragment. Then, the 4.7 Kb fragment was separated and purified from one of these 5 λ clones and subcloned into the HindIII site of pUC119 (Takara Shuzo) to obtain pOHH1. In addition, pOHH1 is set to Hi
After cleaving with ndIII, Southern analysis was repeated again to confirm that the plasmid carries the desired insert.

Next, pOHH1 was digested with various restriction enzymes and analyzed by agarose gel electrophoresis.
A restriction enzyme map of the 4.7 Kb insert shown in Figure 3 was obtained. Then, in order to limit the OAH sulfhydrylase gene coding region present in this fragment, Southern hybridization experiments were performed again between digested fragments of pOHH1 with various restriction enzymes and the OAH probe.
As a result, about 2.8 Kb of PstI in the insert
-It was revealed that the OAH sulfhydrylase gene was present in the SmaI fragment. d. Base sequence of OAH sulfhydrylase gene The entire base sequence of the PstI-SmaI fragment was determined based on the method of Sanger et al. Then, the base sequence and the base sequence of the OAH sulfhydrylase cDNA determined in Example 2 were compared and analyzed. As a result, the PstI-S
The maI fragment is OA derived from Acremonium chrysogenum.
The entire coding region of the H-sulfhydrylase gene (the sequence of the coding region was completely identical to that of cDNA except that it was divided by 5 introns).
It became clear that it included all. It was decided 2
The 860 bp sequence is shown in the sequence listing (SEQ ID NO: 2) together with the expected translation product.

[0046]

Example 4 Construction of pTOAH1 Plasmid pT for introducing an additional copy of the OAH sulfhydrylase gene into Acremonium chrysogenum.
OAH1 was constructed. The process will be described below. First, the plasmid pOHH1 obtained in Example 3 was digested with XbaI and then treated with alkaline phosphatase. On the other hand, p
ACTHY83 was double-cut with XbaI and SpeI and then subjected to agarose gel electrophoresis to separate and purify a fragment of about 3.2 Kb containing a hygromycin B phosphotransferase gene expression unit.

Then, both were ligated with T4-ligase to obtain pTOAH1. The plasmid pACTHY83 used in this example is a hygromycin B phosphotransferase expression unit capable of functioning in Acremonium chrysogenum (promoter and terminator of actin gene derived from Acremonium chrysogenum, hygromycin B phosphotransferase gene derived from bacteria). Is an Acremonium chrysogenum transformation vector having an expression unit bound in an arrangement suitable for expression, and its construction method is described in JP-A-4-588.
No. 91 publication.

[0048]

Example 5 Transformation of Acremonium chrysogenum with pTOAH1 pTOAH1 was transformed into Acremonium chrysogenum IS-5.
By introducing into the strain, a transformant with enhanced OAH sulfhydrylase synthesis ability was obtained. The details will be described below. a. Preparation of Protoplasts 50 ml of CM medium was inoculated with the mycelium of Acremonium chrysogenum IS-5 grown on a CM solid medium at 30 ° C. for 5 days, and a rotary shaker (250 rp.
m. ), And cultured at 30 ° C. for 3 days. Furthermore, 1 ml of the bacterial solution was inoculated into 50 ml of GAG medium,
It was cultured at 30 ° C. for 20 hours. 50 ml of the obtained culture solution was added to 3500 r.p.m. p. m. After precipitating the mycelium by centrifugation for 10 minutes, wash with 0.9% NaCl solution,
It was suspended in 20 ml of McIlvein buffer solution (0.1 M citric acid, 0.2 M sodium phosphate, pH 7.3) containing 0.01 M dithiothreitol, and gently shaken at 30 ° C. for 1 hour.

Next, the mycelium was collected at 3200 r.p. p. m. 1
After precipitation by centrifugation for 0 minutes and washing with P-buffer, the product was suspended in 10 ml of P-buffer containing Novozyme (Novo) at a concentration of 10 mg / ml and gently shaken at 30 ° C for 1 hour. The reaction solution was added to 800 r. p. m. The supernatant obtained by centrifuging for 30 seconds with a filter paper (TOYO FILTER PAPER 5
The mycelium and protoplasts were separated by filtration using A). Then, the filtrate was added to 3000 r. p. m. After centrifuging at 50 ° C. for 5 minutes to precipitate protoplasts, the protoplasts were washed once with P-buffer and suspended in P-buffer so that the concentration of protoplasts was 3 × 10 ⁸ cells / ml. b. Protoplast transformation with pTOAH1. After adding 5 μg (10 microliters) of pTOAH1 to 0.1 ml of the protoplast suspension obtained in the above, 0.05 ml of the PEG solution was added and mixed gently. The mixture was allowed to stand on ice for 25 minutes, then 1 mL of the PEG solution described above was added, and the mixture was allowed to stand at room temperature for another 30 minutes. Each 0.2 ml of the thus-obtained transformed protoplast suspension was referred to as "protoplast regeneration medium" (Isogai et al .: Agric. Biol. Chem.
51 (1987) 2321-2329)) was spread on a plate containing 25 ml of the BRM medium and incubated at 15 ° C. for 20 hours. Then, the plate contained 4.5 mg of hygromycin B, 50 Same as above B kept at ℃
After overlaying 5 ml of RM medium, it was cultured at 28 ° C. for 14 days. As a result, dozens of transformants resistant to hygromycin B (hereinafter abbreviated as HYB transformants) appeared. c. Measurement of OAH sulfhydrylase activity IS-5 strain and the above b. HY by pTOAH1 obtained in
4 strains randomly selected from B transformants (each TOA
H-1, 2, 4, 5) and N3-seed medium 50
The mixture was inoculated into milliliters and shake-cultured at 25 ° C. for 3 days (220 rpm). 1 ml of the culture solution,
The mixture was transferred to a 500 ml flask containing 30 ml of the main medium, and cultured with shaking at 25 ° C. for another 3 days (220 rpm). 1 ml of each culture thus obtained was collected by centrifugation, suspended in 1 ml of TEDPG buffer, and the cells were disrupted by an ultrasonic disruptor. The supernatant obtained by centrifuging the disrupted solution was used as a crude cell extract.

0.1 ml of the extract and 10 mM O-acetylhomoserine at a final concentration, 8 mM sodium sulfide, 0.075 mM pyridoxal phosphate, 100 m
0.15 ml of a solution containing each of M potassium phosphate (pH 7.2) was prepared and reacted at 30 ° C. for 10 minutes, and then the solution of Example 1a. OAH sulfhydrylase activity was determined by quantifying the amount of homocysteine produced by the method described in 1.

As a result, as shown in Table 1, pTOAH1
Most of the HYB transformants of E. coli showed higher (2-6 fold) OAH sulfhydrylase activity than strain IS-5.
That is, this result showed that it was possible to increase the OAH sulfhydrylase activity in Acremonium chrysogenum by using the DNA fragment of the present invention. In addition, the result was obtained by adding O to the above 4.7 Kb DNA fragment.
It is suggested that the promoter and terminator of the AH sulfhydrylase gene are contained. The amount of enzyme that produces 1 nmole of homocysteine in 1 minute of reaction was defined as 1 unit, and the number of units per 1 mg of protein in the crude extract is shown in Table 1 as OAH sulfhydrylase activity.

[0052]

[Table 1]

[0053]

As described in the embodiments, the DN of the present invention
By utilizing the A fragment, it became possible to create a new Acremonium chrysogenum having an enhanced OAH sulfhydrylase activity. Therefore, by enhancing the gene fragment or the cDNA compound corresponding to the gene alone or in combination with the cephalosporin C biosynthesis gene, it is possible to create an Acremonium chrysogenum strain having improved cephalosporin C fermenting ability. Can be expected. Further, the promoter and terminator portion of the present invention can be used as a constituent element of an expression vector for Acremonium chrysogenum.

[0054]

[Sequence Listing] SEQ ID NO: 1 Sequence Length: 1538 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: cDNA to mRNA Origin Biological Name: Acremonium c
hrysogenum) Strain name: IS-5 Sequence features Characteristic symbols: CDS Location: 79. ． 1380 Method of characterizing: P-sequence AGCGTTCTTT GTTGTGGAAC GTAGCTTCCA GATTCCTGTA TACCTACCTG TTGATCAGAG 60 TAGAGACAAT CCAGCGCC ATG TCG GAC CTC AGC AAG AAC TTC GAG ACC CTG 111 Met Ser Asp Leu Ser Lys Asn Phe GluG CTC 1G 10G GAG CCT GAC CCC ACG ACC AAC TCC CGC GCC 159 Gln Leu His Ala Gly Gln Glu Pro Asp Pro Thr Thr Asn Ser Arg Ala 15 20 25 GTC CCT ATC TAC GCG ACC ACG AGC TAC ACC TTC AAT GAC TCG GCC CAC 207 Val Pro Ile Tyr Ala Thr Thr Ser Tyr Thr Phe Asn Asp Ser Ala His 30 35 40 GGC GCG AGG CTC TTC GGC CTC AAG GAG TTC GGC AAC ATC TAC AGC CGC 255 Gly Ala Arg Leu Phe Gly Leu Lys Glu Phe Gly Asn Ile Tyr Ser Arg 45 50 55 ATC ATG AAC CCG ACC GTG GAC GTC TTC GAG AAG CGC ATC GCC GCT CTC 303 Ile Met Asn Pro Thr Val Asp Val Phe Glu Lys Arg Ile Ala Ala Leu 60 65 70 75 GAG GGT GGT GTC GCC GCA CTG GCC GCG TCC TCG GGC CAG GGC GCC CAA 351 Glu Gly Gly Val Ala Ala Leu Ala Ala Ser Ser Gly Gln Gly Ala Gln 80 85 90 TTC ATC GCC ATC GCG ACC CTC GCC CAT GCC GGC GAC AA C ATC GTG TCC 399 Phe Ile Ala Ile Ala Thr Leu Ala His Ala Gly Asp Asn Ile Val Ser 95 100 105 ACC TCC AAT CTC TAC GGC GGC ACC TAC AAC CAG TTC AAG GTC TTC CTC 447 Thr Ser Asn Leu Tyr Gly Gly Thr Tyr Asn Gln Phe Lys Val Phe Leu 110 115 120 CCC CGC CTG GGC ATC AAG ACC AAG TTC GTC GAC GGC GAC AAG CCC GAG 495 Pro Arg Leu Gly Ile Lys Thr Lys Phe Val Asp Gly Asp Lys Pro Glu 125 130 135 GAC ATT GGC GCT GCC ATT GAC GAC AAG ACC AAG GCT GTG TTC ATT GAG 543 Asp Ile Gly Ala Ala Ile Asp Asp Lys Thr Lys Ala Val Phe Ile Glu 140 145 150 155 AGC ATC GGC AAC CCG CGG TAC AAT GTC CCC GAC ATC GAG GCC ATT GCC 591 Ser Ile Gly Asn Pro Arg Tyr Asn Val Pro Asp Ile Glu Ala Ile Ala 160 165 170 AAG GTG GCG CAC GAC AAG GGC GTG CCT CTG ATT GTC GAC AAC ACG TTC 639 Lys Val Ala His Asp Lys Gly Val Pro Leu Ile Val Asp Asn Thr Phe 175 180 185 GGT GCG GGC GGC TAC TTC ATC CGT CCG GTC GAC CAC GGC GCC GAT ATT 687 Gly Ala Gly Gly Tyr Phe Ile Arg Pro Val Asp His Gly Ala Asp Ile 190 195 200 GTC GTC CAC TCG GCC ACC AAG TGG ATC GGC GGC CAC GGC ACC ACC ATT 735 Val Val His Ser Ala Thr Lys Trp Ile Gly Gly His Gly Thr Thr Ile 205 210 215 GGC GGT GTC ATC GTC GAC TCC GGC AAG TTC GAC TGG GGC AAG CAC GGT 783 Gly Gly Val Ile Val Asp Ser Gly Lys Phe Asp Trp Gly Lys His Gly 220 225 230 235 GCG CGC TTC CCC CAA TTC GTC GAG CCG TCC GAG GGC TAC CAC GGC CTG 831 Ala Arg Phe Pro Gln Phe Val Glu Pro Ser Glu Gly Tyr His Gly Leu 240 245 250 AAG TTC TGG GAG ACC TTC GGC CCC ATC TCC TTC ATC ATC CGC GCC CGT 879 Lys Phe Trp Glu Thr Phe Gly Pro Ile Ser Phe Ile Ile Arg Ala Arg 255 260 265 GTC GAG GTC CTG CGC GAC CTC GGC GCC GCC CTC AAC CCC TTC GCC GCC 927 Val Glu Val Leu Arg Asp Leu Gly Ala Ala Leu Asn Pro Phe Ala Ala 270 275 280 CAG CAG CTG CTG CTC GGC ATC GAG ACG CTC AGC CTG CGC GCC GAG CGC 975 Gln Gln Leu Leu Leu Gly Ile Glu Thr Leu Ser Leu Arg Ala Glu Arg 285 290 295 CAC GCC CAG AAC GCC CTC GCC CTG GCC AAG TAC CTC GAG AAC AGC CCC 1023 His Ala Gln Asn Ala Leu Ala Leu Ala Lys Tyr Leu Glu Asn Ser Pro 300 305 310 315 AAC GTC AGC TGG GTC TC TC G TAC CCC GGC CTC GAG AGC CAC GCT TAC CAC 1071 Asn Val Ser Trp Val Ser Tyr Pro Gly Leu Glu Ser His Ala Tyr His 320 325 330 GCC ACG GCC AAG AAG TAC CTG ACC AAG GGC TTT GGC GGC GTC CTG AGC 1119 Ala Thr Ala Lys Lys Tyr Leu Thr Lys Gly Phe Gly Gly Val Leu Ser 335 340 345 TTC GGC GTC AAG GGC GGC GGT GCT GCC GGC AGC AAG ATT GTC GAC GGC 1167 Phe Gly Val Lys Gly Gly Gly Ala Ala Gly Ser Lys Ile Val Asp Gly 350 355 360 TTC AAG CTA ATC TCC AAC CTG GCC AAC GTG GGC GAC GCC AAG ACC CTG 1215 Phe Lys Leu Ile Ser Asn Leu Ala Asn Val Gly Asp Ala Lys Thr Leu 365 370 375 GCC ATT CAC CCT TGG AGC ACG ACG CAC GAG CAG CTA TCG GAC GAG GAG 1263 Ala Ile His Pro Trp Ser Thr Thr His Glu Gln Leu Ser Asp Glu Glu 380 385 390 395 AAG ATC AGC TCC GGT GTC ACC GAA GAC CTC ATC CGT ATC TCG GTC GGA 1311 Lys Ile Ser Ser Gly Val Thr Glu Asp Leu Ile Arg Ile Ser Val Gly 400 405 410 ATC GAG CAC ATT GAC GAC ATC ATT GCC GAT TTC GAT CAG TCG TTC AAG 1359 Ile Glu His Ile Asp Asp Ile Ile Ala Asp Phe Asp Gln Ser Phe Lys 415 420 425 GCG GCC GCT GGC AGC TCC TGAGTGCTGC AGGCGCCGTC TGATTGGAGG 1407 Ala Ala Ala Gla Ser Ser 430 TGGTAATACC CTTATAGATA AGATATGAAT AGCAGCGTTT CCTTTGTAGA AGATGTTTTT 1467 GAGCAATTTG ATAATGAATT GGACGTGATG ATGTAAAAAAAAAAAAAAAAAAAAAAA

[0055]

[Sequence list]

SEQ ID NO: 2 Sequence length: 2860 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Acremonium chrysogenum (Acremonium c)
hrysogenum) Strain name: IS-5 Sequence features Characteristic symbols: intron Location: 496..646, 702..771, 1216..1315, 1915..19
80, a method to determine the 2045..2105 wherein: E SEQ CTGCAGCAAT AGACGGCCAA CGTCGACCGG CGCCGTGATT TCCATGAATA ATGACGCCCA 60 GACGTCGGCG ATGGCGTGCT TACCTGTAGT CGTCGACCCT TGGCCAACCC CACATGCCTT 120 TGCCCGATGG GGACAGCTGC CGGGCTTCAC ATTGGGCCAG CTCCCGCGGG AGTTCGGGTT 180 AGCGCGCGTC GCTCGCATCG AACGGCTGTG CTATCCCACG GTCGCTAGCA AACTGAAATG 240 CTTCCCGCCG CGGTCGGATG GAGGGGGAGG AGGGCGGGGG GAAAAGACAT ATAACGCTAT 300 GGAAGAGTCG GGAACCCCCC AAGGAACCAG TCCCCTACAC CTCCCGCTTC ACTTTTTCCT 360 CCCCTCTTCC AGCGTTCTTT GTTGTGGAAC GTAGCTTCCA GATTCCTGTA TACCTACCTG CCTC CATG CATG CATG CATG CATG CATG CATG CATG CATG CATG CATG CATG CTG CATG CTG ATC ATC ATC ATC TGT 472 Met Ser Asp Leu Gln Leu His Ala Gl 10 15 TCGAAGGGAG AGAAGGAAGG AGGGGGGTCC CATCCCAAAC ACTCGGGAAG AGACGACGTG 585 CCCGAGATAC CTTTATGGAA GTCTGTAGTC GAATCGGGAA AGAGTGCTAA CGACTGGACA 645 GT CAG GCG CAG APR GTC ACG APR GAG CGT G g Ala Val Pro Ile Tyr 20 25 30 GCG ACC ACG GTACGTGTTA CGCCGCGGAC AGACCTGATC AGCGCATTTT 741 Ala Thr Thr CCAGAAGGGT TAACTTGACA TACCTCGCAG AGC TAC ACC TTC AAT GAC TCG GCC 795 Ser Tyr Thr Phe Asn Asp Ser AlaCG 40 CGC CTC GTC AAG GAG TTC GGC AAC ATC TAC AGC 843 His Gly Ala Arg Leu Phe Gly Leu Lys Glu Phe Gly Asn Ile Tyr Ser 45 50 55 CGC ATC ATG AAC CCG ACC GTG GAC GTC TTC GAG AAG CGC ATC GCC GCT 891 Arg Ile Met Asn Pro Thr Val Asp Val Phe Glu Lys Arg Ile Ala Ala 60 65 70 CTC GAG GGT GGT GTC GCC GCA CTG GCC GCG TCC TCG GGC CAG GGC GCC 939 Leu Glu Gly Gly Val Ala Ala Leu Ala Ala Ser Ser Gly Gln Gly Ala 75 80 85 90 CAA TTC ATC GCC ATC GCG ACC CTC GCC CAT GCC GGC GAC AAC ATC GTG 987 Gln Phe Ile Ala Ile Ala Thr Leu Ala His Ala Gly Asp Asn Ile Val 95 100 105 TCC ACC TCC AAT CTC TAC GGC GGC ACC TAC AAC CAG TTC AAG GTC TTC 1035 Ser Thr Ser Asn Leu Tyr Gly Gly Thr Tyr Asn Gln Phe Lys Val Phe 110 115 120 CTC CCC CGC CTG GGC ATC AAG ACC AAG TTC GTC GAC GGC GAC AAG CCC 1083 Leu Pro Arg Leu Gly Ile Lys Thr Lys Phe Val Asp Gly Asp Lys Pro 125 130 135 GAG GAC ATT GGC GCT GCC ATT GAC GAC AAG ACC AAG GCT GTG TTC ATT 1131 Glu Asp Ile Gly Ala Ala Ile Asp Asp Lys Thr Lys Ala Val Phe Ile 140 145 150 GAG AGC ATC GGC AAC CCG CGG TAC AAT GTC CCC GAC ATC GAG GCC ATT 1179 Glu Ser Ile Gly Asn Pro Arg Tyr Asn Val Pro Asp Ile Glu Ala Ile 155 160 165 170 GCC AAG GTG GCG CAC GAC AAG GGC GTG CCT CTG ATT GTGAGTTTTT 1225 Ala Lys Val Ala His Asp Lys Gly Val Pro Leu Ile 175 180 TTTTTTTTTT TTTTTCTTAT TTGCCCTTCC CCTCCCTCGTGTTTTGGAGCCA CCTGGACCGG 1285 GCAGGCAGAG CTGACATCGA GGC AAC ACG GGC AAC ACG GGC AGC AGC AGC ATC CGT CCG GTC GAC CAC GGC GCC GAT ATT GTC GTC CAC 1387 Gly Tyr Phe Ile Arg Pro Val Asp His Gly Ala Asp Ile Val Val His 195 200 205 TCG GCC ACC AAG TGG ATC GGC GGC CAC GGC ACC ACC ATT GGC GGT GTC 1435 Ser Ala Thr Lys Trp Ile Gly Gly His Gly Thr Thr Ile Gly Gly Val 210 215 220 ATC GTC GAC TCC GGC AAG TTC GAC TGG GGC AAG CA C GGT GCG CGC TTC 1483 Ile Val Asp Ser Gly Lys Phe Asp Trp Gly Lys His Gly Ala Arg Phe 225 230 235 CCC CAA TTC GTC GAG CCG TCC GAG GGC TAC CAC GGC CTG AAG TTC TGG 1531 Pro Gln Phe Val Glu Pro Ser Glu Gly Tyr His Gly Leu Lys Phe Trp 240 245 250 GAG ACC TTC GGC CCC ATC TCC TTC ATC ATC CGC GCC CGT GTC GAG GTC 1579 Glu Thr Phe Gly Pro Ile Ser Phe Ile Ile Arg Ala Arg Val Glu Val 255 260 265 270 CTG CGC GAC CTC GGC GCC GCC CTC AAC CCC TTC GCC GCC CAG CAG CTG 1627 Leu Arg Asp Leu Gly Ala Ala Leu Asn Pro Phe Ala Ala Gln Gln Leu 275 280 285 CTG CTC GGC ATC GAG ACG CTC AGC CTG CGC GCC GAG CGC CAC GCC CAC GCC CAC G 1675 Leu Leu Gly Ile Glu Thr Leu Ser Leu Arg Ala Glu Arg His Ala Gln 290 295 300 AAC GCC CTC GCC CTG GCC AAG TAC CTC GAG AAC AGC CCC AAC GTC AGC 1723 Asn Ala Leu Ala Leu Ala Lys Tyr Leu Glu Asn Ser Pro Asn Val Ser 305 310 315 TGG GTC TCG TAC CCC GGC CTC GAG AGC CAC GCT TAC CAC GCC ACG GCC 1771 Trp Val Ser Tyr Pro Gly Leu Glu Ser His Ala Tyr His Ala Thr Ala 320 325 330 AAG AAG TAC CTG ACC AAG GGC TTT GGC GGC GTC CTG AGC TTC GGC GTC 1819 Lys Lys Tyr Leu Thr Lys Gly Phe Gly Gly Val Leu Ser Phe Gly Val 335 340 345 350 AAG GGC GGC GGT GCT GCC GGC AGC AAG ATT GTC GAC GGC TTC AAG CTA 1867 Lys Gly Gly Gly Ala Ala Gly Ser Lys Ile Val Asp Gly Phe Lys Leu 355 360 365 ATC TCC AAC CTG GCC AAC GTG GGC GAC GCC AAG ACC CTG GCC ATT CA 1914 Ile Ser Asn Leu Ala Asn Val Gly Asp Ala Lys Thr Leu Ala Ile Hi 370 375 380 GTAAGTCATG CCAGAAGGGA AACTTGGGGA GGAGAATGAT ACCTTGCTAA TGGTTGTCCC 1974 ATCTAG C CCT TGG AGC ACG ACG CAC GAG CAG CTA TCG GAC GAG GAG AAG 2023 s Pro Trp Ser Thr Thr ACC 385 GTC Glu Glu Glu Ly GAA GTACGAGAAT GACCACGTAA CGTATGCATC 2074 Ile Ser Ser Gly Val Thr Glu 400 TTGTTTCCAT GACTGACACT TCTCCGTGCA G GAC CTC ATC CGT ATC TCG GTC 2126 Asp Leu Ile Arg Ile Ser Val 405 410 GGA ATC GAG CAC ATT GAC GAC GTC TTC ATT GCC GTC ATC TTC GATT 2174 Gly Ile Glu His Ile Asp Asp Ile Ile Ala Asp Phe Asp Gln Ser Phe 415 420 425 AAG GCG GCC GCT GGC AGC TCC TGAG TGCTGC AGGCGCCGTC TGATTGGAGG 2225 Lys Ala Ala Ala Gly Ser Ser 430 TGGTAATACC CTTATAGATA AGATATGAAT AGCAGCGTTT CCTTTGTAGA AGATGTTTTT 2285 GAGCAATTTG ATAATGATAT GGACGTGATG ATGTCGTCAT TTGCCCTTTT GGTCAACATG 2345 AAGTTGTGAC CTCAAGTTGG TTGTCGACTC GAAGTTGCGA TGTCCGAGGG CAGGTGCTGT 2405 ACCGTGTACG TGTACGGAGT GCTGTAGCTG CTCCTGTAAG CTGTACCTGG TATCTGATGT 2465 GGATCTCCGC AGCTGCGCGC TGATTCGTGG TTGCCACTGC CACTACCAAG TACCTACTTG 2525 GTAGTGCCGT ACAAACCACC ACGAACTTCC AACGTAATTG CAGCCCAAAC GCTGACTTCG 2585 ACAATCTCCC GTCCAACAGC AACTCCCCCC TCCCCCTCCC ACATCCGTCC GCCAGATCGC 2645 TCACGATGCG TCTCGACGTC TCGGCCCTCC TGGGCCTCCT CACGGCGCTC ACCCCCGCTC 2705 TTGCCGCCGA CCTCACCCTC TACCTTCCCA CCAGGCCCAA CCCCTTCACC CTACCGCCCA 2765 CGACGCACGC CACGCTCGCT ACCCTCGGCG AGTCGTACTC AGCCCCGCTG TCCGCGGTCA 2825 ACACCTTTGT CTTCCGCAAC GTCTCCGCCC CCGGG 2860

[0056]

[Sequence list]

 SEQ ID NO: 3 Sequence length: 15 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Asp Asn Ile Val Ser Thr Ser Asn Leu Tyr Gly Gly Thr Tyr Asn 1 5 10 15

[0057]

[Sequence list]

 SEQ ID NO: 4 Sequence length: 25 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Asp Lys Gly Val Pro Leu Ile Val Asp Asn Thr Phe Gly Ala Gly Gly 1 5 10 15 Tyr Phe Ile Arg Pro Val Asp His Gly 20 25

[0058]

[Sequence list]

 SEQ ID NO: 5 Sequence length: 15 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Asp Ala Lys Thr Leu Ala Ile His Pro Trp Ser Thr Thr His Glu 1 5 10 15

[0059]

[Sequence list]

 SEQ ID NO: 6 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence AAYYTNTAYG GNGGNACNTA 20

[0060]

[Sequence Listing] SEQ ID NO: 7 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GTNSWCCANG GRTGDATNGC 20

[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.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12R 1: 645) (C12N 9/88 C12R 1: 645)

Claims (6)

[Claims] 1. O derived from Acremonium chrysogenum
-A DNA fragment containing the acetylhomoserine sulfhydrylase gene. 2. The DNA fragment according to claim 1, wherein the O-acetylhomoserine sulfhydrylase gene contains a gene encoding the amino acid sequence shown in the sequence listing (SEQ ID NO: 1). 3. An O-acetylhomoserine sulfhydrylase gene is contained between restriction enzyme sites of PstI-SmaI of about 2.8 Kb, and at least PstI, N
The DNA fragment according to claim 1, which has a restriction enzyme site of heI or NruI. 4. The DNA fragment according to claim 1, wherein the region between the PstI-SmaI restriction enzyme sites is the base sequence shown in the sequence listing (SEQ ID NO: 2). 5. The DNA according to claim 1, 2, 3 or 4.
Recombinant DNA in which the whole fragment or a part thereof is incorporated into a vector. 6. Acremonium chrysogenum transformed with the recombinant DNA according to claim 5.