The present invention relates to the biotechnology field, particularly a DNA fragment encoding a D-amino acid oxidase enzyme. Field of the invention

There is a long standing interest towards the en¬ zyme D-amino acid oxidase (E.C. 1.4.3.3, DAAO) due to its possible applications in the pharmaceutical and the¬ rapeutical fields.

Said enzyme catalyzes the conversion of D-amino acids into the corresponding α-keto acids. α-Keto acids are important therapeutical agents useful in the treatment of chronic uraemia (Mackenzie Walser, M.D., Am. J. Cl. Nutr. 31, 1756-1760 (1978)).

D-amino acid oxidase (hereinbelow defined DAAO for the sake of brevity), obtained from the yeast Rhodoto- rula gracilis. has been used efficiently for the produc¬ tion of keto derivatives in a reactor system (Buto', S. et al., Biotech. Bioeng. , 44, 1288-1294 (1994)).

Furthermore, the same enzyme exhibited a high acti¬ vity on cephalosporin C (Pilone, M.S. et al., Biotech. Appl. Biochem. , 16, 252-262 (1992)) and it can be exploited in the production of 7-aminocephalosporanic acid, which is a key intermediate in the industrial production of se isynthetic cephalosporins.

The activity on the antibiotic substrate cephalorosporin C is much higher than other reported activities of DAAO. (Pilone, ibid.).

The preparation of 7-aminocephalosporanic acid is carried out according to the following scheme:

Scheme

o

t-

co.

__.

7-aminocephalosporanic acid

Another application of the enzyme DAAO isolated from Rhodotorula σracilis is its use in the oxidative therapy for the treatment of tumors; DAAO can be used as a promising enzyme system for the generation of oxygen- reactive species for the in situ treatment of tumors, mainly brain tumors (Ben Yoseph, 0. et al. British J.

Cancer (1995)). Such an oxidase activity is more interesting than others due to its non-physiological substrate (D-amino acids). The genetics of the yeast Rhodotorula αracilis is still completely unknown and, though DAAO produced therefrom has been extensively characterized in its kinetic and functional aspects (Pollegioni, L. et al., J. Biol. Chem., 268, 13580-13587 (1993)), no information at all were reported about a gene encoding for protein.

The availability of the above sequence encoding the DAAO will allow for the ingegnerization of the protein, thus giving higher yields and improved characteristics. Disclosure of the invention Now it has been found, and it is the object of the present invention, a deoxyribonucleic acid (DNA) fragment encoding the gene for D-amino acid oxidase of the yeast R odo orula αracilis.

The DNA fragment according to the present invention has the sequence Id n. 1.

The present invention also relates to the functional analogues of said sequence. By functional analogues, degenerated sequences, allelic variants and mutant sequences are meant. Another object of the present invention is a method for the preparation of said DNA fragment.

According to the present invention said method comprises: a) culture of a Rhodotorula αracil s strain in conditions inducing D-ammo acid oxidase; b) purification of the total fraction of RNA specific for said D-amino acid oxidase; c) synthesis of a first cDNA strand; d) amplification of said cDNA strand by means of the technique known as Polymerase Chain Reaction (PCR), wherein the following synthetic oligonucleotides are used as specific primers: 5'-TCC AAG AAT TCG CGG CCG-3 ^* Sequence Id n. 2 (I) 5'-ATG CAC TCG CAG AAG CGC GTC-3' Sequence Id n. 3 (II) to give said DNA fragment; e) cloning of said fragment in a suitable plasmid; f) isolation of said fragment.

In a first embodiment of the invention, the yeast Rhodotorula σracilis is the strain PAN, ATCC 26217. The DAAO induction is carried out by conventional methods, disclosed in Pilone, S. et al., J. Gen. Microbiol. , 135, 593-600, (1989).

The messenger ribonucleic acid (mRNA) total fraction was purified by conventional techniques, according to Maniatis et al . , Molecular Cloning: A

Laboratory manual, Cold Spring Harbor Laboratory,

(1992) .

In a preferred embodiment of the present invention, the synthesis of the cDNA strand is effected using a Moloney Murine Leukemia Virus reverse transcriptase and a notI-d(T) _j _g synthetic oligo-

nucleotide having the following sequence Id n. 4

(5'd.AAC TGG AAG AAT TCG CGG CCG CAG GAA T ₁₈ _-3'. Both the transcriptase and the synthetic oligonucleotide are commercially available. The preparation of cDNA is described in Maniatis et al. (ibid.).

The method according to the present invention is characterized in that the specific primers used in the

PCR step are those synthetic oligonucleotides reported above (I) and (II). Said synthetic oligonucleotides, defined respectively notl and DAAOn, have been found in the partial protein sequence of DAAO purified from

Rhodotorula crracilis (notl) and the codon usage deriving from the phenylalanine am onia-lyase gene of

Rhodosporidium toruloides (DAAOn), the latter being described by Gilbert, H.J. et al., J. Bacteriol., 161,

314-320, (1985).

The plasmid in which the DNA fragment from PCR is cloned is a conventional vector used for this kind of procedures, for example the plasmid pCR II by Invitrogen.

The isolation and sequencing of the DNA fragment of the present invention are carried out according to conventional methods known to those skilled in the art, for example as described in the above mentioned Maniatis et al.

The following example further illustrate the invention.

_Z__Δ_____ __

Rhodotorula qraci_,is . strain PAN ATCC 26217, was grown in DAAO-inducing conditions, using a culture medium at pH 5.6 containing 30 mM D-alanine, as

described in Pilone, S. et al. , J. Gen. Microbiol. , 135,

593-600, (1989), to obtain the maximum amount of specific DAAO mRNA.

The mRNA total fraction was purified by affinity chromatography on an oligo-dT cellulose column (see

Maniatis et al . , ibid. ).

This sample was used for the preparation of a first strand of CHEDDAR, employing a Moloney Murine Leukemia

Virus reverse transcriptase and a synthetic oligonucleotide notI-d(T) ₁ g, having the following sequence (Sequence Id n. 4)

(5 ^* -d[AAC TGG AAG AAT TCG CGG CCG CAG GAA T ₁₈ 3-3' as the primer.

The single strand CHEDDAR was used for specific PCR amplification reaction using, as specific primers, synthetic oligonucleotides designed from the partial protein sequence of the purified DAAO from Rhodotorula αracilis and the codon usage derived from the phenylalanine ammonia-lyase gene of Rhodosporidium toruloides (Gilbert et al., ibid).

For the amplification of the total fragment of the gene encoding DAAO the following oligonucleotides were used: notl 5'-TCC AAG AAT TCG CGG CCG-3' Sequence Id n. 2 DAAOn 5 ' -ATG CAC TCG CAG AAG CGC GTC-3' Sequence Id n. 3

The amplification conditions were:

94°C x 60"

35 x (94°C x 60"

65 ^* C x 50" 72 ^* C x 60")

72 ^β C x 10' .

The PCR product resulted in a 1.1 kb DNA fragment and was cloned in a commercial plasmid specific for PCR products (pCR II, Invitrogen).

The amplified and cloned DNA fragment was sequenced and its sequence corresponds to the total gene sequence of the gene encoding DAAO from

R P-lOtPrula αracilis.

This DNA fragment was excised from the cloning plasmid pCR II using the corresponding EcoRI sites and inserted into a pKK223-3 commercial plasmid (Pharmacia Biotech) linearized by means of the restriction enzyme EcoRI .

The reco binant plasmid was used to transform competent JM105 E. coli cells. The expression of the protein product of the cloned D-amino acid oxidase gene was obtained with a IPTG {isopropyl β- D-thiogalactopyranoside) induction of transformed E.coli cells by addition of 1 mM IPTG (final concentration) to a cell culture in exponential growth ( ^E go _θ ⁼ °- ⁸ ) conditions.

The cells were grown for additional 6 hours at 37 ^* C before harvesting at 7000 x g. The cell paste was sonicated 4 times for 1 min and the cell extract partially purified according to Pollegioni and Pilone (Prof. Express. Purif. 3, 165-167, (1992)). The protein sample was separated by SDS-PAGE electrophoresis under denaturing conditions and the protein band with a molecular weight of 40 kDa was electrotransferred to a polyvinylidene difluoride membrane, stained and directly used for sequencing analysis. The N-terminal sequence (10 residues determined) corresponds to the primary

sequence of the D-amino acid oxidase protein purified from R. αracilis:

Met-His-Ser-Gln-Lys-Arg-Val-Val-Val-Leu.

The present invention also relates to the plasmids containing the fragment of the gene encoding the DAAO of

Rhodotorula αracilis.

9 SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: (A) NAME: Mirella Pilone

(B) STREET: via Pontaccio 10

(C) CITY: Milan

(E) COUNTRY: Italy

(F) POSTAL CODE (ZIP): 20121 (ii) TITLE OF INVENTION: DNA FRAGMENT ENCODING D-

AMINO ACID OXIDASE

(iii) NUMBER OF SEQUENCES: 4

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Rhodotorula gracilis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

ATGCACTCGC AGAAGCGCGT CGTTGTCCTC GGATCAGGCG TTATCGGTCT GAGCAGCGCC 60

CTCATCCTCG CTCGGAAGGG CTACAGCGTG CATATTCTCG CGCGCGACTT GCCGGAGGAC 120

GTCTCGAGCC AGACTTTCGC TTCACCATGG GCTGGCGCGA ATTGGACGCC TTTCATGACG 180

CTTACAGACG GTCCTCGACA AGCAAAATGG GAAGAATCGA CTTTCAAGAA GTGGGTCGAG 240 TTGGTCCCGA CGGGCCATGC CATGTGGCTC AAGGGGACGA GGCGGTTCGC GCAGAACGAA 300

GACGGCTTGC TCGGGCACTG GTACAAGGAC ATCACGCCAA ATTACCGCCC CCTCCCATCT 360

TCCGAATGTC CACCTGGCGC TATCGGCGTA ACCTACGACA CCCTCTCCGT CCACGCACCA 420

AAGTACTGCC AGTACCTTGC AAGAGAGCTG CAGAAGCTCG GCGCGACGTT TGAGAGACGG 480

ACCGTTACGT CGCTTGAGCA GGCGTTCGAC GGTGCGGATT TGGTGGTCAA CGCTACGGGA 540 CTTGGCGCCA AGTCGATTGC GGGCATCGAC GACCAAGCCG CCGAGCCAAT CCGCGGGCAA 600

ACCGTCCTCG TCAAGTCCCC ATGCAAGCGA TGCACGATGG ACTCGTCCGA CCCCGCTTCT 660

CCCGCCTACA TCATTCCCCG ACCAGGTGGC GAAGTCATCT GCGGCGGGAC GTACGGCGTG 720

GGAGACTGGG ACTTGTCTGT CAACCCAGAG ACGGTCCAGC GGATCCTCAA GCACTGCTTG 780

CGCCTCGACC CGACCATCTC GAGCGACGGA ACGATCGAAG GCATCGAGGT CCTCCGCCAC 840 AACGTCGGCT TGCGACCTGC ACGACGAGGC GGACCCCGCG TTGAGGCAGA ACGGATCGTC 900

CTGCCTCTCG ACCGGACAAA GTCGCCCCTC TCGCTCGGCA GGGGCAGCGC ACGAGCGGCG 960 AAGGAGAAGG AGGTCACGCT TGTGCATGCG TATGGCTTCT CGAGTGCGGG ATACCAGCAG 1020 AGTTGGGGCG CGGCGGAGGA TGTCGCGCAG CTCGTCGACG AGGCGTTCCA GCGGTACCAC 1080 GGCGCGGCGC GGGAGTCGAA GTTGTAG 1107 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

TCCAAGAATT CGCGGCCG 18 (2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

11 ATGCACTCGC AGAAGCGCGT C 21

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

AACTGGAAGA ATTCGCGGCC GCAGGAATTT TTTTTTTTTT TTTTT 45