FORM 2
THE PATENTS ACT 1970
(39 of 1970)
AND
The Patents Rules, 2005
COMPLETE SPECIFICATION
(See section 10 and rulel3)
1. TITLE OF THE INVENTION:
"DNA SEQUENCE ENCODING PENICILLIN ACYLASE, NOVEL
CONSTRUCTS OF A RECOMBINANT DNA AND RECOMBINANT
MICROORGANISMS CARRYING SUCH SEQUENCE"
2. APPLICANTS:


I) (a) NAME: FERMENTA BIOTECH (UK) LIMITED
(b) NATIONALITY: Indian Company incorporated under the Indian
Companies ACT, 1956
(c) ADDRESS: 'DIL' Complex, Ghodbunder Road, Majiwada,
Thane (West) - 400 610, Maharashtra, India.
II) (a) NAME: MTKROBIOLOGICKY USTAV AV CR v.v.i.
(b) NATIONALITY: Czech Republic
(c) ADDRESS: Videnska 1083, Praha 4- KRC -142 20, Czech Republic.
3. PREAMBLE TO THE DESCRIPTION:

The following specification particularly describes the invention and the manner in which it has to be performed.

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DNA sequence encoding penicillin acylase, novel recombinant DNA constructs and recombinant microorganisms carrying this sequence
Technical Field:
The invention relates to a DNA sequence encoding the polypeptide penicillin acylase, novel recombinant DNA constructs and recombinant microorganisms carrying this sequence.
Background Art:
Penicillin acylases (E.C. 3.5.1.11, penicillin amidohydrolase) are produced by bacteria, actinomycetes, fungi and yeast. These important industrial enzymes are on the basis of their substrate specificity divided into three groups: penicillin G acylases (PGA), penicillin V acylases (PVA) and ester hydrolases of a-amino acids (AEH, formerly named ampicillin acylases). The enzymes of the PGA group have broad substrate specificity and catalyse the hydrolysis of the amidic bond of penicillins and cephalosporins. Among the bacterial producers of the PGA belong the species of the genera: Aeromonas, Achromobacter, Alcaligenes, Arthrobacter, Bacillus, Corynebacterium, Escherichia, Erwinia, Flavobacterium, Kluyvera, Micrococcus, Nocardia, Proteus, Providencia, Pseudomonas, Sarcina, Xanthomonas, Xylella (Process Biochem. 24:146-154, 1989, Process Biochem. 27:131-143,1992, Biotechnol. Adv. 18:289-301,2000).
Apart from the production strains expressing PGA, obtained by the mutagenesis, recombinant microorganisms containing recombinant plasmids with the structural gene encoding PGA were prepared from Escherichia coli (CS 244 343 and CS 246 957), Alcaligenes sp. or fecalis (Current Microbiology 39:2444-2448, 1999; EP 638649, Appl. Environ. Microbiol. 63: 3412-3418, 1997), Arthrobacter viscosus (Appl. Environ. Microbiol. 54: 2603-2607, 1988 a 55: 1351-1356, 1989; Gene 143: 79-83,1994), Bacillus megaterium (J. Bacteriol. 93: 302-306, 1967, Appl. Microbiol. Biotechnol. 25: 372-378, 1987; US 3 145 395; Process Biochemistry 29: 263-270, 1994), Kluyvera citrophila (Agric. Biol. Chem. 39: 1225-1232, 1975; Gene 49: 69-80,

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BiotechnoLLetters 14: 285-290, 1992), Providencia rettgeri (Acta Biochim. Polonica
28: 275-284, 1981; J. Bacteriol. 168: 431-433, 1986; DNA sequences 3: 195-200,
1992).
Most prokaryotic producers of the PGA are gram-negative bacteria and the enzyme is
located in the periplasma of the cell. In the Bacillus megaterium culture, the enzyme is
secreted from the cell to the medium.
The penicillin G acylases are industrially used mainly for the hydrolysis of the phenylacetyl derivatives of cephalosporins and penicillin G for the purpose of the preparation of the intermediates 6-APA and 7-ADCA. At present, these enzymes are used also in synthetic reactions, in the acylations of the above-mentioned intermediates, leading to the preparation of semi-synthetic antibiotics (e.g., US 5 753 458 and US 5 801011; WO 98/04732; WO 97/04086; Enzyme Microb. Technol. 25: 336-343, 1999; Synthesis of (3-Iactam antibiotics: Chemistry, Biocatalysis and Process Integration, Ed.: A. Bruggink, Kluwer Academic Publishers, Dordrecht/Boston/London, 2001).
For a repeated, long-term use of the PGA in the catalysis of the enzyme reaction, the enzyme is stabilized by immobilization or encapsulation, forming an enzyme catalyst. In this form, the enzyme shows a higher pH stability, temperature stability and a longer half-life period under the reaction conditions, under which it catalyzes the course of the reaction.
Disclosure of the Invention:
Object of the present invention is a nucleotide sequence having the size of 2646 bp, wherein the order of nucleotides is at least 95% identical to the order of nucleotides shown in Fig. 5,
The aspect of the present invention are further fragments of the nucleotide sequence of the present invention, encoding penicillin acylase, having the length of at least 150 nucleotides.

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A further aspect of the present invention is a nucleic acid construct containing the nucleotide sequence of the present invention or the fragment of the nucleotide sequence of the present invention, having at least one regulatory sequence regulating the expression of the gene and the production of the polypeptide having the penicillin acylase activity.
Another aspect of the present invention is a recombined plasmid containing the sequence of nucleotides of the present invention or the fragment of the sequence of the present invention.
A further aspect of the present invention is a recombinant expression vector consisting of the nucleic acid construct of the present invention, promoter, translational start signal and translational and transcriptional stop signal.
Another aspect of the present invention is a host cell containing the nucleic acid construct of the present invention. In this host cell, the nucleic acid construct can be carried in the recombinant expression vector of the present invention or it can be integrated in the ceil genome.
Yet another aspect of the present invention are recombined plasmids pKXIPl, pKLP3 and pKLP6, characterized by the inserted nucleotide sequence of the present invention, isolated from the strain Achtomobacter sp., and by the restriction map according to Fig. 1 and 2..
A further aspect of the present invention is a strain Escherichia coli BL21 containing the sequence of the present invention, carried by the plasmids pKXIPl, pKLP3 or pKXP6.
The basis of the present invention is the nucleotide sequence having the size of 2646 bp, wherein the order of nucleotides is identical to the order of nucleotides shown in Fig. 5, eventually the fragments of this sequence, encoding penicillin acylase, having the length of at least 150 bases. Said sequence can form part of DNA constructs, recombinant plasmids and vectors. By a suitable vector, said sequence can be inserted

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into the genome of a bacterial or yeast host, which can then be used for the production of penicillin acylase.
One of the possible embodiments of the present invention is the strain Escherichia coli BL21(pKXlPl) (CCM 7394) containing the recombinant plasmid pKXIPl, prepared on the basis of the nucleotide sequence of the newly isolated structural gene having the penicillin acylase activity. Said plasmid is characterized by the insertion of the DNA fragment having the size of 2646 bp (including the region with the SD sequence) into the plasmid vector pK19 (R. D. Pridmore, Gene 56: 309-312,1987), and the restriction map shown hi Fig. 1. The preparation of this recombinant microorganism consisted in the isolation of the chromosomal DNA from the cells of the strain Achromobacter sp. CCM 4824 (originally Comamonas testosteroni CCM 4824; Plh£5kova et al., Appl. Microbiol. Biotechnol. 62: 507-516, 2003) and the preparation of the recombinant microorganism E. coli TOP10(pKLP3) carrying a 5.1 kb fragment of the chromosomal DNA. The nucleotide sequence of the structural gene pga was obtained by the PCR technique (polymerase chain reaction) using the DNA of the plasmid pKLP3. In accordance with the determined NT sequence of the gene, DNA primers were proposed (Table 1), which were used for determining the complete nucleotide sequence of the gene, including the regulatory region with the SD sequence by the same PCR-sequencing technique. Based on the complete nucleotide sequence, the primers providing for the PCR-amplification of the whole structural gene for the penicillin acylase were proposed (PCR; W. Rychlik: Methods in Molecular Biology 15, 31-39,1993). Chromosomal DNA of the strain Achromobacter sp. CCM 4824 was used as the template. The resulting PCR products were isolated and ligated into the multicopy plasmid pK19 (R. D. Pridmore, Gene: 309-312,1987), yielding recombinant plasmids, used subsequently for the transformation of the host strain Escherichia coli TOP10 (Invitrogen, USA.).
The preparation of the competent cells and the transformation of the host strains by the population of the recombinant plasmids was carried out according to H. Hanahan, J. Mol. Biol. 166: 557-580, 1983. From the grown colonies, the recombinant plasmids were isolated by the alkaline lysis method (HC. Birnboim a J. Doly: Methods in

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Enzymology 100, 243-254, 1983) and the size of the inserted fragment and its orientation were determined.
In selected recombinant clones of the host TOP10, the activity of the enzyme penicillin acylase was tested in batch cultures in the LB medium. The recombinant plasmid isolated from the strain with the highest overall activity of the PGA was designated pKXIPl. From this plasmid construct, thepga gene is expressed constitutively. The host strain BL21 was subsequently transformed by this plasmid.
The resulting recombinant strain Escherichia coli carrying the plasmid pKXIPl and producing penicillin acylase was cultivated in a stirred bioreactor. The optimum procedure is the cultivation of the strain on the mineral medium M9 supplemented with casein hydrolysate and glycerol as the source of carbon and energy. The fed batch cultivation was carried out at the. temperature in the range of from 20 to 30 °C, while the pH was maintained in the range of from 5.5 to 7.5 and the concentration of the oxygen dissolved in the medium was 10 to 40 %.
Brief Description of Drawings: .
Fig. 1 represents the restriction map of the recombinant plasmid pKXIPl.
Fig. 2 represents plasmids pKAGSaa401, pKLP3 and pKLP6.
Fig. 3 represents the course of the optical density of the culture (OD600) and the dry
weight of the cells (dry mass) during the fed batch cultivation of the strain
BL21(pKXlPl) in the stirred bioreactor.
Fig. 4 represents the course of the total activity (TA) and the specific activity (SA)
during the fed batch cultivation of the strain BL21(pKXlPl) in the stirred bioreactor.
Fig. 5 represents the nucleotide sequence of the structural genepga and the adjacent
regions.
Examples:
Example 1
Cultivation of A. sp. and isolation of the chromosomal DNA

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The soil isolate of Achromobacter sp. CCM 4824 (Skrob et al., Enzyme Microbiol. Technol. 32:738-744, 2003; PlhdCkova et al, Appl. Microbiol. Biotechnol. 62: 507-516,2003) was cultivated in 50 ml of LB medium (in g/1: tryptone 10, yeast extract 5, NaCl 5; pH 7.2-7.5) at 37 °C on the rotating shaker (200 rpm) for 12 - 16 h. The biomass from 2 ml of the culture was isolated from the medium by centrifligation, washed with saline solution and stored at -20 °C.
The chromosomal DNA was isolated from the de-frosted biomass by the commercially available columns GENOMIC-TIP 100/G (Qiagen, Switzerland) and the appropriate buffer kit GENOMIC BUFFER SET (Qiagen).
Example 2
Technology of the recombinant DNA
All the methods of the DNA cleavage by the restriction enzymes (Fermentas, Lithuania), the analysis of the DNA molecules in 1.0% agarose gel (USB, U.S.A.) in Hie TBE buffer, the ligation by the T4 DNA ligase (Fermentas, Lithuania) were carried out in accordance with the standard protocols (J, Sambrook, 1989). For the PCR reactions and the DNA sequencing, the primers (Tab. 1) were synthesized by the companies MWG-Biotech AG (Germany) and Metabion (Germany). For carrying out the PCR reactions, the Thermocycler PTC-200 (MJ-Research, Inc., U.S.A.) was used, and all the reactions were carried out with Pwo SuperYield DNA Polymerase in the presence of GC-RICH Solution (ROCHE, Switzerland). The size of the products of the PCR technique was determined in 1 % agarose gel in the TBE buffer, wherein the DNA of the phage X cleaved by the restriction endonuclease Pstl was used as the molecular weight standard. The specific products of the PCR intended for further subcloning or sequencing were isolated from the agarose gel with the QIAEX II Gel Extraction Kit (Qiagen, Germany) according to the manufacturer's instructions. The unique DNA fragments were purified by the High Pure PCR Product Purification Kit (ROCHE, Switzerland) according to the manufacturer's instructions. The preparation of competent cells and the transformation of the host strains were carried out according to J. Hanahan, Mol. Biol. 166: 557-580, 1983, wherein the competent cells, transformed

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by the plasmids carrying the desired PCR fragments, were cultivated for 16 h at 37 °C on the solid cultivation medium Luria-Bertani (LB, in g/1: tryptone 10, yeast extract 5, NaCl 5, agar 15; pH 7.2-7.5) supplemented with the antibiotic kanamycin (Km, 50 ug/ml), eventually in 50 ml of liquid LB medium supplemented with Km on the orbital shaker Gallenkamp (200 rpm). For subsequent analyses, the recombinant plasmids were isolated with the Qiagen Plasmid Midi Kit (Qiagen, Germany) and the High Pure Plasmid Isolation Kit (ROCHE, Switzerland) according to the manufacturer's instructions. The determination of all NT DNA sequences was carried out at the Institute of Microbiology AS CR automatically on the 3100 DNA Sequencer (Perkin-Elmer, U.S.A.). The obtained sequences were analyzed by the software Lasergene (DNASTAR Inc., U.S.A.). The homology of the NT sequences was verified by the software BLAST (National Center for Biotechnology Information, U.S.A.; S. F. Altschul et al.: Nucleic Acids Res. 25: 3389-3402,1997).

3?
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Table 1 DNA primers and their location with regard to the pga gene »
00


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Example 3
Determination of the nucleotide sequence of the pga gene and preparation of the
expression system
The chromosomal DNA of the strain Achromobacter sp. CCM 4824 was partially cleaved by the enzyme &zu3AI. These DNA-fragments were ligated with the BamHI-linearized plasmidDNA of the vector pK19 (RD. Pridmore: Gene 56, 309-312,1987). The resulting constructs were subsequently used for the transformation of the host strain E. colt TOP10. The plasmid pKAGSaw401 (Fig. 2), isolated from the recombinant strain E. coli showing the penicillin acylase phenotype (PGA*), was subjected to the restriction by PstL The largest pstf-fragment, ca 5.1 kb, was subsequently subcloned into the Pj/I-finearized vector pK19, forming 2 types of constructs, pKLP3 and pKLP6 (Fig. 2), with inversely oriented Psfl-inserts. Hie recombinant strains E. coli TOP10(pKXP3) and E. coli TOP10(pKXP6) had the phenotype PGA*.
The isolated plasmids pKLP3 and pKLP6 were used as the template for obtaining the complete nucleotide sequence of the pga gene with both universal M13/pUC sequencing primers and subsequently derived pga-strictly specific primers (list of the primers - see Tab.l). The nucleotide sequence of the structural gene pga having 2592 nucleotides (including the termination triplet TAA) shows in the region defined by the nucleotides 63 to 2592 92% identity to the pga gene of the related microorganism Achromobacter xylosoxidans ssp. xylosoxidans (GenBank AF490005).
Based on the thus obtained nucleotide sequence of the pga gene including the adjacent regions, the primers were proposed, enabling the PCR amplification of the structural gene pga with the chromosomal DNA of the strain Achromobacter sp. CCM 4824 as the template. The proposed two primers UPACYLXbal and KEVACYLPstl from the regions adjacent to the structural gene pga with inserted restriction sites Xba\ resp. PsA (Tab. 1) were used in the PCR reaction comprising the following steps: 1) 5 min at 94 °C; 2) 30 cycles with the denaturation at 94 °C for 45 s, binding of the primers at 60 °C for 45 s and the polymerization at 72 °C for 3 min; 3) completion of the polymerization at 72 °C for 10 min. Under these conditions, the specific PCR product of the size 2663 bp was prepared, carrying the whole structural gene pga including the preceding part of the

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regulatory region with the Shine-Dalgamo sequence. This pga-specific PCR product was subjected to the cleavage by the restriction endonuclease Xbal (target sequence in the UPACYLXbal primer) and subsequently ligated into the vector pK19, cleaved by two polylinker enzymes Xbal and Smal. The obtained recombinant construct was designated pKXIPl plasmid (Fig. 1). The analysis of the DNA sequence of this plasmid has shown that during the insertion of the PCR-prqduct, no insertion-deletion mutations have occurred, but the site mutation C -> T at the 99th nucleotide of the structural gene pga in comparison with the originally postulated nucleotide sequence was found. However, this mutation is silent, because it does not change the amino acid type encoded by the triplet The prototrophic host strain E. coli BL21 (Invitrogen, USA) was transformed by the isolated recombinant plasmids pKLP6 and pKXIPl and the prokaryotic expression system BL21(pKXlPl) for PGA was prepared.
Example .4
Expression of the pga in Escherichia coli
Preparation of the inoculum for the cultivation of the Escherichia coli BL21 (pKXIPl)
strains
From a glycerol conserve (grown culture mixed with glycerol according to J. Sambrook,1989) stored at -70 °C, 50 ml of LB medium supplemented with kanamycin (Km, 50 p.g/ml) was inoculated. The inoculum was cultivated for 16 h at 28 °C in the orbital incubator shaker Gallenkamp (200 rpm).
Cultivation in the bioreactor
The strain BL21 (pKXIPl) was cultivated under strictly defined conditions in the M9 medium (Tab. 2) supplemented with glycerol (5-25 g/1) and casein hydrolysate (5-25 g/1) as the carbon and energy sources in the stirred bioreactor Biostat MD (B. Braun Biotech Intl., Melsungen, Germany): working volume 8.2 1, air flow rate 8-91 of air/min, initial stirring frequency 200 to 300 rpm, at the temperature in the range of from 20 to 28 °C. The pH of the medium was maintained in the range of from 7.5 to 5.5 and the concentration of the dissolved oxygen (p02) was automatically maintained in the range of

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from 5 to 30 % of the maximum oxygen saturation of the medium by adjusting the
stirring frequency in the range of from 200 to 840 rpm. The cultivation ran for 20 to 25
hours as fed batch cultivation (Fig. 3 and 4). At the end of the cultivations, the following
parameters were determined: the biomass concentration (cell dry weight, cdw), the total
activity (TA) and the specific activity (SA) of penicillin acylase.
The parameters were determined as follows:
biomass concentration: 28 g cdw/1 of the cultivation medium
total activity 18 000 U/l of the cultivation medium
specific activity 670 U/g cdw
Enzyme activity determination
For determining the PGA enzyme activity, samples of the culture of the volume of 1 to 2
ml were taken from Hie production cultivation. The biomass was isolated by
centrifugation and after washing with 1 to 2 ml of distilled water and further
centrifiigation it was stored at
-20 °C. The de-frosted biomass was resuspended in 0.005 M phosphate buffer (pH 8.0).
The activity of the PGA was determined by the titration at 37 °C with penicillin G as the
substrate.
Table 2 The components of the cultivation medium M9

Industrial Use
The recombinant strains of the microorganisms containing the nucleotide sequence of the present invention can be used in the production of penicillin acylase for various applications in the chemical and the pharmaceutical industry.

We claim,

1. A nucleotide sequence having the size of 2646 bp, wherein the order of nucleotides is at least 95% identical to the order of nucleotides shown in Fig. 5.
2. Novel fragments of the nucleotide sequence according to claim 1, encoding penicillin acylase, having the length of at least 150 nucleotides.
3. A nucleic acid construct containing the nucleotide sequence according to claim 1 or the fragment of the nucleotide sequence according to claim 2, having at least one regulatory sequence regulating the expression of the gene and the production of the polypeptide having the penicillin acylase activity.
4. A recombined plasmid containing the nucleotide sequence according to claim 1 or the fragment of the sequence according to claim 2.
5. A recombinant expression vector consisting of the construct according to claim 3, promoter, translational start signal and translational and transcriptional stop signal.
6. A host cell containing the nucleic acid construct according to claim 3.
7. The host cell according to claim 6, in which the nucleic acid construct is carried by the recombinant expression vector according to claim 5.
8. The host cell according to claim 6, in which the nucleic acid construct is integrated into the cell genome.
9. Recombined plasmids pKXIPl, pKLP3 and pKLP6, characterized by the inserted nucleotide sequence according to claim 1, isolated from the strain Achromobacter sp., and by the restriction map according to Fig. 1 and 2.
10. A strain Escherichia coli BL21 containing the sequence according to claim 1 carried by the plasmids pKXIPl, pKLP3 or pKLP6 according to claim 9.