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WO1986000929A1 - Recombinant dna molecule, transformed microorganisms and process for producing penicillin v amidase - Google Patents

Recombinant dna molecule, transformed microorganisms and process for producing penicillin v amidase Download PDF

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Publication number
WO1986000929A1
WO1986000929A1 PCT/SE1985/000287 SE8500287W WO8600929A1 WO 1986000929 A1 WO1986000929 A1 WO 1986000929A1 SE 8500287 W SE8500287 W SE 8500287W WO 8600929 A1 WO8600929 A1 WO 8600929A1
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Prior art keywords
amidase
penicillin
dna
recombinant dna
dna molecule
Prior art date
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PCT/SE1985/000287
Other languages
French (fr)
Inventor
Sten Gatenbeck
Björn Nilsson
Anders Olsson
Matthias Uhlen
Original Assignee
Sten Gatenbeck
Nilsson Bjoern
Anders Olsson
Matthias Uhlen
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Application filed by Sten Gatenbeck, Nilsson Bjoern, Anders Olsson, Matthias Uhlen filed Critical Sten Gatenbeck
Publication of WO1986000929A1 publication Critical patent/WO1986000929A1/en
Priority to DK141586A priority Critical patent/DK141586A/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C12N9/84—Penicillin amidase (3.5.1.11)

Definitions

  • the present invention relates to recombinant deoxy- ribonucleic acid with codes for penicillin V amidase or an active derivative thereof as well as a process for the pre ⁇ paration thereof.
  • the invention also relates to a transfor- mant microorganism including such recombinant deoxyribonucleic acid and the preparation thereof.
  • the invention further relates to the use of such a transformant microorganism for the production of penicillin .V amidase or an active derivative thereof.
  • Penicillin amidases, (E.C.3.5.1.11) catalyzing the hydrolysis of penicillins to 6-aminopenicillanic acid (6-APA) and the specific side chain, are widely distributed among microorganisms (see e.g.
  • Penicillin V amidases specific for phenoxymethyl- penicillin, are mainly found in fungi, although several strains of Bacillus species, including B. sphaericus (see Carlsen and Emborg ), produce penicillin V amidase.
  • a recombinant DNA molecule which includes a deoxyribonucleotide sequence con ⁇ taining the genetic information for penicillin V amidase.
  • a recombinant DNA molecule or so-called hybrid vector can be introduced into any suitable host microorganism, which can then be cultured to produce the desired enzyme material.
  • a transformed microorganism can be obtained, which will be nonpathogenic and also will produce substantial quantities of penicillin V amidase. Due to self- replication of the vector DNA in the host cells producing an amplified quantity of genes coding for penicillin V amidase, the production of this amidase will be substantially greater than that from hitherto used B. sphaericus strains.
  • the relatively new recombinant DNA technology or genetic engineering referred to above permits the introduction of a specific nucleotide sequence coding for a desired protein or polypeptide into a bacterial or other appropriate host cell thereby conferring the desired property thereto.
  • the DNA may
  • transformant microorganisms may comprise the steps of producing a double-stranded DNA sequence coding for the desired protein or polypeptide; link ⁇ ing the DNA to an appropriate site in an appropriate cloning vehicle or vector to form recombinant DNA molecules, some of which will contain the desired protein coding gene; transform ⁇ ing an appropriate host microorganism with the recombinant DNA molecules; screening the resulting clones for the presence of the protein or polypeptide coding gene by suitable means; and selecting and multiplying one or more of the positive clones.
  • one or more re- or subclonings may be performed, comprising the extraction and cleavage of the protein coding DNA, insertion of the cleaved fragments into a second cloning vehicle or vector and screening for the presence of the protein or polypeptide coding gene.
  • the aim of the present invention is to achieve a pro ⁇ duction of penicillin V amidase with the use of recombinant DNA technology. This has been accomplished by the finding of a DNA sequence of at least partially unknown structure, i.e. the DNA sequence coding for penicillin V amidase in a suitable host, and the separation of the sequence from a highly complex mixture of DNA sequences, as will be further explained below.
  • the recombinant DNA technology offers the possibility of preparing microorganisms capable of producing not only penicillin V amidase but also modified enzyme products having penicillin V amidase activi ⁇ ty, such as fragments of penicillin V amidase or fusion proteins which consists of penicillin V amidase and another protein or oligopeptide, as also will be described in more detail hereafter.
  • one aspect*of the present invention relates to a novel microorganism transformed by recombinant DNA technolo- gy, which microorganism is capable of producing penicillin V amidase.
  • the invention also relates to the preparation of such a microorganism through transformation of a host organism with a recombinant DNA molecule.
  • Another aspect of the invention relates to such a re- combinant DNA molecule, which comprises at least one DNA sequence coding for penicillin V amidase, and to a process for its preparation.
  • Still another aspect of the invention relates to a process for preparing penicillin V amidase by culturing a transformed microorganism of the invention.
  • penicillin V amidase means any protein acromolecule which can catalyze the hydro ⁇ lysis of phenoxymethyl penicillin to 6-aminopenicillanic acid (6-APA) and phenoxy-acetic acid.
  • the expression comprises penicillin V amidase molecules which may have a non-penicillin V amidase peptide sequence linked thereto, e.g. due to insertion of chemically synthetized oligonucleo- tides when constructing the cloning vehicle.
  • Nucleotide A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose) , a phosphate, and a nitrogenous hetero- cyclic base.
  • the base is linked to the sugar moiety via the glycosidic carbon (1 • carbon of the pentose) and that combi ⁇ nation of base and sugar is a nucleoside.
  • the base characte- rizes the nucleotide.
  • the four DNA bases are adenine ("A"), guanine ("G”), cytosine (“C”) and thymine (“T”).
  • the four RNA bases are A, G, C and uracil (“U”).
  • DNA sequence A linear series of nucleotides connected one to the other by phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses.
  • Codon A DNA sequence of three nucleotides (a triplet) S which encodes through messenger RNA "mRNA") an amino acid, a translational start signal or a translational termination signal.
  • mRNA messenger RNA
  • the nucleotide triplets TTA, TTG, CTT, CTC, CTA and CTG encod-e for the amino acid leucine ("Leu"), TAG, TAA and TGA are translational stop signals and ATG is 10 a translational start signal.
  • Plasmid A non-chromosomal double-stranded DNA sequence comprising an intact "replicon" such that the plasmid.is replicated in a host cell.
  • the characteristics of that or- 15 ganism are changed or transformed as a result of the DNA of the plasmid.
  • Tet tetracycline resistance
  • a host cell transformed by a plasmid is called a "trans- 20 formant”.
  • Phage or Bacteriophage Bacterial virus many of which in ⁇ clude DNA sequences encapsidated in a protein envelope or coat.
  • Cloning Vehicle A plasmid, phage DNA or other DNA sequence 25. which is able to replicate in a host cell, characterized by one or a small number of endonuclease recognition sites, or restriction sites, at which its DNA sequence may be cut in a determinable fashion without attendant loss of an essential biological function of the DNA, e.g., replication, production 0 of coat proteins or loss of promoter or binding sites, and which contains a marker suitable for use in the identifica ⁇ tion of transformed cells, e.g., tetracycline resistance or ampicillin resistance.
  • a cloning vehicle is also known as a vector.
  • Host An organism which on transformation by a cloning ve ⁇ hicle enables the cloning vehicle to replicate and to accomp ⁇ lish its other biological functions, e.g., the production of polypeptides or proteins through expression of the genes of a plasmid.
  • Cloning The- process of obtaining a population of organisms or DNA sequences derived from one such organism or sequence by asexual reproduction.
  • Expression The process undergone by a gene to produce a polypeptide or protein. It is a combination of transcription and translation.
  • Transcription The process of producing mRNA from a gene.
  • Translation The process of producing a protein or poly- peptide from mRNA.
  • Promoter The region of the DNA of a gene at which RNA poly- merase binds and initiates transcription.
  • a promoter is loca ⁇ ted before the ribosome binding site of the gene.
  • Ribosome Binding Site The region of the DNA of a gene which codes for a site on mRNA which helps the mRNA bind to the ribosome, so that translation can begin. The ribosome binding site is located after the promoter and before the translational start signal of the gene.
  • Gene A DNA sequence which encodes, as a template for mRNA, a sequence of amino acids characteristic of a specific poly ⁇ peptide or protein.
  • a gene includes a promoter, a ribosome binding site, a translational start signal and a structural DNA sequence.
  • the gene also includes a signal DNA sequence.
  • Expression Control Sequence A DNA sequence in a cloning vehicle that controls and regulates expression of genes of the cloning vehicle when operatively linked to those genes.
  • Signal DNA Sequence A DNA sequence within a gene for a polypeptide or protein which encodes, as a template for mRNA, a sequence of hydrophobic amino acids at the amino terminus of the polypeptide or protein, i.e., a "signal sequence” or "hydrophobic leader sequence” of the polypeptide or protein.
  • a signal DNA sequence is located in a gene for a polypeptide or protein immediately before the structural DNA sequence of the gene and after the translational start signal (ATG) of the gene.
  • a signal DNA sequence codes for the signal sequence of a polypeptide or protein which (signal sequence) is characteristic of a precursor of the polypeptide or pro ⁇ tein.
  • Precursor A polypeptide or protein as synthetized within a host cell with a signal sequence.
  • Downstream and Upstream On a coding DNA sequence downstream is the direction of transcription, i.e. in the direction 5 from 5 r to 3*. Upstream is the opposite direction.
  • Recombinant DNA Molecule or Hybrid DNA A molecule consis ⁇ ting of segments of DNA from different genomes which have been joined end-to-end " outside- of living cells and have the capacity to infect some host cell and be maintained therein.
  • Structural DNA Sequence A DNA sequence within a gene which encodes, as a template for mRNA, a sequence of amino acids characteristic of a specific mature polypeptide or protein, i.e., the active form of the polypeptide or protein.
  • One aspect of the invention is to provide a recombinant 15 DNA molecule comprising a deoxynucleotide sequence coding for penicillin V amidase or all active derivatives thereof.
  • the origin of said deoxynucleotide sequence is not critical to the invention, but any source can be used, including natural, synthetic or semi-synthetic DNA sources.
  • the source 20 of DNA coding for penicillin V amidase is in most cases a bacterial donor, such as a strain of B. sphaericus, although synthetically produced molecules also are possible.
  • a suitable cloning vehicle or 25. vector may be cleaved by means of a restriction enzyme and the DNA sequence or fragment coding for the penicillin V amidase inserted into the cleavage site to form a recombinant DNA molecule.
  • This general procedure is known per se and various techniques or methods to link the DNA sequence to 0 the cleaved cloning vehicle are described in the literature
  • B. sphaericus chromosomal DNA is used as the source of the deoxynucleotide sequence to be inserted into the selected vector, it may be obtained by treating the B. sphaericus strain with the enzyme lysozyme to form proto-.
  • a DNA preparation 5 plasts, which are then lysed and the DNA extracted and isolat ⁇ ed to form a DNA preparation.
  • the DNA preparation can be cleaved into fragments of an appropriate size.
  • the transformed cells which contain either a vector or a vector/Bacillus DNA combination, can be selected on the basis of a suitable marker included in the vector, e.g. antibiotic resistance, such as tetracycline resistance.
  • a suitable marker included in the vector e.g. antibiotic resistance, such as tetracycline resistance.
  • antibiotic resistance such as tetracycline resistance.
  • a "gene bank" of the B. sphaericus strain can be obtained, consisting of a great number, e.g. several thousands, of clones which contain Bacillus DNA.
  • the clone or clones containing the penicillin V amidase producing gene can then be found by any suitable assay for penicillin V amidase activity, e.g. sensitivity to the product 6-APA.
  • fragments of the DNA of penicillin V amidase producing clones may be subcloned into the same or another host micro ⁇ organism.
  • the cloning vehicles or vectors that can be used in the present invention depend i.a. on the nature of the host cell to be transformed, i.e. whether it is a bacterium, yeast or other fungi, etc.
  • Useful cloning vehicles may, for example, consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known bacterial plasmids, e.g., plasmids from E.
  • coli including pBR 322 and their derivatives, phage DNA, such as derivatives of phage lambda, vectors derived from combinations of plasmids and phage DNAs, yeast plasmids, specially constructed composite plasmids, etc.
  • phage DNA such as derivatives of phage lambda
  • vectors derived from combinations of plasmids and phage DNAs yeast plasmids, specially constructed composite plasmids, etc.
  • the particular selection of cloning vehicle with regard to a particular host may be made by a person skilled in the art, who also can select the site for the insertion of the DNA within each specific cloning vehicle, the cleavage sites being dependent on the restriction enzyme used. It is not necessary for a cloning vehicle useful in this invention to have a restriction site for insertion of the chosen DNA fragment, but the cloning vehicle could instead be joined to the fragment by alternative means, which 8
  • Hosts to be used in the present invention include bacterial hosts, such as strains of Escherichia, Bacillus, Staphylococcus, and Streptomvces, e.g. E. coli and Bacillus subtilis, yeasts and other fungi, plant cells in culture or other hosts. Due to its non-pathogenic nature the soil-bacterium Bacillus subtilis is considered as particularly useful as ' a final host in the present invention. It is, however, also within the scope of the present inven- tion to transform already penicillin V amidase producing strains of e.g.
  • B.sphaericus with the penicillin V amidase coding recombinant molecule of the invention to multiply the number of penicillin V amidase coding genes in the B.sphaericus cells.
  • Yeast (whose genetics is fairly well known) is also a preferred final host to be transformed into a penicillin V amidase producing microorganism in accordance with the present invention.
  • the promoter sequence of the donor Bacillus strain into the cloning vehicle together with the penicillin V amidase coding sequence. This is the case when e.g. the whole penicillin V amidase coding gene, from promoter to the stop codon, of the Bacillus strain is inserted into the cloning vehicle.
  • Other promoters may also be used, such as the promoter of a naturally occurring vector or of a vector modified by the insertion of strong external promoter.
  • DNA coding for an active derivative of penicillin V amidase as defined above may be obtained by introducing site specific changes in the DNA sequence coding for penicillin V amidase. It is also possible to use the technique called gene fusions (described i.a. by Uhlen et al ) in which the coding sequence of two or more genes are spliced together to form a combined gene which on expression in a suitable host organism will produce a fusion product wherein the separate proteins or polypeptides coded by the respective genes are fused together into a single molecule.
  • gene fusions described i.a. by Uhlen et al
  • gene fusions are.used to combine a DNA sequence coding for penicillin V amidase activity with a DNA sequence coding for a desired protein or polypeptide into a functional gene capable of expressing the fusion product of said desired protein or polypeptide and the penicillin V amidase.
  • the penicillin V amidase product produced according to the present invention can be recovered by conventional techniques.
  • the protein, or polypeptide product is retained within the cell, as is the case when no signal peptide for the product is synthetized, the cells must be ruptured before isolation can be effected.
  • Such rupture of the cell walls may e.g. be done by pressing, ultras ⁇ nication, homogenization, shaking with glass-beads, etc.
  • Fig. 1 is a SPS-PAGE of minicell preparations and purified penicillin V amidase from B. sphaericus. The samples were applied to the same gel and the gel divided in two halves and bands visualized by auto radiography and Comassie staining, respectively.
  • Fig. 2 is the pH optimum of penicillin V amidase.
  • Fig. 3 is a Lineweaver-Burke plot for penicillin V amidase ( * ) , with 50 mM 5-APA ( o ) and with 20 mM phenoxy- acetic acid ( + ) .
  • Fig. 4 is a schematic presentation of plasmid construct ⁇ ion used to subclone the gene coding for penicillin V amidase. Thick lines indicate plasmid vectors and thin lines indicate inserted fragment. Relevant restriction sites are indicated. In the Examples the starting the starting materials, buffers, cell media and routine method steps were as follows. 10
  • the bacterial strains and plasmids used are listed in the following table.
  • Bacillus sphaericus Penicillin V amidase ATCC 14577 producer Bacillus sphaericus Penicillin V amidase ATCC 14577 producer
  • Luria-broth 10 g Difco tryptone, 5 g Difco yeast extract, 0.5 g NaCl, 2 ml 1M NaOH; adjusted to pH 7.0 with 1M NaOH; 10 ml 201 glucose added after autoclaving.
  • LA-medium Luria-broth supplemented with 11 Difco agar.
  • Tris-EDTA buffer (TE) 0.001 M EDTA and 0.01 M Tris (pH 7.8)
  • NY-medium 8 g Difco nutrient broth, 5 g Difco yeast extract, 5 ml 1M MgSO. and 2 ml 10 ml mM MnCl 2 added after autoclaving.
  • Antibiotics were obtained from Pfizer (tetracycline), Astra, Sweden (ampicillin) , Sigma (chloramphenicol) and Fermenta, Sweden (penicillin V, 6-APA and phenoxyacetic acid). S-methionine was obtained from New England Nuclear.
  • Transformations Transformation of E. colj ⁇ C12, with plasmid DNA, was performed exactly as described by Morrison .
  • the 20 transformed cells were selected in a conventional manner on plates by plating for single colonies on LA plates containing suitable antibiotics, i.e. 10 ⁇ g/ml of tetracycline.
  • Proto ⁇ plasts of _B. subtil-is were transformed exactly as described by Chang and Cohen .
  • DNA fragments All DNA fragments were ligated at 14°C over-night with T4 DNA ligase purchased from New England Bio Labs, Waltham, MA., USA, in a buffer recommended by the supplier.
  • Agarose gel electrophoresis 0. 7% agarose gel electro- phoresis for separating cut plasmid fragments, supercoiled plasmids, and DNA fragments 1000 to 10,000 nucleotides in
  • Polyacryla ide gel electrophoresis 131 polyacrylamide gel electrophoresis for the separation of proteins of molecular weights of 5,000 to 120,000 was performed exactly as described by Laemmli .
  • Chromosomal DNA from B. sphaericus was prepared by total lysis followed by sodium dodecyl sulfate (SDS) treatment and extraction with phenol.
  • the DNA was frac ⁇ tionated after digestion with restriction enzyme on a sucrose gradient 10-30 (w/v) in TE buffer (according to Maniatis et al. ) containing 1 M NaCl.
  • Plasmid DNA from E. coli was prepared by the alkaline lysis method as described by Birnboim and Doly ⁇ .
  • Plasmids from B. subtilis were prepared with a modified alkaline lysis method as follows.
  • a 5 ml overnight culture was centrifuged and the pellet resuspended in a total volume of 500 yl lysozyme solution (0.3 M sucrose, 25 mM Tris/HCl pH 8.0, 0.02 bro phenolblue, 25 mM EDTA and 2 mg/ml lysozyme).
  • 500 yl lysozyme solution 0.3 M sucrose, 25 mM Tris/HCl pH 8.0, 0.02 bro phenolblue, 25 mM EDTA and 2 mg/ml lysozyme.
  • the suspension was incubated for 30 min at 37°C.
  • 2S0 ⁇ l of NaOH/SDS solution 0.3 M NaOH and 2 SDS
  • the solution was then extracted with 80 ⁇ l phenol/ chloroform, (500 g phenol, 500 ml chloroform and 200 ml water) , the phases separated and the aqueous phase containing plasmid DNA precipitated with 0.3 M sodium acetate and 1 volume isopropanol.
  • E. coli. clones carrying B.sphaericus DNA were replicated to LA plates and incubated overnight. They were then overlayed with 5 ml of soft agar containing Serratia arcesens (0.5 ml of an over 13
  • Minicells were puri ⁇ fied from the E. coli strain M2141 by two sucrose gradient centrifugations as described by Kennedy . Labelling of mini- cells with 35S -methionine was carried out in minimal medium 0 supplemented with glucose, thiamine and methionine assay medium
  • Assay of penicillin V amidase activity Cells were grown in liquid medium containing the appropriate antibiotic and har ⁇ vested after overnight growth. The cells were resuspended in S 1/10 vol of 0.1 M sodium citrate buffer (pH 5.8) and dis ⁇ rupted by sonication. The homogenate was centrifuged and the supernatant used for assay of penicillin V amidase activity.
  • the standard assay mixture (total volume 500 yl) contained 3% (w/v) penicillin V (potassium salt) in 0.1 M sodium citrate 0 buffer pH 5.8. The reaction was started by adding enzyme
  • 6-APA was also identified by thin layer chromatography according to
  • B. sphaericus was grown in a 12 1 fermentor (Chemoferm) in NY-medium without regulation of pH with an aeration of 1 vvm, stirring at 700 rpm and at temperature of 30°C. After 10-11 hrs of growth, the cells were harvested in a CEPA centrifuge. The yield was 85 g wet weight from 10 1 culture medium. Cell paste was suspended (16. g wet weight/60 ml) in 50 mM potassium phosphate buffer (pH 6.8) and the cells were disrupted by soni- cation for 5x45 s at 0°C (70W).
  • the extract was centrifugated at 15 OOOxg for 30 min at 4°C and the suspernatant used for purification of the enzyme essentially as described by Carlsen and Emborg . Following a 401 ammonium sulfate precipitation the enzyme was precipitated with 70 ammonium sulfate.
  • the pre ⁇ cipitate was resuspended in a small volume " of 0.1 M Tris/HCl and 10 mM EDTA pH 8.0 and placed on a Sephadex G-200 gel column equilibrated with 0.1 M Tris/HCl, 10 mM EDTA pH 8.0.
  • the column was eluted with the same buffer and fractions with high activity were pooled and immediately chromatographed on a DEAE-Sephadex A-50 column. Analysis of the purified enzymes: The protein concentration was measured by the method of Bradford with bovine albumin as standard. Polyacrylamide gel electrophoresis (SDS-PAGE) was carried out as described by Laemmli . Isoelectric focusing was carried out using LKB ampholine PAGplates pH 4.0-6.5. The molecular weight of the native enzyme was determined by HPLC using a LKB 2135 Ultropac TSK column packed with G3000 SYI gel. The solvent was 50 mM potassium phosphate buffer pH.7.0 with 0.2 M NaCl.
  • One unit (U) is defined as the amount of enzyme which catalyzed the formation of 1 ⁇ mol of 6-APA per min. Determined according to Ko ⁇ feld1
  • the purity of the final preparation was analysed by SDS- -PAGE (see Fig. 1 ) revealing a major, band with a molecular weight of 35 000 , corresponding to more than 95! of the total protein content .
  • the molecular weight of the native enzyme was determined using two independent methods . Gradient electrophoresis followed by enzyme staining gave one band with a molecular weight of 140 000 and gel filtration in a HPLC-system gave a molecular weight of 135 000. This suggests that the native enzyme consists of four identical subunits , each with a molecular weight of 35 000.
  • the isoelectric point of the native enzyme was determined by isoelectric focusing which gave a major band corresponding to a pi of 4.8.
  • the enzyme activity at different pH values was mea ⁇ sured giving the pH-profile shown in Fig. 2 with an optimum around pH 5.8.
  • the kinetic properties are shown in Fig. 3.
  • the K of fi mM is significantly lower than that previously published .
  • both enzyme products inhibit the reaction.
  • Phenoxyacetic acid is a non-competitive inhibi ⁇ tor with K. of 25 mM and 6-APA acts as a competitive inhibitor Cloning and expression of the gene for penicillin V amidase:
  • a gene bank of B. sphaericus DNA was constructed. Chromo ⁇ somal DNA was partially digested with Mbol and fractionated by sucrose gradient centrifugation (10-30%) and fractions containing approximately 10 kilobases (kb) fragments were mixed and ligated with Bell-cleaved pTR262 21 and used to transform E. coli HB10T. About 2200 tetracycline resistant clones were transferred to microtiterplates, grown overnight, and stored in a freezer at -80°C. Approximately 1000 clones were screened with the Serratia overlay technique and two positive clones, pOH2 and pOH3, were found.
  • FIG. 4 A restriction map of pOH3, the smaller of the two plasmids, is shown in Fig. 4.
  • This E. coli strain with the plasmid pOH3 is denomi ⁇ nated PVA-3 and has been deposited at Deutsche Sammelung von Mikroorganismen and given the accession number DSM 2982.
  • the size of the insert in pOH3 was shown to be 9.1 kb.
  • Various subclones were constructed in order to determine the minimum size of inserted DNA that is necessary for amidase activity (Fig. 4).
  • the plasmid pOH31 obtained by digestion with HindiII and religation has no functional amidase activity (Table 3) , in contrast to plasmid pOH35 containing a 2.2.
  • pOH36 kb fragment obtained by digestion with PstI and religation.
  • Clal fragment was removed from pOH35 the activity was lost indicating that all or part of the gene is within this fragment.
  • the resulting plasmid pOH36 was, in all clones isolated, found to be exclusively a dimer (Fig.4).
  • a shuttle vector for B. subtilis and E. coli was constructed by inserting HindiII cleaved pC194 (which has a gene for chloramphenicol acetyl transferase) in the Hindlll site of pOH35. The result ⁇ ing plasmid is called pOH38 (Fig. 4).
  • pOH38 is denominated PVA 38 has been deposited at Deutsche Sammelung von Mikroorganismen and given the accession number DSM 2983.
  • the specific amidase activity in cell-free extracts °f E « coli and B.subtilis are shown in Table 3.
  • the activities in the different E. coli clones are lower than in B.sphaericus B.subtilis (pOH38) gives an expression that is two times higher than in B. sphae.ricus.
  • the plasmids pOH35, pBR322 and pUN101 were used to transform the E.coli minicell strain M2141.
  • the minicells were labelled with S -methionine and the plasmid coded polypeptides were identified after separation on SDS-PAGE (Fig. 1).
  • a protein coded by pOH35 which comigrates with purified penicillin V amidase from B.sphaericus was found. While embodiments of the invention have been presented above, the invention is not restricted thereto, but many variations and modifications of the processes and recombinant 18
  • the invention is, e.g., also meant to encompass cloning vehicles containing more than one separate deoxy- nucleotide sequence coding for the desired protein or poly ⁇ peptide.
  • the invention is intended to comprise also recombinant DNA molecules containing deoxynucleotide sequences, from whatever source obtained, including natural, synthetic or semisynthetic sources, which are related to the " deoxynucleotide sequences coding for penicillin V amidase or an active fragment thereof, as defined above, by muta ⁇ tion, including single or multiple base substitutions, deletions, insertions and inversions.

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Abstract

A recombinant DNA molecule comprising at least one DNA sequence coding for penicillin V amidase or a derivative thereof, and to a process for its preparation. The invention also relates to microorganisms transformed by said recombinant DNA molecule and to a porocess for their preparation. The invention further relates to a process for preparing penicillin V amidase and to the amidase produced thereby.

Description

Recombinant DNA molecule, transformed microorganisms and process for producing penicillin V amidase
The present invention relates to recombinant deoxy- ribonucleic acid with codes for penicillin V amidase or an active derivative thereof as well as a process for the pre¬ paration thereof. The invention also relates to a transfor- mant microorganism including such recombinant deoxyribonucleic acid and the preparation thereof. The invention further relates to the use of such a transformant microorganism for the production of penicillin .V amidase or an active derivative thereof. Penicillin amidases, (E.C.3.5.1.11) , catalyzing the hydrolysis of penicillins to 6-aminopenicillanic acid (6-APA) and the specific side chain, are widely distributed among microorganisms (see e.g. Vandamme and Voets 23). These enzymes are commercially important in the production of semisynthetic penicillins. Penicillin V amidases, specific for phenoxymethyl- penicillin, are mainly found in fungi, although several strains of Bacillus species, including B. sphaericus (see Carlsen and Emborg ), produce penicillin V amidase.
In order to produce this enzyme in a commcercial scale it would be highly desirable to use a microorganism which has an increased production of penicillin V amidase. Such a micro¬ organism is provided by the present invention.
Thus, by the present invention, which relates to so- called genetic engineering, a recombinant DNA molecule is provided which includes a deoxyribonucleotide sequence con¬ taining the genetic information for penicillin V amidase. Such a recombinant DNA molecule or so-called hybrid vector can be introduced into any suitable host microorganism, which can then be cultured to produce the desired enzyme material. By selecting the DNA transfer vector and the host micro¬ organism in an optimal way a transformed microorganism can be obtained, which will be nonpathogenic and also will produce substantial quantities of penicillin V amidase. Due to self- replication of the vector DNA in the host cells producing an amplified quantity of genes coding for penicillin V amidase, the production of this amidase will be substantially greater than that from hitherto used B. sphaericus strains.
The relatively new recombinant DNA technology or genetic engineering referred to above permits the introduction of a specific nucleotide sequence coding for a desired protein or polypeptide into a bacterial or other appropriate host cell thereby conferring the desired property thereto. The DNA may
* be prepared in different routes, such as by chemical synthesis or by extraction from another bacterial strain or other orga¬ nism. The construction of such transformant microorganisms may comprise the steps of producing a double-stranded DNA sequence coding for the desired protein or polypeptide; link¬ ing the DNA to an appropriate site in an appropriate cloning vehicle or vector to form recombinant DNA molecules, some of which will contain the desired protein coding gene; transform¬ ing an appropriate host microorganism with the recombinant DNA molecules; screening the resulting clones for the presence of the protein or polypeptide coding gene by suitable means; and selecting and multiplying one or more of the positive clones. Optionally, one or more re- or subclonings may be performed, comprising the extraction and cleavage of the protein coding DNA, insertion of the cleaved fragments into a second cloning vehicle or vector and screening for the presence of the protein or polypeptide coding gene.
Using such recombinant DNA technology several bacterial as well as non-bacterial proteins have been obtained and are described in the literature (this technology is mostly accom¬ plished in Escherichia coli) . None of the previously known recombinant DNA processes is, however, directed towards the synthesis of penicillin V amidase.
The aim of the present invention is to achieve a pro¬ duction of penicillin V amidase with the use of recombinant DNA technology. This has been accomplished by the finding of a DNA sequence of at least partially unknown structure, i.e. the DNA sequence coding for penicillin V amidase in a suitable host, and the separation of the sequence from a highly complex mixture of DNA sequences, as will be further explained below. Once the DNA sequence has been identified the recombinant DNA technology offers the possibility of preparing microorganisms capable of producing not only penicillin V amidase but also modified enzyme products having penicillin V amidase activi¬ ty, such as fragments of penicillin V amidase or fusion proteins which consists of penicillin V amidase and another protein or oligopeptide, as also will be described in more detail hereafter.
Thus, one aspect*of the present invention relates to a novel microorganism transformed by recombinant DNA technolo- gy, which microorganism is capable of producing penicillin V amidase. The invention also relates to the preparation of such a microorganism through transformation of a host organism with a recombinant DNA molecule.
Another aspect of the invention relates to such a re- combinant DNA molecule, which comprises at least one DNA sequence coding for penicillin V amidase, and to a process for its preparation.
Still another aspect of the invention relates to a process for preparing penicillin V amidase by culturing a transformed microorganism of the invention.
The expression "penicillin V amidase" ,. as used herein, means any protein acromolecule which can catalyze the hydro¬ lysis of phenoxymethyl penicillin to 6-aminopenicillanic acid (6-APA) and phenoxy-acetic acid. Thus the expression comprises penicillin V amidase molecules which may have a non-penicillin V amidase peptide sequence linked thereto, e.g. due to insertion of chemically synthetized oligonucleo- tides when constructing the cloning vehicle.
Other expressions used herein are as defined below: Nucleotide: A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose) , a phosphate, and a nitrogenous hetero- cyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1• carbon of the pentose) and that combi¬ nation of base and sugar is a nucleoside. The base characte- rizes the nucleotide. The four DNA bases are adenine ("A"), guanine ("G"), cytosine ("C") and thymine ("T"). The four RNA bases are A, G, C and uracil ("U"). DNA sequence: A linear series of nucleotides connected one to the other by phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses.
Codon: A DNA sequence of three nucleotides (a triplet) S which encodes through messenger RNA "mRNA") an amino acid, a translational start signal or a translational termination signal. For example, the nucleotide triplets TTA, TTG, CTT, CTC, CTA and CTG encod-e for the amino acid leucine ("Leu"), TAG, TAA and TGA are translational stop signals and ATG is 10 a translational start signal.
Plasmid: A non-chromosomal double-stranded DNA sequence comprising an intact "replicon" such that the plasmid.is replicated in a host cell. When the plasmid is placed within a unicellular host organism, the characteristics of that or- 15 ganism are changed or transformed as a result of the DNA of the plasmid. For example, as plasmid carrying the gene for tetracycline resistance (Tet) transforms a host cell previous¬ ly sensitive to tetracycline into one which is resistant to it. A host cell transformed by a plasmid is called a "trans- 20 formant".
Phage or Bacteriophage: Bacterial virus many of which in¬ clude DNA sequences encapsidated in a protein envelope or coat.
Cloning Vehicle: A plasmid, phage DNA or other DNA sequence 25. which is able to replicate in a host cell, characterized by one or a small number of endonuclease recognition sites, or restriction sites, at which its DNA sequence may be cut in a determinable fashion without attendant loss of an essential biological function of the DNA, e.g., replication, production 0 of coat proteins or loss of promoter or binding sites, and which contains a marker suitable for use in the identifica¬ tion of transformed cells, e.g., tetracycline resistance or ampicillin resistance. A cloning vehicle is also known as a vector. 5 Host: An organism which on transformation by a cloning ve¬ hicle enables the cloning vehicle to replicate and to accomp¬ lish its other biological functions, e.g., the production of polypeptides or proteins through expression of the genes of a plasmid.
Cloning: The- process of obtaining a population of organisms or DNA sequences derived from one such organism or sequence by asexual reproduction. Expression: The process undergone by a gene to produce a polypeptide or protein. It is a combination of transcription and translation.
Transcription: The process of producing mRNA from a gene. Translation: The process of producing a protein or poly- peptide from mRNA.
Promoter: The region of the DNA of a gene at which RNA poly- merase binds and initiates transcription. A promoter is loca¬ ted before the ribosome binding site of the gene. Ribosome Binding Site: The region of the DNA of a gene which codes for a site on mRNA which helps the mRNA bind to the ribosome, so that translation can begin. The ribosome binding site is located after the promoter and before the translational start signal of the gene. Gene: A DNA sequence which encodes, as a template for mRNA, a sequence of amino acids characteristic of a specific poly¬ peptide or protein. A gene includes a promoter, a ribosome binding site, a translational start signal and a structural DNA sequence. In the case of an exported or secreted protein or polypeptide, the gene also includes a signal DNA sequence. Expression Control Sequence: A DNA sequence in a cloning vehicle that controls and regulates expression of genes of the cloning vehicle when operatively linked to those genes. Signal DNA Sequence: A DNA sequence within a gene for a polypeptide or protein which encodes, as a template for mRNA, a sequence of hydrophobic amino acids at the amino terminus of the polypeptide or protein, i.e., a "signal sequence" or "hydrophobic leader sequence" of the polypeptide or protein. A signal DNA sequence is located in a gene for a polypeptide or protein immediately before the structural DNA sequence of the gene and after the translational start signal (ATG) of the gene. A signal DNA sequence codes for the signal sequence of a polypeptide or protein which (signal sequence) is characteristic of a precursor of the polypeptide or pro¬ tein. Precursor: A polypeptide or protein as synthetized within a host cell with a signal sequence.
Downstream and Upstream: On a coding DNA sequence downstream is the direction of transcription, i.e. in the direction 5 from 5r to 3*. Upstream is the opposite direction.
Recombinant DNA Molecule or Hybrid DNA: A molecule consis¬ ting of segments of DNA from different genomes which have been joined end-to-end" outside- of living cells and have the capacity to infect some host cell and be maintained therein. 10" Structural DNA Sequence: A DNA sequence within a gene which encodes, as a template for mRNA, a sequence of amino acids characteristic of a specific mature polypeptide or protein, i.e., the active form of the polypeptide or protein.
One aspect of the invention is to provide a recombinant 15 DNA molecule comprising a deoxynucleotide sequence coding for penicillin V amidase or all active derivatives thereof. The origin of said deoxynucleotide sequence is not critical to the invention, but any source can be used, including natural, synthetic or semi-synthetic DNA sources. The source 20 of DNA coding for penicillin V amidase is in most cases a bacterial donor, such as a strain of B. sphaericus, although synthetically produced molecules also are possible.
When preparing a recombinant DNA molecule in accordance with the present invention a suitable cloning vehicle or 25. vector may be cleaved by means of a restriction enzyme and the DNA sequence or fragment coding for the penicillin V amidase inserted into the cleavage site to form a recombinant DNA molecule. This general procedure is known per se and various techniques or methods to link the DNA sequence to 0 the cleaved cloning vehicle are described in the literature When B. sphaericus chromosomal DNA is used as the source of the deoxynucleotide sequence to be inserted into the selected vector, it may be obtained by treating the B. sphaericus strain with the enzyme lysozyme to form proto-. 5 plasts, which are then lysed and the DNA extracted and isolat¬ ed to form a DNA preparation. By partial digestion with a suitable restriction enzyme the DNA preparation can be cleaved into fragments of an appropriate size. After treat- ment of the vector with the same or another restriction enzyme the vector cleavage products and the above fragmented DNA preparation are admixed and randomly combined under the ligating action of a ligase enzyme. Screening of the ligated DNA fragment combinations may be effected by making them biologically active in a suitable microorganism cell, e.g. a bacteria. The transformed cells, which contain either a vector or a vector/Bacillus DNA combination, can be selected on the basis of a suitable marker included in the vector, e.g. antibiotic resistance, such as tetracycline resistance. In this way a "gene bank" of the B. sphaericus strain can be obtained, consisting of a great number, e.g. several thousands, of clones which contain Bacillus DNA. The clone or clones containing the penicillin V amidase producing gene can then be found by any suitable assay for penicillin V amidase activity, e.g. sensitivity to the product 6-APA. As mentioned above fragments of the DNA of penicillin V amidase producing clones may be subcloned into the same or another host micro¬ organism. The cloning vehicles or vectors that can be used in the present invention depend i.a. on the nature of the host cell to be transformed, i.e. whether it is a bacterium, yeast or other fungi, etc. Useful cloning vehicles may, for example, consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known bacterial plasmids, e.g., plasmids from E. coli including pBR 322 and their derivatives, phage DNA, such as derivatives of phage lambda, vectors derived from combinations of plasmids and phage DNAs, yeast plasmids, specially constructed composite plasmids, etc. The particular selection of cloning vehicle with regard to a particular host may be made by a person skilled in the art, who also can select the site for the insertion of the DNA within each specific cloning vehicle, the cleavage sites being dependent on the restriction enzyme used. It is not necessary for a cloning vehicle useful in this invention to have a restriction site for insertion of the chosen DNA fragment, but the cloning vehicle could instead be joined to the fragment by alternative means, which 8
are wellknown in the art
Hosts to be used in the present invention include bacterial hosts, such as strains of Escherichia, Bacillus, Staphylococcus, and Streptomvces, e.g. E. coli and Bacillus subtilis, yeasts and other fungi, plant cells in culture or other hosts. Due to its non-pathogenic nature the soil-bacterium Bacillus subtilis is considered as particularly useful as 'a final host in the present invention. It is, however, also within the scope of the present inven- tion to transform already penicillin V amidase producing strains of e.g. B.sphaericus with the penicillin V amidase coding recombinant molecule of the invention to multiply the number of penicillin V amidase coding genes in the B.sphaericus cells. Yeast (whose genetics is fairly well known) is also a preferred final host to be transformed into a penicillin V amidase producing microorganism in accordance with the present invention.
In the present invention it is most convenient to insert the promoter sequence of the donor Bacillus strain into the cloning vehicle together with the penicillin V amidase coding sequence. This is the case when e.g. the whole penicillin V amidase coding gene, from promoter to the stop codon, of the Bacillus strain is inserted into the cloning vehicle. Other promoters may also be used, such as the promoter of a naturally occurring vector or of a vector modified by the insertion of strong external promoter.
DNA coding for an active derivative of penicillin V amidase as defined above may be obtained by introducing site specific changes in the DNA sequence coding for penicillin V amidase. It is also possible to use the technique called gene fusions (described i.a. by Uhlen et al ) in which the coding sequence of two or more genes are spliced together to form a combined gene which on expression in a suitable host organism will produce a fusion product wherein the separate proteins or polypeptides coded by the respective genes are fused together into a single molecule. In accordance with the present invention gene fusions are.used to combine a DNA sequence coding for penicillin V amidase activity with a DNA sequence coding for a desired protein or polypeptide into a functional gene capable of expressing the fusion product of said desired protein or polypeptide and the penicillin V amidase.
The penicillin V amidase product produced according to the present invention can be recovered by conventional techniques. When, e.g., the protein, or polypeptide product is retained within the cell, as is the case when no signal peptide for the product is synthetized, the cells must be ruptured before isolation can be effected.
Such rupture of the cell walls may e.g. be done by pressing, ultrasσnication, homogenization, shaking with glass-beads, etc.
The invention will now, by way of illustration only, be described in more detail in the following non-limiting examples, showing the cloning into B. subtilis and E. coli of penicillin V amidase gene from a Bacillus strain. Reference will be made to the accompanying drawings, on which:
Fig. 1 is a SPS-PAGE of minicell preparations and purified penicillin V amidase from B. sphaericus. The samples were applied to the same gel and the gel divided in two halves and bands visualized by auto radiography and Comassie staining, respectively.
Fig. 2 is the pH optimum of penicillin V amidase. Fig. 3 is a Lineweaver-Burke plot for penicillin V amidase ( * ) , with 50 mM 5-APA ( o ) and with 20 mM phenoxy- acetic acid ( + ) .
Fig. 4 is a schematic presentation of plasmid construct¬ ion used to subclone the gene coding for penicillin V amidase. Thick lines indicate plasmid vectors and thin lines indicate inserted fragment. Relevant restriction sites are indicated. In the Examples the starting the starting materials, buffers, cell media and routine method steps were as follows. 10
STARTING MATERIALS
The bacterial strains and plasmids used are listed in the following table.
Table 1
Strains and Relevant Reference plasmids characteristics
Escherichia coli HB 101 Host for gene Boyer et al constructions
Bacillus sphaericus Penicillin V amidase ATCC 14577 producer
Serratia marcescens 6-APA sensitive ATCC 271 77 Bacillus- subtilis 168 Type strain ATCC 6051 Escherichia coli M2141 Minicell producer Dougan et al. pTR262 Positive selection Roberts at al 21 vector pC194 : QπlR Horinouchi et al. 10
2 pBR322 Aπ-pR, TcR Bolivar et al. pUN101 AmpR Nilsson et al. 20
pOH3 TcR, penicillin V aimidase + pOH31 TcR, penicillin V amidase - pOH35 TcR, penicillin V amidase + pOH36 TcR, penicillin V amidase - pOH38 TcR, CmlR, penicillin V amidase +
Described later in this application. 11
BUFFERS AND MEDIA
Luria-broth (LB): 10 g Difco tryptone, 5 g Difco yeast extract, 0.5 g NaCl, 2 ml 1M NaOH; adjusted to pH 7.0 with 1M NaOH; 10 ml 201 glucose added after autoclaving.
5 LA-medium: Luria-broth supplemented with 11 Difco agar.
Tris-EDTA buffer (TE) : 0.001 M EDTA and 0.01 M Tris (pH 7.8)
NY-medium: 8 g Difco nutrient broth, 5 g Difco yeast extract, 5 ml 1M MgSO. and 2 ml 10 ml mM MnCl2 added after autoclaving.
10 Soft agar: Luria broth supplemented with 0.6% Difco agar.
Antibiotics were obtained from Pfizer (tetracycline), Astra, Sweden (ampicillin) , Sigma (chloramphenicol) and Fermenta, Sweden (penicillin V, 6-APA and phenoxyacetic acid). S-methionine was obtained from New England Nuclear.
15 METHODS
The different procedures were, unless otherwise stated, carried out as follows.
Transformations: Transformation of E. colj ΪC12, with plasmid DNA, was performed exactly as described by Morrison . The 20 transformed cells were selected in a conventional manner on plates by plating for single colonies on LA plates containing suitable antibiotics, i.e. 10 μg/ml of tetracycline. Proto¬ plasts of _B. subtil-is were transformed exactly as described by Chang and Cohen .
_ *,c Isolating plasmids: Large scale plasmid preparation22was per- formed exactly as described by Tanaka and Weisblum . For scoring a large number of clones for plasmids the "mini alkali method" was used exactly as described by Birnboim and Doly .
Restriction enzyme digestion of DNA: DNA was cleaved with 30 conventional restriction enzymes purchased from New England
Bio Labs, Waltham MA, USA. The restriction enzymes were added to DNA at conventional concentrations and temperatures and with buffers as recommended by the supplier. 12
Ligating DNA fragments: All DNA fragments were ligated at 14°C over-night with T4 DNA ligase purchased from New England Bio Labs, Waltham, MA., USA, in a buffer recommended by the supplier. Agarose gel electrophoresis: 0. 7% agarose gel electro- phoresis for separating cut plasmid fragments, supercoiled plasmids, and DNA fragments 1000 to 10,000 nucleotides in
9 length was performed exactly as described by Helling et al.
Polyacryla ide gel electrophoresis: 131 polyacrylamide gel electrophoresis for the separation of proteins of molecular weights of 5,000 to 120,000 was performed exactly as described by Laemmli .
Preparation of DNA: Chromosomal DNA from B. sphaericus was prepared by total lysis followed by sodium dodecyl sulfate (SDS) treatment and extraction with phenol. The DNA was frac¬ tionated after digestion with restriction enzyme on a sucrose gradient 10-30 (w/v) in TE buffer (according to Maniatis et al. ) containing 1 M NaCl. Plasmid DNA from E. coli was prepared by the alkaline lysis method as described by Birnboim and Doly^ . Plasmids from B. subtilis were prepared with a modified alkaline lysis method as follows. A 5 ml overnight culture was centrifuged and the pellet resuspended in a total volume of 500 yl lysozyme solution (0.3 M sucrose, 25 mM Tris/HCl pH 8.0, 0.02 bro phenolblue, 25 mM EDTA and 2 mg/ml lysozyme). The suspension was incubated for 30 min at 37°C. After addition of 2S0 μl of NaOH/SDS solution (0.3 M NaOH and 2 SDS) the mixture was incubated at 65°C for 20 min. The solution was then extracted with 80 μl phenol/ chloroform, (500 g phenol, 500 ml chloroform and 200 ml water) , the phases separated and the aqueous phase containing plasmid DNA precipitated with 0.3 M sodium acetate and 1 volume isopropanol.
Screening for penicillin V amidase positive clones: E. coli. clones carrying B.sphaericus DNA were replicated to LA plates and incubated overnight. They were then overlayed with 5 ml of soft agar containing Serratia arcesens (0.5 ml of an over 13
night culture/100 ml) and penicillin V (4 mg/ml). The plates were incubated over night at 28°C and positive clones detected as described by Mayer 17. An alternative and quick method of detecting positive clones was also developed. The cells were
5 resuspended in a 2 mM solution of 6-nitro-3-phenylacetamido-
-bensoic acid and the hydrolysis was measured. The product of this reaction, 5-amino '2-nitrobensoic acid, was quantitatively
- determined by recording the increase in optical density at 405 nm or qualitatively by visual inspection. This substrate 0 was also used for enzymatic staining in the gradient gel elektrophoresis. 6-Nitro-3-phenoxyacetamidobensoic acid was synthetized following a method previously described by Kutzbach and Rausenbusch 13, except that the. phenylacetate was replaced by phenoxyacetic acid. The product was finally purifie 5 on a Florisil column.
Purification and labelling of minicells: Minicells were puri¬ fied from the E. coli strain M2141 by two sucrose gradient centrifugations as described by Kennedy . Labelling of mini- cells with 35S -methionine was carried out in minimal medium 0 supplemented with glucose, thiamine and methionine assay medium
18 according to Molin et al .
Assay of penicillin V amidase activity: Cells were grown in liquid medium containing the appropriate antibiotic and har¬ vested after overnight growth. The cells were resuspended in S 1/10 vol of 0.1 M sodium citrate buffer (pH 5.8) and dis¬ rupted by sonication. The homogenate was centrifuged and the supernatant used for assay of penicillin V amidase activity. The standard assay mixture (total volume 500 yl) contained 3% (w/v) penicillin V (potassium salt) in 0.1 M sodium citrate 0 buffer pH 5.8. The reaction was started by adding enzyme
(50-250 μl) and the mixture was incubated at 37°C for up to
50 minutes. The reaction was terminated by heating in a boiling water bath for 90 seconds. The amount of 6-APA was determined as described by Kornfeld 12. The reaction product, 6-APA, was also identified by thin layer chromatography according to
Lowe et al . The pH-optimum was determined using 0.1 M sodium citrate (pH 4.5-6.2) and 0.1 M sodium phosphate (pH 14
Isolation of penicillin V amidase from B.sphaericus: B. sphaericus was grown in a 12 1 fermentor (Chemoferm) in NY-medium without regulation of pH with an aeration of 1 vvm, stirring at 700 rpm and at temperature of 30°C. After 10-11 hrs of growth, the cells were harvested in a CEPA centrifuge. The yield was 85 g wet weight from 10 1 culture medium. Cell paste was suspended (16. g wet weight/60 ml) in 50 mM potassium phosphate buffer (pH 6.8) and the cells were disrupted by soni- cation for 5x45 s at 0°C (70W). The extract was centrifugated at 15 OOOxg for 30 min at 4°C and the suspernatant used for purification of the enzyme essentially as described by Carlsen and Emborg . Following a 401 ammonium sulfate precipitation the enzyme was precipitated with 70 ammonium sulfate. The pre¬ cipitate was resuspended in a small volume "of 0.1 M Tris/HCl and 10 mM EDTA pH 8.0 and placed on a Sephadex G-200 gel column equilibrated with 0.1 M Tris/HCl, 10 mM EDTA pH 8.0. The column was eluted with the same buffer and fractions with high activity were pooled and immediately chromatographed on a DEAE-Sephadex A-50 column. Analysis of the purified enzymes: The protein concentration was measured by the method of Bradford with bovine albumin as standard. Polyacrylamide gel electrophoresis (SDS-PAGE) was carried out as described by Laemmli . Isoelectric focusing was carried out using LKB ampholine PAGplates pH 4.0-6.5. The molecular weight of the native enzyme was determined by HPLC using a LKB 2135 Ultropac TSK column packed with G3000 SYI gel. The solvent was 50 mM potassium phosphate buffer pH.7.0 with 0.2 M NaCl.
Purification and characterization of penicillin V amidase from B. sphaericus: The penicillin V amidase from B. sphaeri¬ cus was purified and the results of the purification procedure are summarized in Table 2. 15
Table 2. Summary of the purification of penicillin V amidase from B. sphaericus .
Purifica¬ Vol Total acti¬ Total pro¬ Specific Purifica¬ Yield tion U)a tein(mg) activi¬ tion step (ml) vity( ty(U/mg) factor O)
Sonicatiσn 44 1540 ' 1170 1.31 1 100
Ammonium sulfate 16 874 333 2.62 2.0 56
Sephadex G-200 120 328- 121 2.71 2.1 21
DEAE- Sephadex 81 139 6.89 20.2 .15.4 9
One unit (U) is defined as the amount of enzyme which catalyzed the formation of 1 μmol of 6-APA per min. Determined according to Koπιfeld1
Protein determined according to Bradford .
The purity of the final preparation was analysed by SDS- -PAGE (see Fig. 1 ) revealing a major, band with a molecular weight of 35 000 , corresponding to more than 95! of the total protein content . The molecular weight of the native enzyme was determined using two independent methods . Gradient electrophoresis followed by enzyme staining gave one band with a molecular weight of 140 000 and gel filtration in a HPLC-system gave a molecular weight of 135 000. This suggests that the native enzyme consists of four identical subunits , each with a molecular weight of 35 000.
The isoelectric point of the native enzyme was determined by isoelectric focusing which gave a major band corresponding to a pi of 4.8. The enzyme activity at different pH values was mea¬ sured giving the pH-profile shown in Fig. 2 with an optimum around pH 5.8. The kinetic properties are shown in Fig. 3. The K of fi mM is significantly lower than that previously published . As shown in Fig. 3 both enzyme products inhibit the reaction. Phenoxyacetic acid is a non-competitive inhibi¬ tor with K. of 25 mM and 6-APA acts as a competitive inhibitor Cloning and expression of the gene for penicillin V amidase:
A gene bank of B. sphaericus DNA was constructed. Chromo¬ somal DNA was partially digested with Mbol and fractionated by sucrose gradient centrifugation (10-30%) and fractions containing approximately 10 kilobases (kb) fragments were mixed and ligated with Bell-cleaved pTR262 21 and used to transform E. coli HB10T. About 2200 tetracycline resistant clones were transferred to microtiterplates, grown overnight, and stored in a freezer at -80°C. Approximately 1000 clones were screened with the Serratia overlay technique and two positive clones, pOH2 and pOH3, were found. A restriction map of pOH3, the smaller of the two plasmids, is shown in Fig. 4. This E. coli strain with the plasmid pOH3 is denomi¬ nated PVA-3 and has been deposited at Deutsche Sammelung von Mikroorganismen and given the accession number DSM 2982. The size of the insert in pOH3 was shown to be 9.1 kb. Various subclones were constructed in order to determine the minimum size of inserted DNA that is necessary for amidase activity (Fig. 4). The plasmid pOH31 obtained by digestion with HindiII and religation has no functional amidase activity (Table 3) , in contrast to plasmid pOH35 containing a 2.2. kb fragment obtained by digestion with PstI and religation. When the Clal fragment was removed from pOH35 the activity was lost indicating that all or part of the gene is within this fragment. For unknown reasons, the resulting plasmid pOH36 was, in all clones isolated, found to be exclusively a dimer (Fig.4). A shuttle vector for B. subtilis and E. coli was constructed by inserting HindiII cleaved pC194 (which has a gene for chloramphenicol acetyl transferase) in the Hindlll site of pOH35. The result¬ ing plasmid is called pOH38 (Fig. 4). Protoplasts of B.subtilis were transformed with the resulting plasmid pOH38. Transfor ants were selected on regeneration plates containing chloramphenicol. Plasmid purification and enzyme digestion showed that all chloramphenicol resistant clones contained pOH38. Expression of amidase activity was confirmed in cellfree extracts. This B. subtilis strain with the plasmid 17
pOH38 is denominated PVA 38 has been deposited at Deutsche Sammelung von Mikroorganismen and given the accession number DSM 2983. The specific amidase activity in cell-free extracts °f E« coli and B.subtilis are shown in Table 3. The activities in the different E. coli clones are lower than in B.sphaericus B.subtilis (pOH38) gives an expression that is two times higher than in B. sphae.ricus.
Table 3. Specific penicillin V amidase activity of different strains and clones.
Strain Plasmid Specific activity (U/g)'
E.coli HB101 pOH3 110 pOH31 0 pOH35 110 pOH36 0 pOH38 82
B.subtilis pOH38 410 pC194 0
B.sphaericus 240
determined according to Kornfeld . Zero values correspond to less than 1 U/g.
Identification of plasmid coded penicillin V amidase. The plasmids pOH35, pBR322 and pUN101 were used to transform the E.coli minicell strain M2141. The minicells were labelled with S -methionine and the plasmid coded polypeptides were identified after separation on SDS-PAGE (Fig. 1). A protein coded by pOH35 which comigrates with purified penicillin V amidase from B.sphaericus was found. While embodiments of the invention have been presented above, the invention is not restricted thereto, but many variations and modifications of the processes and recombinant 18
matter of this invention are possible without departing from the scope thereof as defined by the subsequent claims. Thus, the invention is, e.g., also meant to encompass cloning vehicles containing more than one separate deoxy- nucleotide sequence coding for the desired protein or poly¬ peptide. Further, the invention is intended to comprise also recombinant DNA molecules containing deoxynucleotide sequences, from whatever source obtained, including natural, synthetic or semisynthetic sources, which are related to the" deoxynucleotide sequences coding for penicillin V amidase or an active fragment thereof, as defined above, by muta¬ tion, including single or multiple base substitutions, deletions, insertions and inversions.
LITERATURE CITED
1 Birnboira,: H.C. , and J. Doly. 1979. Nucl. Acid Res. 7:1513-1523
2 Bolivar, F. , R.L. Rodriquez, P.J. Greene, M.C.Betlach, H.L.Heyneker, H.W.Boyer, J.H.Crosa, and S.Falkow. 1977. Gene 2:95-113 Boyer, H.W. , and D.Roulland-Dussoix. 1969. J.Mol.Biol. 41:459-472 Bradford, M.M. 1976. Anal.Bioche . 75:248-254 Carlsen, F. , and C. E borg. 1981. Biotechnology Letters, 3:375-378 Carlsen, F. , and C. Emborg. 1982. J.Chem.Tech.Biotechnol. 32:808-811 Chang, S. , and S.N. Cohen. 1979. Mol.Gen.Genet. 168:111-115 Dougan, G. , and D. Sherratt. 1979. Mol.Gen.Genet. 151:151-160 Helling, R.B., H.M. Goodman and H.W.Boyer 1974. J.Virol. 14 1235-1244 Hourinouchi, S., and B.Weisblum. 1982. J.Bacteri l. 150:815-825 Kennedy, N. , L.Beutin, M.Actman, R.Skurray, U.Rahmsdorf, and P.Herrlich. 1977. Nature 270:580-585 Kornfeld, J.M. 1978. Anal.Biochem. 86:118-126 Kutzbach, C. , and E. Rauenbusch. 1974. Hoppe-Seylers Z Physiol.Chem. 354:45-53 Laemmli, U.K. 1970. Nature 227:680-685 Lowe, D.A. , G. Romancik, and R.P.Elander. 1981. Dev.Ind.Microbiol. 22:163-180 Maniatis, T. , E.F.Fritsch, and J.Sambrook. 1982. Molecular cloning. Cold Spring Harbor Laboratory Mayer, H., J.Collins, and F. ager. 1979. p 459-470 in K.N.Timmis and A.Puhler (eds) Plasmids of medical, environmental and commercial importance Bio edical Press, Elsevier/North Holland Molin, S. , P.Stougaard, J. Light, M.Nordstrδm, and K.Nordstrom. 1981. Mol.Gen.Genet. 181:123-130 Morrison, D.A. 1979. p.326-331 in R.Wu (ed) ,
Methods in enzymology. Vol 68, Academic Press, New York Nilsson, B. , M. Uhldn, S. Josephs'sonv S. Gatenbeck, and L.Philipsson. 1983. Nucl.Acid.Res. 11:8019-8030 Roberts, T.M. , S.L.Swanberg, A.Poteete, G.Riedel, and K.Backman. 1980. Gene 12:123-127 Tanaka, T. and B. Weisblum. 1975. J.Bacterial 121 , 354-362 Vandamme, E.J. , and J.P.Voets. 1974. p. 311-369 in Adv.Appl.Microbiol. Uhlέn, M. , B.Nilsson, B.Guss, M.Lindberg, S.Gatenbeck, and L.Philipsson. 1983 "Gene fusion vectors based on the gene for Staphylococcal protein A." Gene.23:369-378.

Claims

20CLAIMS
1. A recombinant DNA molecule, characterized in that it comprises a deoxynucleotide sequence coding for penicillin V amidase or a derivative thereof capable of catalysing the hydrolysis of phenoxymethylpenicillin to 6-aminopenicillanic acid and phenoxy-acetic acid.
2. The recombinant DNA molecule of claim 1, characterized in that said deoxynucleotide sequence coding for penicillin V amidase or a derivative thereof is derived from a bacterial donor.
3. The recombinant DNA molecule of claim 2 characterized in that said bacterial donor is a strain of Bacillus sphaericus and that said deoxynucleotide sequence comprises the promotor sequence of the penicillin V amidase gene of the Bacillus donor.
4. The recombinant DNA molecule of any one of claims 1-3, characterized in that it is a recombinant plasmid.
5. A process for preparing the recombinant DNA molecule of any one of claims 1-4, characterized by inserting at least one deoxynucleotide sequence coding for penicillin V amidase or a derivative thereof into a cloning vehicle.
6. The process of claim 5, characterized in that said deoxynucleotide sequence is obtained by digesting Bacillus DNA with a restriction enzyme.
7. The process of any of claims 5 or 6, characterized in that it comprises one or more subcloning steps.
8. A microorganism transformed by the recombinant DNA molecule of any of claims 1-4.
9. The microorganism of claim 8, characterized in that it is a bacterium or yeast.
10. The microorganism of claim 8, characterized in that it is a Escherichia, Bacillus, Staphylococcus or Streptomyces strain, preferably a strain of Bacillus subtilis.
1 1 . The microorganism of claim 8, characterized in that it is Escherichia coli PVA 3 (DSM 2982) or Bacillus subtilis PVA 38 (DSM 2983).
12. A process for preparing the transformed microorganism of any one of claims 8-11, characterized by introducing into a host organism the recombinant DNA molecule of any one of claims 1-4.
13. The process of claim 12, characterized in that it comprises at least one subcloning step.
14. A process for preparing penicillin V amidase or a derivative thereof, characterized by culturing the transformed microorganism of any of claims 8-11 in a suitable nutrient medium and isolating the desired product formed.
15. Penicillin V amidase or a derivative thereof, characterized by being produced by the process of claim 14.
PCT/SE1985/000287 1984-07-31 1985-07-19 Recombinant dna molecule, transformed microorganisms and process for producing penicillin v amidase WO1986000929A1 (en)

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DK141586A DK141586A (en) 1984-07-31 1986-03-26 RECOMBINANT DNA MOLECULE, PROCEDURES FOR PREPARING THEREOF, MICROORGANISMS TRANSFORMED THEREOF AND PROCEDURE FOR PENICILLIN-V-AMIDASE PREPARATION

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SE8403929A SE8403929L (en) 1984-07-31 1984-07-31 RECOMBINANT DNA MOLECULE, TRANSFORMED MICROORGANISMS AND PROCEDURE FOR PENICILLIN V-AMIDAS PREPARATION
SE8403929-6 1984-07-31

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995019371A3 (en) * 1994-01-14 1995-11-16 Solvay Expression of surface layer proteins
US5516679A (en) * 1994-12-23 1996-05-14 Bristol-Myers Squibb Company Penicillin V amidohydrolase gene from Fusarium oxysporum
US6403356B1 (en) 1996-11-05 2002-06-11 Bristol-Myers Squibb Co. Mutant penicillin G acylases
WO2015151118A1 (en) 2014-04-04 2015-10-08 Council Of Scientific & Industrial Research A recombinant penicillin v acylase and process for the prepartion thereof

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EP0021468A1 (en) * 1979-05-11 1981-01-07 Gist-Brocades N.V. Plasmid, host containing it, its preparation and use
EP0036259A2 (en) * 1980-03-10 1981-09-23 Cetus Corporation Method,vectors and organisms for transported cloned heterologous gene products in bacillus subtilis and E.coli
EP0060057A1 (en) * 1981-02-25 1982-09-15 Genentech, Inc. Expression of polypeptides in yeast
EP0107823A2 (en) * 1982-10-21 1984-05-09 Gesellschaft für Biotechnologische Forschung mbH (GBF) DNA sequences and DNA structures and process for the preparation of penicillin acylase using them

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
EP0021468A1 (en) * 1979-05-11 1981-01-07 Gist-Brocades N.V. Plasmid, host containing it, its preparation and use
EP0036259A2 (en) * 1980-03-10 1981-09-23 Cetus Corporation Method,vectors and organisms for transported cloned heterologous gene products in bacillus subtilis and E.coli
EP0060057A1 (en) * 1981-02-25 1982-09-15 Genentech, Inc. Expression of polypeptides in yeast
EP0107823A2 (en) * 1982-10-21 1984-05-09 Gesellschaft für Biotechnologische Forschung mbH (GBF) DNA sequences and DNA structures and process for the preparation of penicillin acylase using them

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995019371A3 (en) * 1994-01-14 1995-11-16 Solvay Expression of surface layer proteins
US5874267A (en) * 1994-01-14 1999-02-23 Solvay (Societe Anonyme) Expression of surface layer proteins
US5516679A (en) * 1994-12-23 1996-05-14 Bristol-Myers Squibb Company Penicillin V amidohydrolase gene from Fusarium oxysporum
EP0718402A3 (en) * 1994-12-23 1997-12-17 Bristol-Myers Squibb Company Penicillin V amidohydrolase gene from fusarium oxysporum
CN1100877C (en) * 1994-12-23 2003-02-05 布里斯托尔-迈尔斯斯奎布公司 Pencillin V. amidohydrrolase gene from fusarium oxysporum
US6403356B1 (en) 1996-11-05 2002-06-11 Bristol-Myers Squibb Co. Mutant penicillin G acylases
WO2015151118A1 (en) 2014-04-04 2015-10-08 Council Of Scientific & Industrial Research A recombinant penicillin v acylase and process for the prepartion thereof

Also Published As

Publication number Publication date
DK141586D0 (en) 1986-03-26
JPS61502799A (en) 1986-12-04
SE8403929L (en) 1986-02-21
DK141586A (en) 1986-03-26
EP0190252A1 (en) 1986-08-13
SE8403929D0 (en) 1984-07-31

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