SK126992A3 - Recombination plasmides with g-penicillin amides gene and strains of microorganism escherichia coli, containing this plasmides - Google Patents

Abstract

Recombinated plasmides code the synthesis of G-peniciline of amidase, their derivatives originate by deletion of noncoding areas and they are deductived from basic plasmide pK19 and modificated plasmide pBRa4 deductived from basic plasmide pBR332, are labelled pKA718 and pBH7. Recombinated plasmide pKA716 is derivative deductived from recombinated plasmides pKA18 and pBH7. Strain of microorganism Escherichia coli CCM 4228, which contain any of these recombinated plasmides, can be used as source of enzyme for preparation of 6-aminopenicilane acid (6-APK) from G-peniciline or 7-aminodeacetoxycefalosporane acid (7-ADCK) from deacetoxycephalosporine C.

Description

The present invention relates to recombinant plasmids pKA18, pBH7 and pKA716 and a derivative thereof carrying the G-penicillin amidase gene and a microorganism strain Escherichia coli CCM 4228 (pKA18), cCM 4228 (pBH7) and CCM 4228 (pKA716) containing these plasmids.

BACKGROUND OF THE INVENTION

It is known that some strains of microorganisms, especially of the genera Escherichia, Proteus and Bacillus, e.g. strains of Escherichia coli CCM 2843 and CCM 3759 obtained according to U.S. Pat. of author's certificates no. 188 631 and 244 343, produce the enzyme G-penicillin amidase hydrolyzing the amidic bond of G-penicillin (benzylpenicillin) to 6-aminopenicillanic acid (hereinafter referred to as 6-APK) or the amidic bond of deacetoxycephalosporin G (N-phenylacetyl-7-aminodorosporeceto) 7-aminodeacetoxycephalosporanoic acid (hereinafter 7-ADCK).

The transfer of chromosomal encoded information for the synthesis of G-penicillin amidase (PAG) to a plasmid or phage molecule has been described in the literature for several PAG structural genes derived from a microorganism of the genus Escherichia (Mayer et al.,

In: K.N. Timmis and A. Puhler (eds), Plasmid of Medical, Environmental and Commercial Importance, pp. 459-470, Elsevier / North Holland Biomedical Press, Amsterdam 1979; Oh et al., Gene 56, 87-92, 1987) of the genus Proteus (Daumy et al., J.Bacteriol. 163, 925-936, 1985) of the genus Arthrobacter (Ohashi et al., Appl. Environm. Microbiol., Pp. 2603-2615,

1988) of the genus Bacillus (Meevootis and Saunders, Appl. Microbiol. Biotechnol. 25, 372-386, 1987) and of the genus Kluyvera (Garcia and Buess. J. Biotechnol. 3, 187-199, 1986). Escherichia coli strains which did not carry genetic information for PAGs were used as host microorganisms for the recombinant plasmids in all of the above. In all cases, cloning of the PAG gene led to an increase in PAG synthesis compared to the production of starting microorganisms, and in some cases the regulation of this enzyme was changed (constitutive synthesis, change in susceptibility to catabolic repression, etc.).

-2V čs. A process for the preparation of recombinant plasmids (U.S. Patent Application No. PV 978-91) has also been described, wherein the strains carrying these plasmids (pAAA, pAA6, pBA4, pBA6) were characterized by PAG synthesis. Especially when using a recipient Escherichia coli strain carrying in addition a chromosomal determined PAG gene (RE3, see below), the sum of the production of the PAG gene, chromosomal and extrachromosomal, was determined, thereby increasing PAG synthesis. The specific activity of PAG in these strains was 460-550 µg dry matter in the cultivation of these strains in mineral soil, in the presence of phenylacetic acid (FOK) as an inducer of PAG synthesis and a single carbon source.

The patent application PV 978-91 discloses the properties of the Escherichia coli RE3 recipient strain used which carries the chromosomal determined PAG gene. This strain is derived from the strain Escherichia coli CCM 3759, a regulatory hyperinducible mutant obtained according to U.S. Pat. copyright certificate no. 162,274, wherein the strain RE3 does not contain the greater of the two original extrachromosomal DNA elements of more than 30 kb. Said patent application discloses a method of removing this extrachromosomal element.

SUMMARY OF THE INVENTION

The present invention provides recombinant plasmids pKA18, pBH7 and pKA716, characterized by a restriction map on the st. 1,2,3, encoding the synthesis of G-penicillin amidase in the inserted fragment of chromosomal DNA of Escherichia coli strain 3.9 kb for pKA18, 3.1 kb for pBH7 and pKA716, and derivatives of these plasmids produced by deletions of non-coding regions. The invention furthermore relates to Escherichia coli strains comprising these plasmids pKA18, pBH7 and pKA716 and their derivatives formed by deletion of non-coding regions deposited in the collection of microorganisms of the Institute of Microbiology C3AV in Prague under the designation CCM 4228 (pKA18), CCM 4228 (pBH7) and CCM 4228 (pKA716) ).

A small multicopy plasmid pK19 carrying a gene responsible for kanamycin resistance (Km gene) and a functional part was used as a starting vector for the preparation of recombinant plasmids pKA18 and pKA716.

-3 Lac operon (Pridmore R.G., Gene 56, 309-312, 1987). For the preparation of the recombinant plasmid pBH7, the plasmid pBRa4, which is a derivative of plasmid pBR332 having a lower molecular weight of 4.2 kb, carrying the gene determining resistance to the antibiotic tetracycline (Tc) obtained according to patent application PV 978-91, was used as starting vector.

The DNA fragment carrying the gene encoding PAG synthesis was isolated from the recombinant plasmid pAA6, which was derived from the recombinant plasmid pAA1 obtained by inserting the chromosomal DNA of the Escherichia coli RE3 strain into the pACYC184 vector as described in patent application PV 978-91.

For the isolation and detection of plasmid DNA, the Birmboin and Doly method (Methods in Enzymology 100, 243-254, Grosman and Moldave (eds.), Acad. Press N.Y., 1983) were used.

Preparation of competent cells and transformation of host strains was performed according to Hanahan (J. Mol. Biol. 166, 557-580, 1983) and isolation of chromosomal DNA was performed according to Au and T's (Proc. Natl. Acad. Sci., 86: 5507-5511 (1989).

As a recipient strain for transformation with said recombinant plasmids, Escherichia coli RE3 (CCM 4228) with a chromosomal determined PAG gene and an extrachromosomal element removed, as described in the prior art part of the present patent application, can be used.

A modified method (Zhang et al., Analyt. Biochem. 156, 413-416, 1986) based on the detection of enzyme active colonies using a colorless substrate of 2-nitro-5-phenylacetamidobenzoic acid was used to screen for a clone containing recombinant plasmids carrying the PAG gene. (hereinafter NIPAB), wherein contact with the colony surface results in enzymatic hydrolysis of the amide bond of NIPAB to form intense yellow 2-nitro-5-aminobenzoic acid as the product of the enzyme reaction. The procedure is as follows: filter paper saturated with fresh NIPAB solution (1.8 mg / ml 0.05 M phosphate buffer pH 7.5) was pressed onto colonies grown on solid nutrient media in Petri dishes, and then the filter papers with printed colonies were incubated at 37 ° C in empty sealed petri dishes for 30 to 60 minutes. Positive colonies intensify yellow and diffuse color around the paper.

The amount of G-penicillin amidase in the cells was determined by measuring the initial rate of hydrolysis of penicillin G in 0.05 M phosphate buffer at pH 8.0 at 37 ° C in the region of zero kinetics

-zídu. The reaction product, 6-APK, was determined spectrophotometrically using p-dimethylaminobenzaldehyde (Balasingham et al. Biochim. Biophys. Acta 276, 250-260, 1972). One unit (U) is the amount of enzyme that corresponds to the formation of one micromole of 6-APK per minute. Specific activity is defined as Ug ^{1 of} dry matter.

The subject strain Escherichia coli CCM 4228 (RE3) and the strain containing the recombinant plasmid pKA18, pBH7 or pKA716 and / or derivatives thereof are useful for the enzymatic hydrolysis of penicillin G to 6-APK or deacetoxycephalosporin to 7-ADCK. Preferably, this strain can be used as a starting source for the production of immobilized whole cells, their parts after disintegration, or it can be used for isolation of the crude enzyme and its subsequent immobilization.

The following summary summarizes the basic properties of the obtained Escherichia coli strains containing the recombinant plasmids as compared to the parent strain and the strains carrying the recombinant plasmids, which are the subject of patent application PV 978-91. The strains were cultured in 500 ml beakers with 50 ml soil on a rotary shaker at 30 ° C. The cultivation was always completed at the beginning of the stationary phase of growth and took place in the mineral soil M1 (composition: 0.3% FOK in the form of ammonium salt; 1.46% Na ₂ HPC> ₄ .12H ₂ O; 0.3% KH ₂ PO ₄ ; 0.05% NaCl; 0.1% NH ₄ Cl; 0.025% MgSO ₄ .7H ₂ 0; 0.0015% CaCl ₂ ; 0.02% yeast extract, pH 7.2) or MV mineral composition (composition 2% glycerol, 0.4% (NH ₄ ) ₂ SO ₄ , 1.36% KH ₂ PO ₄ , 0.3% NaOH;

0.001% FeSO ₄ .7H ₂ O; 0.165% MgSO ₄ .7H ₂ O; 0.008% CaCl ₂ .6 H ₂ O; pH 7.2-7.3) as described in Example 3.

Patent registration tribe Escherichia coli Specific PAG activity -1 (U.g dry matter) medium M ₁ inductor (F0K) + MV medium FOK - PV 978-91 CCM 4228 450 50 CCM 4228 460 73 CCM 4228 500 130 Patents in question- CCM 4228 500 620 your application CCM 4228 180 255 CCM 4228 (pKA716) 96 160 plasmids size chro- total mosomálního size (kb) fragment (kb)

PV 978-91 pAA6 PBA4 pBA6 3.9 3.9 2.9 10.5 ¾ \> 7.9 6.6 Concerned ρΚΛ18 3.9 6.56 Patent pBH7 3.1 7.43 registration pKA716 3.1 5.76

From this overview, the strains obtained according to the application PV 978-91 containing recombinant plasmids required a synthesis inducer (FOK) for maximum enzyme production (PAG), whereas strains containing the new prepared plasmids according to the present patent application (pKA18, pBH7 and pKA716) do not require FOK as an inducer of enzyme synthesis, they are therefore constitutive and in some cases synthesize more enzyme than inducible strains. In addition, in the presence of FOK, PAG synthesis was less than or equal to the new strains obtained. FOK inhibited growth. Thus, the advantage of using the new strains obtained is not only to achieve higher PAG production, but to eliminate the presence of an economically disadvantageous inducer from the culture soil or to eliminate FOK dosing during strain growth.

The invention is further illustrated by the following non-limiting examples.

Example 1

The recombinant plasmid pKA18 was constructed from the vector pK19 and the P fragment, which was obtained from the recombinant plasmid pAA6 as follows. The previously obtained pAA6 was digested with two restriction endonucleases Eco RI and Hind III. The restriction mixture contained a 3.9 kb fragment carrying the gene encoding G-penicillin amidase and 2 smaller fragments (2.7 and 1.3 kb) derived from the vector molecule. These fragments were separated electrophoretically in an agarose gel. A portion of the gel containing the 3.9 kb fragment (designated P fragment) was excised from the gel, and the DNA was isolated and purified using the Geneclean II Kit (Bio.ioi, USA) as described by the manufacturer. The vector pK19 was also digested with the restriction enzymes Eco RI and Hind III. These restriction sites are located at the multicloning site of this vector. After restriction inactivation, the linearized vector DNA was precipitated with ethanol and dissolved in CDTA-Tris buffer (1 mM CDTA and 10 mM Tris-HCl).

The thus prepared P fragment and the pK19 vector were joined by treatment with T4 DNA ligase (4 hours, room temperature). After inactivation of T4 DNA ligase, the E. coli strain HB101 was transformed with the ligation mixture. Competent cells were prepared by the Hanahan method (1983, cited above). After transformation, the cell suspension was sown on solid SOB (2% Trypton Difco; 0.5% yeast Difco extract; 10 mM NaCl; 2.5 mM KCl; 10 mM MgSO 4) containing 20 µg kanamycin. ml < 1 > After cultivation at 37 [deg.] C. for 24 hours, kanamycin-resistant clones were inoculated on LB soil (1% Trypton; 0.5% yeast extract; 1% NaCl; pH 7.2-7.5) and after 24 hours incubation at 25 ° C tested for the ability to synthesize PAG by the NIPAB cleavage method as described above. Positive clones were tested for the presence of recombined plasmids as follows. 1 ml of LB soil in microtubes (Eppendorf) was inoculated with a loop of test clones and the tubes incubated stationary overnight. After cell separation by centrifugation, plasmids were isolated by Birmboin and Doly (see above) and detected by agarose gel electrophoresis. PAG positive strains contained recombinant plasmids) of 6.5 kb. The monocolonate isolate selected was designated HB 101 (pKA18) and the recombinant plasmid pKA18 contained therein was isolated. Restriction analysis of this plasmid confirmed that it contained the P fragment and the vector pK19. Plasmid pKA18 was transformed with an E. coli strain RE3 to obtain a strain carrying the recombinant plasmid pKA18, which was designated CCM 4228 (pKA18).

Example 2

The recombined 7.43 kb plasmid pBH7 results from spontaneous changes in the structure of the plasmid molecule that occurred during the first replication of the plasmid in transformed E. coli HB101 strains. The plasmid resulting from the ligation of the P fragment with the Hind III linearized pBRα4 vector and treated with alkaline phosphatase (CIP) was used to transform the cells.

-Fragment P is flanked by Eco RI and Hind III restriction enzyme recognition sequences and can only be inserted into a vector linearized by the restriction endonuclease Hind III, only after joining the cohesive ends of the 2 P fragments generated by Eco RI. In the resulting double fragment, characterized by a sequence of Hind III-EcoRI-Hind III restriction sites, the PAG genes are mirror-oriented to each other and their promoters are located near the Hind III end of the fragment. However, this double fragment was not detected between tetracycline resistant transformants and synthesizing PAG. As a result of its structural instability, a clone carrying plasmids with fragments of 7 to 3.1 kb was selected which is structurally stable. In this way, a P 'fragment was spontaneously reduced to

3.1 kb, with the same terminal recognition sequences for Eco RI and Hind III as for the P fragment.

Plasmid pAA6 with P fragment and pBH7 with P 'fragment (as shown in FIGS. 1 and 2) were found to be reduced by the PAG non-coding region with one restriction enzyme site Sma I and Xba I by size 0,8 kb. The P 'fragment from the recombinant plasmid pBH7 was cloned directly into the pK19 vector, so that both pBH7 and pK19 were digested with a mix of restriction endonucleases Eco RI and Hind III, mixed and ligated to transform E. coli HB101 strain (as described above). in Example 1).

The obtained transformed strains grown on LB soil with kanamycin were tested for their ability to synthesize PAG. A 5.76 kb plasmid pKA716 was obtained from the positive clone, and the restriction analysis confirmed it to consist of the pKI9 vector and the inserted P 'fragment. Plasmid pKA716 was further transformed as in Example 1 into E. coli strain RE3 to obtain strain 4228 (pKA716).

Example 8

To compare the specific activity of the PAG strain RE3 carrying the recombinant plasmids obtained, cultivation was performed in mineral medium MV containing: 2% glycerol; 0.4% (NH4 J3 SO4);

1.36% KH ₂ PO ₄ ; 0.3% NaOH; 0.001% FeSO ₄ .7H ₂ O; 0.008% CaCl ₂ · 6 H ₂ O; 0.165% MgSO ₄ .7H ₂ O; pH 7.2-7.3. For solid culture in Petri dishes, this medium was supplemented with 2% agar and kanamycin was added at a concentration of 20 µg / ml. Strains from canned glycerol stored at -20 ° C were seeded on this solid soil and the plates were incubated at 30 ° C for 48 hours. The grown strains were inoculated with 50 ml of liquid MV soil with kanamycin (the same concentration as in solid soil) in 500 ml beakers and incubated on a rotary shaker (240 rpm 25 mm eccentric) for 20 hours at 30 ° C.

Then, 1 ml of the inoculum was seeded with 50 ml MV soil in a 500 ml flask. Cultivation was carried out under the same conditions for 14 hours (to stationary phase). Determination of PAG activity of the penicillin G substrate cells was performed as described above. The specific activities of the PAG strain are shown in Table 1 of the disclosure.

Example 4

Structural and segregation stability was studied in plasmid pKA18 in strain RE3. Strain cultivation was performed as described in Example 3. The culture in the stationary phase of growth was monitored for PAG production, the presence of plasmid cells and possible structural changes of the plasmid by the mini-preparative method of Birmboin and Doly (1983; cited in Example 1). This culture was also used as an inoculum for further cultivation. In a number of subsequent passages in the absence of kanamycin, the specific activity of PAG cells was unchanged in the first 5 passages. Over the next three passages, enzyme activity decreased slightly to 77% of baseline. No changes in plasmid size and structure were detected electrophoretically.

Plasmid-free cells were detected in a set of 100 monoclonal isolates that were cultured in parallel on solid

-9 currents in the presence and absence of kanamycin. 100% kanamycin resistant colonies were always isolated from the sowing of cell suspensions from each of the 8 passages. These results indicate that the plasmid in the cells is stable, with structural changes resulting in decreased activity only after the fifth passage in the absence of kanamycin.

Industrial usability

The plasmids and strains of the invention can be used to prepare intermediates for the production of β-lactam antibiotics, semisynthetic penicillins and deacetoxycephalosporins.

Claims (3)

PATENT NEEDS 1. Recombinant plasmids ρΚΑ18, ρΒΗ7 and ρΚΑ716 and their derivatives resulting from the deletion of non-coding regions, encoding G-penicillin amidase synthesis, characterized by the insertion of the 3.9 kb Escherichia coli DNA fragment for pKA18, 3.1 kb for pBH7, 3.1 kb in pKA716 and a restriction map. 2. Escherichia coli strains containing plasmids according to item 1 deposited in the collection of the microorganism of the Institute of Microbiology of the Czechoslovak Academy of Sciences under the designation CCM 4228 (pKA18), CCM 4228 (pBH7) and CCM 4228 (pKA716). 7V Fig. 1 Fig. 2 W -11- Fig. 3 The legends of the invention Recoabinated plasids carrying the G-penicillin aaidase gene and Escherichia coli likroorganis syrups containing these plasids Fig Recoabinated plasmid id pKA18 G-penicillin aaldase. Size Cloned DNX fragment (3.9 kb). it harbored the 6.56 kb plasmid gene. marked with double Fig. 2 The recoabinated plasmid pBH7 carried the G-penicillin aaldase gene. Plasmid size 7.43 kb. The cloned DNA fragment (3.1 kb) is indicated by a double line. Fig. 3 Recombinantly plasmid pKA716 carrying the G-penicillin aaldase gene. Plasmid size 5.76 kb. The cloned DNA fragment (3.1 kb) is indicated by a double line.