RU2300566C2 - RECOMBINANT PLASMID pSVH0106 PROVIDING SYNTHESIS OF Gl7ACA ACYLASE IN Escherichia coli CELLS, RECOMBINANT STRAIN OF Escherichia coli BL21(DE3)/pSVH0106 AS PRODUCER OF Gl7ACA ACYLASE - Google Patents

Abstract

FIELD: biotechnology, genetic engineering, biochemistry, antibiotics.

SUBSTANCE: invention proposes a constructed recombinant plasmid pSVH0106 comprising the natural gene sequence for acylase of glutaryl-7-aminocephalosporinic acid of the strain Brevundimonas diminuta BKM B-1297 that encodes a full-scale enzyme precursor. As result of transformation of E. coli strain with the proposed recombinant plasmid and selection of transformed clones the novel strain of E. coli BL21(DE3)/pSVH0106 is obtained that represents a producer of G17ACA acylase providing high yield of active enzyme. Owing to the effective and stable expression of recombinant G17ACA acylase in the proposed system using the proposed invention allows scaling a process for preparing this enzyme used broadly in manufacturing antibiotics.

EFFECT: valuable properties of plasmid.

2 cl, 4 dwg, 2 tbl, 9 ex

Description

The present invention relates to the field of biotechnology, in particular to genetic engineering, and can be used in the microbiological industry for the production of semi-synthetic beta-lactam antibiotics of a new generation.

A recombinant plasmid pSVH0106 is proposed that contains a DNA fragment that encodes the complete amino acid sequence of the acylase of glutaryl-7-aminocephalosporic acid of the strain Brevundimonas diminuta BKM B-1297, and provides a high level of its expression in Escherichia coli cells, and recombinant strain DE3 (E. 3) / pSVH0106- producer of Gl7ACA acylase

State of the art

The most important precursor compound for the preparation of antibiotics from the cephalosporin family is 7-aminocephalosporic acid (7-ACA) [1]. The conversion of cephalosporin C to 7-ACA can be carried out either by chemical hydrolysis, which requires the use of extremely toxic compounds and special incubation conditions at extremely low temperatures, or by enzymatic conversion. Known enzymatic transformation processes can be divided into 2 groups: a) one-stage using, for example, cephalosporin C-acylase from Pseudomonas [2] and b) two-stage, including the conversion of cephalosporin C to glutaryl-7-ACA (Gl7ACA) using the enzyme oxidase D-aminoxylot (DAO; EC-1.4.3.3), in particular obtained from Trigonopsis variabilis, Aspergillus, Penicillium, Neurospora, Pseudomonas, Cephalosporium, and subsequent hydrolysis of glutaryl-7-ACA to form 7-ACA or under the action of specific 7-beta - (4-carboxylbutanamino) acylases (Gl7ACA-acylases) [3], or under the action of fe other classes of gamma-glutamyl-transferase or cephalosporin-C-deacetylase [4].

Of greatest practical interest for the biocatalytic synthesis of 7-ACA is the enzyme Cl7ACA acylase [5], which belongs to the extensive family of penicillin amidases (EC 3.5.1.11), capable of converting penicillins and cephalosporins into valuable intermediates used for the production of new antibiotics [6, 7]. Gl7ACA acylases are heterotetramers consisting of 2 α and 2 β subunits [8] and are characterized by high activity with respect to Gl7ACA with low activity with cephalosporin C [3].

To date, the primary structures of the bacterial Gl7ACA acylase genes from various Pseudomonas strains are known, recombinant plasmid DNAs that synthesize these enzymes in E. coli and Bacillus subtilis cells, as well as methods for producing recombinant forms of enzymes using them are described [4, 8-14 ].

Common disadvantages of the known methods of heterologous expression of Gl7ACA acylases are the low level of synthesis of the recombinant product [15] and the complexity of the processes for scaling the fermentation of strains [16, 17], which are related to this, as well as the need to release the obtained Gl7ACA acylases from impurities of non-specific beta-lactamases [3]. In this regard, the challenge remains to develop new tools that increase the yield of Gl7ACA acylase when it is produced by recombinant DNA technology and simplifies the purification of these enzymes.

Disclosure of invention

The solution to the problem of increasing the yield of active recombinant Gl7ACA acylase involves the identification of bacterial strains that efficiently synthesize this enzyme in vivo, the isolation of genes encoding the enzyme from them, and the creation of optimal vector constructs for their efficient and stable expression in a heterologous bacterial cell. These aspects were the goal of the invention.

The goal was achieved due to the fact that

1) a strain (Brevundimonas diminuta BKM B-1297) was found in the collection of microorganisms that actively produces Gl7ACA acylase (BrdGl7ACA);

2) the gene of Gl7ACA acylase was obtained from this strain and the DNA sequence encoding the complete amino acid sequence of the enzyme was determined;

3) a recombinant plasmid DNA pSVH0106 was designed to synthesize BrdGl7ACA in E. coli cells in high yield;

4) as a result of cell transformation with the expressing plasmid pSVH0106, a recombinant strain Escherichia coli BL21 (DE3) / pSVH0106 with a high level of inducible synthesis of active Gl7ACA acylase was obtained.

The strain Brevundimonas diminuta VKM B-1297 was previously deposited in the All-Russian collection of microorganisms and selected by us as the most active producer of Gl7ACA acylase (BrdGl7ACA).

The complete coding sequence of the BrdGl7ACA gene was obtained by polymerase chain reaction (PCR) using the chromosomal DNA isolated from the Brevundimonas diminuta strain VKM B-1297 as a template, and the synthetic oligonucleotides with nucleotide sequences SEQ ID No. 12 (primer 7-aca) as primers -F) and SEQ ID NO: 13 (primer 7-aca-R), specific for the N- and C-terminal regions of the coding part (from position 103 to position 2265) of the isolated BrdGL7ACA gene fragment with SEQ ID N06.

This sequence encodes a 74 kDa protein (SEQ ID N0 7), which has a high degree of homology with other known bacterial precursors of Gl7ACA acylases and includes an N-terminal secretion signal sequence and a sequence encoding two enzyme subunits separated by a spacer peptide.

At the first stage of creating a recombinant plasmid for expression of a DNA fragment encoding Gl7ACA acylase, a carrier vector pAST7 was constructed, the physical and genetic maps of which are presented in Fig. 3. This vector includes the promoter and terminator of T7 phage RNA polymerase separated by a polylinker site, the replicon p15A and the kanR gene encoding aminoglycoside-3-phosphotransferase and ensuring the resistance of E. coli cells carrying the plasmid to kanamycin. The use of the kanamycin resistance gene as a marker, rather than the ampicillin resistance gene traditionally used in such designs, eliminates the need for subsequent complex purification of the target protein to remove beta-lactamase impurities.

The expressing recombinant plasmid pSHV0106 was obtained by inserting the coding sequence of the BrdGl7ACA gene into the pAST7 vector at the EcoRI and Sac I restriction sites (Fig. 4).

The recombinant producer strain BrdGL7ACA was obtained by transformation of Escherichia coli BL21 (DE3) cells with the constructed plasmid pSVH0106. The choice of the recipient strain is due to the fact that it carries the T7 phage RNA polymerase gene, which is necessary to ensure efficient transcription of the target genes in the vector construct under the control of the T7 phage promoter, as well as the fact that this strain is defective in protease synthesis, which is essential increases the yield of synthesized heterologous proteins. The synthesis of BrdGL7ACA in strain BL21 (DE3) / pSVH0106 is carried out by cultivation on conventional selective media with the addition of an isopropyl-D-thiogalactoside inducer (IPTG) or lactose.

The resulting recombinant strain Escherichia coli BL21 (DE3) / pSVH0106 is characterized by high physiological stability (the decrease in the activity of the enzyme produced after 50 generations does not exceed 20%) and a high yield of the active product, which is no less than 1.5 times the productivity of the known strains.

Thus, the present invention includes two objects.

The first object is the recombinant plasmid pSVH0106, which provides the synthesis of glutaryl-7-aminocephalosporin acid acylase (Gl7ACA-acylase) in Escherichia coli cells, which is characterized by the fact that it contains a DNA fragment with the nucleotide sequence of SEQ ID No 6 from the nucleotide at position 103 to the nucleotide at 2265 encoding the complete amino acid sequence of the Gl7ACA acylase of the strain Brevundimonas diminuta VKM B-1297 inserted at the EcoRI and SacI restriction sites into the polylinker region of the pAST7 vector, consisting of a fragment modified in the region of ilinkera rET21d plasmids containing promoter and terminator of RNA polymerase of T7, separated by a portion of the polylinker, and a fragment of plasmid rSYC117 containing r15A replicon and the kanamycin resistance gene, combined with each other as shown in Figure 3.

The second object of the invention is a recombinant strain of Escherichia coli BL21 (DE3) / pSVH0106-producer BrdGL7ACA.

The technical result achieved by the implementation of the present invention is to create new tools (expression vector and recombinant strain) that increase the quantity (yield) and quality (absence of beta-lactamase impurities) of the resulting recombinant Gl7ACA acylase.

Features of the present invention are illustrated by the best options for its implementation with reference to the figures.

A brief description of the figures.

Figure 1 Comparison of the nucleotide sequences of the Central coding part of the Gl7ACA gene of strain Brevundimonas diminuta VKM-1267 (Query) and homologous region of the gene for glutaryl acylase Pseudomonas SY-77 (Sbjct) using the BLAST program. The degree of sequence similarity is 98%.

Figa and 2b Comparison of the amino acid sequences of known Gl7ACA acylases from Pseudomonas sp.130 (AAC34685), Pseudomonas sp.SY-77 (AAN39264), Pseudomonas sp.THA1 (AAP68796) with the sequence BrdGl7ACA. The degree of sequence similarity is 98%.

Figure 3 Physical and genetic maps of the vector pACT7. The positions of the indicator restriction sites, the regions of the promoter and terminator of the T7 phage RNA polymerase (T7prom and T7 term), the region of the onset of replication (p15Aori), the kanamycin resistance gene (designated KN (R), black) are indicated.

Figure 4 Physical and genetic map of plasmid pSVH0106. The position of the BrdGl7ACA gene, (gray filling) is indicated, the remaining designations are as in FIG. 3.

The implementation of the invention

When carrying out the invention, in addition to the methods described in detail in the following examples, the methods well-known to those skilled in the art described in the manuals of molecular biology and genetic engineering were used [18, 19].

Example 1. Identification of a strain of Brevundimonas with a high level of biosynthesis of Gl7ACA acylase

The isolate cultures of various strains of the laboratory collection were grown in 100 ml Erlenmeyer flasks in 15 ml of medium containing 2% casein hydrolyzate, 0.5% monosodium glutamate, 0.5% yeast extract, 0.2% corn extract, 0.1% glutaric acid (pH 8.2) at 28 ° C with constant stirring (150 rpm) for 16 hours. When the culture density reached 2 OE at a wavelength of 600 nm, the cells were collected by centrifugation (6000 rpm; 10 min; + 4 ° C), washed in TE buffer (50 mM Tris pH 8.0 50 mM EDTA), suspended in the same buffer (0.1 g of wet cells per 200 μl of buffer) and stored frozen until use.

Glutaryl acylase activity was determined in each of the obtained samples. For this, a colorimetric method was used similar to the method proposed for the determination of 6-aminopenicillic acid [20]. For this, 100 μl of a 65 mM solution of glutaryl-7-ACA in 0.1M phosphate buffer (pH 7.0) is added to 900 μl of a cell suspension prepared in the same (0.1M phosphate) buffer containing 3 mg / ml potassium clavulinate, and incubate the mixture at 37 ° C for 30 minutes to 4 hours. At certain time intervals, the mixture is centrifuged, 500 μl of the supernatant are taken and mixed with 3 ml of a mixture (2: 1) of 20% acetic acid and 0.5 M NaOH solution to stop the reaction. 0.5 ml of p-dimethylaminobenzaldehyde (PDAB) was added to the sample, and after color development, absorbance was measured at 415 nm. For one unit of activity of the enzyme, the amount required for the conversion of 1 μmol of the substrate under the indicated conditions for 1 minute of incubation is taken.

The level of acylase activity determined in this way in 64 analyzed strains ranged from 0.01 U / L to about 4 U / L of culture. The strains Brevundiminas diminuta VKM B-1297 and Brevundimonas vesicularis VKM B-974- obtained from the All-Russian collection of microorganisms were most active. The strain Brevundimonas diminuta VKM B-1297 was selected for further work.

Example 2. Obtaining chromosomal DNA from a strain of Brevundimonas diminuta VKM B-1297

To 200 μl of a suspension of cells of B.diminuta strain VKM B-1297 obtained as described in example 1, add 10% SDS to a final concentration of 2% and 20 μl of proteinase K (3000 u / ml) and incubated for 30 min at 37 ° C, and then 15 min at 75 ° C. 200 μl of acid washed glass beads (0.1 mm diameter) were added to the suspension and vortexed vigorously for 5 minutes. Then, 300 μl of a phenol-saturated TE buffer solution was added to the suspension and vortexed for 30 seconds. The mixture was centrifuged, the supernatant was removed and extracted twice with a water-saturated chloroform solution. The aqueous phase was removed and a 5M potassium acetate solution (pH 6.0) was added to a final concentration of 1.5 M. The solution was centrifuged and the SDS potassium salt precipitate was discarded. An equal volume of isopropanol is added to the supernatant. The nucleic acid precipitate was collected by centrifugation, dissolved in 300 μl of 0.5 M potassium acetate, reprecipitated by adding 3 volumes of ethanol and dissolved in 100 μl of deionized water with heating for 30 minutes at 65 ° C. The chromosomal DNA of the strain Brevundiminas diminuta VKM B-1297 obtained by this method was used to obtain a gene encoding Gl7ACA acylase.

Example 3. Isolation of the central fragment of the BrdGl7ACA gene

A preliminary comparison was made of the amino acid and nucleotide sequences of bacterial Gl7ACA acylases using information available on the Internet at the National Center for Biotechnological Information of the United States. The most conserved regions of GL7ACA acylases of bacterial origin were identified and two synthetic oligonucleotides brd1 (SEQID N01) and brd2 (SEQ ID N02) were synthesized that bound the central coding sequence of the Gl7ACA acylase gene:

For PCR, the chromosomal DNA of Brevundiminas diminuta VKM B-1297, isolated as described above, is denatured by heating at 100 ° C for 5 minutes, placed in ice, subjected to 30 cycles in 50 μl of a mixture of the following composition:

5 μl 10-fold Taq-SE PCR buffer (Sibenzyme)

5 μl of genomic DNA (200 ng / μl)

5 μl of 3 μm brd1 primer

5 μl 3 μM brd2 primer

5 μl 2.5 mM dNTP (a mixture of all four types of deoxynucleotide triphosphates)

25 μl deionized water

1 μl Taq-SE polymerase (5 u / μl, Sibenzyme)

Reaction conditions: 94 °, 5 '(denaturation), 94 °, 30' ', 50 °, 30' ', 72 °, 1' (amplification). After amplification, 5 μl of PCR mixture was analyzed by electrophoresis in 1% agarose gel. Upon separation in electrophoresis, a homogeneous fragment of about 1 kb was identified. The fragment was isolated from the gel using the Wizard PCR Preps Kit (Promega, USA) (according to the manufacturer's instructions) and subjected to automatic sequencing on an ABIPrizm 3100 DNA Sequencer using brd1, brd2 primers and the Applera fluorescent Big dye Cycle sequencing kit .

Comparison of the obtained nucleotide sequence with the GenBank database using the BLAST program showed that it has a high degree of homology with the corresponding coding sequences of other bacterial Gl7ACA acylases (Fig. 1). Based on this, it was concluded that the obtained fragment corresponds to the central part of the Brd Gl7ACA gene, the acylase of the strain Brevundiminas diminuta VKM B-1297.

Example 4. Obtaining, sequencing and analysis of a DNA fragment comprising the complete coding sequence of the BrdGl7ACA gene

A DNA fragment comprising the complete coding sequence is obtained by PCR amplification with primers (BrdGl7ACA_F with SEQID N03 and BrdGl7ACA_R with SEQ ID N04) specific for conserved regions flanking the complete coding sequences of the known Gl7ACA acylase genes of pseudomonads.

1 μg of genomic DNA was subjected to 25 cycles of PCR amplification with BrdGl7ACA_F / BrdGl7ACA_R primers and using the Expand Long Template PCR system kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions.

The reaction mixture was separated by electrophoresis on a 1% agarose gel and the reaction product was revealed — a single fragment of about 2.25 kb in size. The fragment was isolated from the gel using the Wizard PCR preps Purification System kit (Promega, USA) and sequenced using the BrdGl7ACA_F, BrdGl7ACA_R, brd1, brd2 and brd 3 primers (SEQ ID N05).

The nucleotide sequence of the BrdGl7ACA gene thus established is listed under SEQ ID N06. The BrdGl7ACA protein amino acid sequence derived from it (SEQ ID N07) is 98% similar to the amino acid sequences of other known bacterial GL7ACA acylases (Fig. 2), while the differences are localized at sites remote from the sites of the active center of the enzyme and the areas of intersubunit contacts [8]. These data make it possible to conclude with a high degree of probability that the BrdGl7ACA enzyme corresponds to other bacterial Gl7ACA acylases both in their enzymatic and physicochemical properties.

Example 5. Construction of a vector carrier pACT7

Phage T7 promoter vectors provide high expression of heterologous genes in E. coli strains synthesizing T7 polymerase [21] and are commercially available [22]. At the same time, it is known that the immobilized preparations of Gl7ACA-acylases used for biotransformation of cephalosporin antibiotics should not contain beta-lactamase impurities. The presence of beta-lactamase also complicates the quantitative analysis of Gl7ACA acylase production levels in recombinant producer strains. Therefore, for the expression of BrdGl7ACA and other biotransformation enzymes of antibiotics free of beta-lactamase impurities, it is advisable to use vectors carrying plasmid resistance markers other than AmpR. Such vectors also have higher segregation stability [22], which can be further enhanced by using the p15A replicon instead of the ColE1 replicon [23].

The construction of the pACT7 vector satisfying these conditions was carried out in several stages.

A) Construction of the intermediate plasmid pET2ldRI.

M15 Tn10 (Tet^r)] (Stratagene, США), затем из полученных ампициллин-устойчивых трансформантов выделяют препараты плазмидной ДНК с использованием набора Wizard MiniPreps Kit (Promega, США). Полученные образцы ДНК анализируют совместным гидролизом рестриктазами EcoRI/PstI и BamHI/PstI и отбирают "положительные" клоны, содержащие EcoRI/PstI фрагменты размером 1300 и 4100 п.н. При этом за счет делеции сайта BamHI в BamHI/PstI гидролизате ДНК плазмидных клонов присутствует уникальный фрагмент размером 5400 пн, в то время как в препарате плазмидной ДНК pET21d, гидролизованной BamHI/PstI, обнаруживаются фрагменты размером 1300 и 4100 п.н. Несколько "положительных" клонов проверяют секвенированием с использованием праймеров Т7prom (SEQ ID N0 10) и T7term (SEQ ID N0 11) и отбирают клон с модифицированным таким образом участком полилинкера, не содержащий неспецифических мутаций, который обозначают как pET21dR1.Using the inverted PCR method and primers pETR1_F (SEQ ID N08) and pETR1_R (SEQ ID N09) on the plasmid matrix pET21d [21] using the Expand Long Template PCR system, a unique PCR fragment of 5.4 kb is obtained which is isolated from agarose gel, as described in example 4. 100 ng of the obtained fragment is hydrolyzed with 5 units of restriction enzyme EcoRI (Fermentas) and ligated with T4 DNA ligase. The competent ligase mixture was transformed with competent cells of the Escherichia coli strain XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F'proAB lacI ^q Z

M15 Tn10 (Tet ^r )] (Stratagene, USA), then plasmid DNA preparations were isolated from the obtained ampicillin-resistant transformants using the Wizard MiniPreps Kit (Promega, USA). The obtained DNA samples were analyzed by co-hydrolysis with restriction enzymes EcoRI / PstI and BamHI / PstI and "positive" clones containing EcoRI / PstI fragments of 1300 and 4100 bp were selected. Moreover, due to the deletion of the BamHI site in the BamHI / PstI plasmid clone DNA hydrolyzate, a unique fragment of 5400 bp is present, while fragments of 1300 and 4100 bp are detected in the preparation of plasmid DNA pET21d hydrolyzed by BamHI / PstI. Several “positive” clones were verified by sequencing using T7prom primers (SEQ ID N0 10) and T7term (SEQ ID N0 11) and a clone was selected with the polylinker section thus modified without nonspecific mutations, designated pET21dR1.

B) Construction of the plasmid pET21dRl / Tet.

To remove the AmpR gene from the plasmid pET21dR1 and replace it with the tetracycline resistance gene, the plasmid pET21dR1 / Tet was constructed. For this, in order to obtain an “insert” that carries the tetracycline resistance gene, 2 μg of plasmid DNA pKRP12 [24] was hydrolyzed with PstI restriction enzyme and a 1200 bp fragment was isolated from the agarose gel. The plasmid pET21dR1 hydrolyzed at the PstI site was used as a vector. The following ligation conditions were used: 1 μl vector (20 ng / μl), 1 μl fragment (50 ng / μl), 2 μl 5x ligase buffer (Gibco-BRL), 5 μl water and 1 μl T4 DNA ligase (1 unit / μl, Sibenzyme). The mixture was incubated for 14 hours at 12 ° C, then heated for 15 min at 65 ° C, cooled in ice, and 5 μl of the mixture was used to transform competent cells of E. coli strain JM109 [19]. Tetracycline resistant ampicillin sensitive clones were selected. Additional selection of the desired clones was carried out using restriction analysis of plasmid DNA preparations (by the formation of fragments of 1200, 1300, 4300 bp in size during EcoRI + PstI hydrolysis). One of the selected clones was designated as pET21dR1 / Tet and used in further work.

B) Construction of the plasmid pACYCpET.

To obtain a derivative of pET21dRI vector with replicon p15A, plasmid DNA pACYC177 [20] was hydrolyzed together with BamHI + PstI restriction enzymes and a 3000 bp fragment was included from the agarose gel, including the p15A replicon region, the kanamycin resistance gene and the C-terminal coding part of the beta gene -lactamases (resistance to ampicillin). The plasmid pET21dR1 was hydrolyzed together with BglII + PstI and a 1.5 kb fragment was isolated from the agarose gel, including the polylinker site, the T7 phage promoter and terminator, and the N-terminal coding part of the beta-lactamase gene. Two fragments were ligated; the E. coli Xl1Blue strain was transformed with the obtained ligase mixture and transformants resistant to ampicillin and kanamycin were selected.

Plasmid DNAs isolated from cultures of the obtained clones were analyzed by restriction with SmaI endonuclease and clones were selected to form 3.2 and 1.2 kb fragments. One of the selected clones was designated as pACYCpET.

D) Construction of the vector pACT7.

To remove the beta-lactamase gene from the pACYCpET vector, the plasmid DNA preparation is hydrolyzed together with NaeI / FspI restriction enzymes, a 3.4 kb fragment is isolated from the agarose gel, ligated "onto itself" at the "blunt ends" and the competent cells of strain E are transformed with the ligase mixture. coli JM109. Plasmid DNA is isolated from the selected kanamycin-resistant and ampicillin-sensitive transformants and the desired clone is identified by restriction analysis (the presence of fragments of 1.2 and 2.2 bp in SmaI-hydrolysates of plasmid DNA preparations).

Example 6. Construction of plasmids for the expression of BrdGL7ACA in E. coli.

First, a DNA fragment of the strain Brevundiminas diminuta VKM B-1297 containing the complete coding sequence of BrdGl7ACA (without adjacent non-coding sequences) and flanked by unique restriction sites is obtained by PCR.

For this, 1 μg of genomic DNA of VKM strain B-1297 was subjected to 25 cycles of PCR amplification with 7-aca_F / 7aca_R primers (SEQ ID N012,13) using the Expand Long Template PCR system kit ("Roche Diagnostics GmbH", Mannheim, Germany) according to the manufacturer's instructions. The primers used limit the BrdGL7ACA gene fragment, including the translation initiation codon located at position (+103) of SEQID N06, and the TGA termination codon located at position + 2265 of the same sequence and carry EcoRI and SacI restriction enzyme sites that are not in the gene coding sequence BrdGl7ACA.

The reaction mixture was separated by electrophoresis on a 1% agarose gel and the reaction product was revealed — a single fragment of about 2.16 kbp. The fragment isolated from the agarose gel was digested with EcoRI / SacI restriction enzymes and the vector pET21dR1 / Tet was cloned into EcoRI / SacI (see Example 5 “B”). Tetracycline-resistant transformants of E. coli strain JM109 were selected according to the criterion for the formation of fragments of 2.2 and 6.5 kb in EcoRI / SacI hydrolysates of the obtained plasmid DNA preparations.

In order to identify a clone carrying an insert with the intact BrdGL7ACA gene without possible mutations introduced by PCR amplification, the ability of the selected plasmids to direct the synthesis of functionally active BrdGL7ACA when transferred to E. coli strain BL21 (DE3) is tested [22].

For this, individual transformants of E. coli strain BL21 (DE3) carrying the obtained plasmids are grown on LB medium containing 10 μg / ml tetracycline in a volume of 5 ml to an OD600 ~ 1.0. After that, IPTG is added to the cultures to a final concentration of 1 μM to induce the T7 phage promoter and cultivation is continued for 18 hours at a temperature of 25-30 ° C. Cells are harvested by centrifugation and Gl7ACA acylase activity is determined as described in Example 1. Selected “positive clones” are sequenced using T7prom, T7term, brd1, brd2, brd3 primers and a clone carrying the insert with the intact BrdGl7ACA gene is identified, the coding sequence of which is identical the sequence of the DNA fragment from position + 103 to position + 2265 of the specific coding sequence of the BrdGl7ACA gene shown in the sequence list under the number SEQ ID N07. The plasmid isolated from the selected clone is designated pSVH18.

To obtain a BrdGl7ACA-expressing plasmid with p15A EcoR1 / Sac1 replicon, a fragment with the BrdGl7ACA gene from plasmid pSVH18 is cloned into pACT7 vector to form plasmid pSVH0106.

Example 7 Obtaining recombinant E. coli strains using plasmids pSVH18 and pSVH0106.

To obtain producer strains of recombinant BrdGl7ACA using standard methods [18], the recipient strain of E. coli BL21 (DE3) [22] is transformed with the obtained recombinant plasmid DNA. On plates with LB medium containing 10 mg / L tetracycline, individual tetracycline-resistant transformants obtained using pSVH18 plasmid DNA were selected. Primary transformants were cultured in LB liquid medium containing 10 mg / l of tetracycline, BrdGl7ACA synthesis was induced using IPTG as described in Example 6. One of the selected clones was designated as E. coli BL21 (DE3) / pSVH18, grown in 5 ml liquid LB medium containing 10 mg / l tetracycline to OD600 ~ 5.0, add an equal volume of sterile 30% glycerol to the culture, pour 500 μl into sterile 2 ml tubes, freeze and store at -70 ° C.

In a similar manner, primary transformants of E. coli strain BL21 (DE3) are obtained with plasmid pSVH0106. Individual kanamycin-resistant transformants are grown in LB liquid medium with the addition of 30 mg / L kanamycin, tested for the ability to synthesize functionally active BrdGl7ACA, a separate clone of E. coli BL21 (DE3) / pSVH0106 is selected and stored at - 70 ° C.

Example 8 Determination of production levels of recombinant BrdGl7ACA and physiological stability of the producing ability of recombinant strains.

For comparative quantitative determination of the BrdGl7ACA expression level in the obtained recombinant E. coli BL21 (DE3) / pSVH18 and E.coli BL21 (DE3) / pSVH0106 recombinant strains, cells are grown in 300 ml Erlenmeyer flasks with the addition of an appropriate antibiotic at a temperature of 25 ° C to A ₆₀₀ 1.0 OE. Next, make the inductor IPTG to a concentration of 0.2mM and incubate for 22 hours. Aliquots of cell suspensions are periodically taken and used to determine culture growth and enzyme activity, as described in Example 1.

As can be seen from the data presented (table 1), the enzyme activity reaches a maximum after 20 hours of incubation; while in E. coli strain BL21 (DE3) / pSVH0106 it is significantly higher (the maximum value is 1200 mU / ml culture) than in E. coli strain BL21 (DE3) / pSVH18.

Table 1
The dynamics of accumulation of BrdGl7ACA during the cultivation of recombinant strains of E. coli BL21 (DE3) / pSVH18 and E. coli BL21 (DE3) / pSVH0106 The cultivation time after induction, (hour) Activity, IU / ml culture The density of the culture, A ₆₀₀ , nm pSVH18 pSVH0106 pSVH18 pSVH0106 0
5
10
twenty one hundred
200
450
800 70
150
400
1200 1,0
1,5
2.1
4.8 1,0
1.7
2.5
5,6

Verification of the physiological stability of the producing activity of recombinant strains under continuous cultivation is as follows.

1. The first culture cycle is carried out on LB medium as described above, and at the end of the culture, the acylase activity is determined (the first culture cycle corresponds to about 30 generations).

2. 1 ml of the culture obtained after the first cycle is used as seed, which is added to 50 ml of fresh LB medium and the whole cycle is repeated once more (2nd cultivation cycle, approximately 5 generations).

3. Repeat the procedure 4 more times - up to 50 generations.

As follows from the data in Table 2, strain BL21 (DE3) / pSVH0106 has a higher physiological stability compared to strain BL21 (DE3) / pSVH18.

table 2
Physiological stability of BrdGl7ACA production in cells of recombinant E. coli BL21 (DE3) / pSVH18 and E. coli BL21 (DE3) / pSVH0106 strains Strain The activity of BrdGl7ACA (IU / ml culture) depending on the number of generations thirty 35 40 45 fifty E.coli BL21 (DE3) / pSVH18 800 ± 15 750 ± 12 700 ± 20 650 ± 15 600 ± 20 E.coli BL21 (DE3) / pSVH0106 1200 ± 30 1150 ± 25 1120 ± 20 1100 ± 20 1070 ± 15

Example 9. Characterization of the recombinant E. coli strain BL21 (DE3) / pSVH0106

Given that the specific activity of acylases homologous to BrdGL7ACA is on average about 10,000 mU / mg protein, the yield of recombinant BrdGL7ACA in strain BL21 (DE3) / pSVH0106 is approximately 100 mg of protein per liter of producer culture. A comparison of the obtained data with the previously known literature on the characteristics of recombinant E. coli strains producing bacterial Gl7ACA acylases suggests that the production level of recombinant Gl7ACA acylase in strain BL21 (DE3) / pSVH0106 is at least 1.5 times higher than the productivity of known strains [9 ,12].

Morphological features

Cells are elongated, rod-shaped, do not bud when dividing.

Cultural signs

Cells grow well on commonly used culture media. The generation time is about 30 minutes in a liquid LB medium. On 2-2.5% nutrient agar "Difco" round, smooth, yellowish colonies with smooth edges are formed. When grown on liquid LB and YT media, intense even turbidity forms.

Physiological and biochemical characteristics

The optimum cultivation temperature is from 25 to 30 ° C, the optimum pH is 7.6. The source of nitrogen is organic compounds (in the form of tryptone, yeast extract).

The level of synthesis of BrdGL7ACA (according to the determination of activity in samples of the biomass of the producer strain) is about 100 mg / L with a culture titer of 1 × 10 ⁹ cells / ml.

Claims (2)

1. Recombinant plasmid pSVH0106, providing synthesis of glutaryl-7-aminocephalosporin acid acylase (Gl7ACA-acylase) in Escherichia coli cells, which is characterized by the fact that it contains a DNA fragment with nucleotide sequence SEQ ID No. 6 from nucleotide at position 103 to nucleotide at position 2265 encoding the complete amino acid sequence of the Gl7ACA acylase of the Brevundimonas diminuta BKM B-1297 strain inserted at the EcoRI and SacI restriction sites into the polylinker region of the pAST7 vector consisting of a fragment of the plasmid pET21d modified in the polylinker region containing the promoter and terminator of RNA polymerase of phage T7, separated by a polylinker site, and a fragment of plasmid pCYC117 containing the replicon p15A and the kanamycin resistance gene, interconnected, as shown in Fig.3. 2. Recombinant strain of Escherichia coli BL21 (DE3) / pSVH0106-producer of Gl7ACA acylase.

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