RU2499830C1 - RECOMBINANT STRAIN Escherichia coli-PRODUCER OF HYDROLASE OF ALPHA-AMINO ACIDS ESTERS FROM Xanthomonas rubrilineans, METHOD FOR MICROBIOLOGICAL SYNTHESIS OF HYDROLASE OF ALPHA-AMINO ACIDS ESTERS BASED ON ABOVE SAID STRAIN - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: strain is built by transformation of strain-recipient Escherichia coli BL21 (DE3) with plasmid DNA containing the coding area of gene aehR of hydrolase of alpha-amino acids esters Xanthomonas rubrilineans All-Russian collection of industrial microorganisms B-9915 under control of promoter T7 and gene of stability to kanamycin Kan of hydrolase of alpha amino acids esters from Xanthomonas rubrilineans All-Russian collection of industrial microorganisms B-9915. The proposed synthesis method of hydrolase of alpha-amino acids esters is implemented by cultivation of strain-producer Escherichia coli All-Russian collection of industrial microorganisms B-11246 with addition to the composition of environment of kanamycin and isopropyl-"в"-D-tiogallactoside as an expression inducer of the obtained product.

EFFECT: high biosynthesis level of target product, which can be used for development of methods for obtaining biocatalysts of synthesis processes of aminopenicillins and aminocephalosporins.

2 cl, 3 dwg, 1 tbl, 5 ex

Description

The group of claimed inventions relates to biotechnology, in particular to the biosynthesis of hydrolase of alpha-amino acid esters, and is a recombinant strain of bacteria Escherichia coli (E. coli) BL21 (DE3), containing the alpha-amino acid ester hydrolase gene, capable of synthesizing alpha-amino acid ester hydrolase , as well as a method of microbiological synthesis of a hydrolase of alpha-amino acid esters based on this strain.

Alpha-amino acid esther hydrolase (AEH) hydrolases are a class of enzymes that catalyze the hydrolysis of alpha-amino acids, hydrolysis of the acylamide bonds of various cephalosporins and penicillins, as well as N-acylation of 7-aminochepheme and 6-aminopenam with alpha-esters amino acids. Such substrate specificity allows the use of these enzymes as biocatalysts for the synthesis of cephalosporins and penicicillins containing in the side chain an amino group in the alpha position - aminocephalosporins and aminopenicillins (Figure 1; structures I and II, respectively), which make up the group of aminobeta-lactam antibiotics [1 ].

Semisynthetic penicillins and cephalosporins account for approximately 65% of the total global antibiotic market worth $ 15 million [2]. On an industrial scale, the most promising alternative to the chemical synthesis of antibiotics from key intermediates is biocatalysis. Advances in genetic engineering to create recombinant producer strains of various beta-lactam transformation enzymes provide the basis for the introduction of biocatalysis in the industrial production of semisynthetic penicillins and cephalosporins.

The information sources contain information about microorganisms producing intracellular AEH, which are natural strains or strains obtained by selection methods: Acetobacter turbidans [3-5], Pseudomonas melanogenum [6-12], Flavobacterium [13], Xanthomonas citri [14-18-18 ], Xanthomonas rubrilineans [19]. Genetic engineering strains of E. coli, producers of intracellular recombinant AEHs from Acetobacter turbidans, Zymomonas mob His Xanthomonas citri, Xanthomonas campestris pv, are also described. campestris [20-26]. In various sources, the quantitative characterization of the producers was carried out according to different types of enzymatic activity (hydrolysis of esters; hydrolysis of aminobeta-lactams, for example cephalexin, synthesis of antibiotics, usually cephalexin). A comparison of the available data showed that in all the cases described, the specific (in terms of protein content) enzymatic activity of the cell biomass in the synthesis of cephalexin from 7-aminodetoxycephalosporanic acid and D-phenylglycine methyl ester does not exceed 2 IU / mg protein (per international unit (IU) the enzymatic activity of the drug in relation to any biocatalytic transformation take such an amount of the drug that catalyzes the conversion of 1 μmol of substrate (or the formation of 1 μmol of product) in dard conditions for 1 minute). The comparison was carried out taking into account the approximate ratio of the rates of the above enzymatic reactions, as well as the fact that, using the soft methods of cell destruction, the specific activity of the initial cell biomass is not higher than the specific activity of the cell-free extract obtained from it.

Known work on the production of genetic engineering strains of Escherichia

coli - AEN producers are aimed primarily at producing a pure enzyme in order to study its structure and properties and did not lead to a significant increase in the productivity of recombinant strains compared to the original producers. For example [20], when the cells are destroyed by ultrasound and AEN is removed from Acetobacter turbidans ATCC 9325, a cell-free extract with a specific activity of 1.3 IU / mg of protein is obtained, and the gene-engineered AEN producer from Acetobacter turbidans based on E. coli provides 2 IU / mg protein.

Increasing the level of production of alpha-amino acid ester hydrolases is a necessary step in creating highly effective technological biocatalysts for industrial processes for the production of semi-synthetic aminopenicillins and aminocephalosporins and can be achieved by using heterologous microorganisms, which is the aim of the claimed group of inventions.

The closest analogue of the claimed strain is E. coli strain B834 (DE3), containing the aehX gene from Xanthomonas citri on plasmid pEC [27] under the control of the tac promoter and expressing the alpha-amino acid ester hydrolase [23, 25]. Strain E. coli B834 (DE3) was created with the aim of obtaining a pure enzyme and studying its structure and properties and was not characterized by the level of AEN synthesis. According to the results of a comparative analysis (http://blast.ncbi.nlm.nih.gov), the homology level of aehR genes from Xanthomonas rubrilineans VKPM B-9915 and aehR from Xanthomonas citri is 85%.

The closest analogue of the proposed method of microbiological synthesis of hydrolase of alpha-amino acid esters from Xanthomonas rubrilineans is a method using the mutant strain X. rubrilineans VKPM B-9915 producing this enzyme [28, 29]. Enzymatic activity of cell biomass in the synthesis of cephalexin from 7-aminodetoxic-cephalosporanic acid (7-ADCA), taken at a concentration of 0.04 M, and methyl ester of D-phenylglycine (MEFG), taken at a concentration of 0.08 M, at 40 ° C and pH 6.0, is 83.3 IU / g wet., 450 IU / g dry. The specific activity of the cells can be roughly estimated as 1 IU / mg of protein, given that the total protein is about half the dry weight of the biomass of this strain.

The task of the claimed group of inventions is to develop a method of microbiological synthesis of a hydrolase of alpha-amino acid esters from X. rubrilineans VKPM B-9915 with increased productivity.

The problem is solved by:

- designing a recombinant strain of bacteria Escherichia coli VKPM B-11246

- a producer of alpha-amino acid ester hydrolase from Xanthomonas rubrilineans VKPM B-9915, obtained by transformation of the recipient strain Escherichia. coli BL21 (DE3) plasmid DNA corresponding to the nucleotide sequence of SEQ ID NO 1 containing the coding region of the aehR gene for alpha-amino acid ester hydrolase from Xanthomonas rubrilineans VKPM B-9915 under the control of the T7 promoter, as well as the Kanamycin resistance gene.

- development of a method for microbiological synthesis of a hydrolase of alpha-amino acid esters from Xanthomonas rubrilineans VKPM B-9915 by culturing a recombinant producer strain Escherichia coli VKPM B-11246 in a suitable nutrient medium, which includes kanamycin, and the expression of the target product is carried out by adding isopropyl-β- D-thiogalactoside as an inducer.

Work includes:

- cloning of the aehR gene encoding the hydrolase of alpha-amino acid esters from X. rubrilineans VKPM B-9915;

- construction of recombinant plasmid DNA (plasmid) pAEN containing the gene for the hydrolase of alpha-amino acid esters from X. rubrilineans VKPM B-9915 (aehR) under the control of the T7 promoter;

- the construction of a recombinant bacterial strain of E. coli BL21 (DE3) / pAEH containing the plasmid pAEN and capable of synthesizing the hydrolase of alpha-amino acid esters from A: rubrilineans VKPM B-9915;

- development of a method for microbiological synthesis of a hydrolase of alpha-amino acid esters from X. rubrilineans VKPM B-9915 based on recombinant E. coli BL21 (DE3) / pAEH bacteria with an increased level of production of the target enzyme.

The process of constructing the inventive strain consists of several stages.

Step 1. Construction of recombinant plasmid DNA pAHE containing the aehR gene encoding the hydrolase of alpha-amino acid esters from X. rubrilineans VKPM B-9915.

The coding part of the aehR gene is amplified using X. rubrilineans VKPM B-9915 chromosomal DNA as a template. The plasmid pAHEN is constructed by cloning a PCR fragment containing the aehR gene into the pET-28a expression vector (Novagen) (Figure 2). The pET-28a vector of 5369 bp contains the T7 promoter (370-386 bp); a sequence encoding His-Tag (270-287); a sequence encoding a T7-Tag (207-239); polylinker BamHI-XhoI (158-203); a sequence encoding His-Tag (140-157); T7 terminator (26-72); the coding region of the lad gene (773-1852); pBR322 replication initiation site (3286); the coding region of the Kan gene, which provides resistance of E. coli strains to kanamycin (3995-4807); the site of replication initiation P (4903-5358) (Figure 2).

Plasmid pAHE with a size of 7210 base pairs along with the genes of the vector pET-28a contains the coding region of the adhR gene of the alpha amino acid ester hydrolase from X. rubrilineans under the control of the T7 promoter. The adhR gene is inserted into the pET-28a plasmid vector at the NdeI and XhoI restriction sites (Figure 3). The hydrolase of alpha-amino acid esters from X. rubrilineans expressed by this plasmid from the N-terminus is modified by the polyhistidine sequence.

Stage 2. Transformation of the plasmid pAEN strain E. coli BL21 (DE3)

Competent cells of E. coli strain BL21 (DE3) are transformed with the plasmid pAHEN. The result is a recombinant strain of E. coli BL21 (DE3) / pAEH, which contains the aehR gene of alpha-amino acid ester hydrolase from X. rubrilineans VKPM B-9915 and is capable of synthesizing alpha-amino acid ester hydrolase from A "rubrilineans VKPM B-9915.

The strain E. coli BL21 (DE3) / pAEH is deposited in the All-Russian collection of industrial microorganisms as E. coli VKPM B-11246.

The inventive strain VKPM B-11246 has the following morphological and physiological-biochemical characteristics.

Morphological features

Cells are straight, rod-shaped, motile, gram-negative,

non-spore forming. When grown for 24-72 hours at a temperature of 30-37 ° C on agar media (LB, 2YT) containing kanamycin (17 mg / l), the colonies are smooth, round, shiny, the edge is even. The composition of the medium LB, wt.%:

Bactotryptone - 1

yeast extract - 0.5

NaCl-1,

water is the rest.

 The composition of the medium 2YT, wt.%:

Bactotryptone - 1.6

yeast extract - 1.0

NaCl-0.5

water is the rest.

Physiological and biochemical characteristics

The strain grows at a temperature of 25 to 40 ° C (optimum 37 ° C). As a source of nitrogen, uses organic nitrogen in the form of peptone, amino acids.

The E. coli strain VKPM B-11246 synthesizes the hydrolase of alpha-amino acid esters from X. rubrilineans after induction with 1 mM IPTG (isopropyl-β-D-thiogalactoside).

Genotypic traits

The E. coli strain VKPM B-11246 is resistant to kanamycin.

Strain E. coli VKPM B-11246 contains on the plasmid pAHEN the gene for the hydrolase of alpha-amino acid esters (aehR) of X. rubrilineans VKPM B-9915 under the control of the T7 promoter.

Method for microbiological synthesis of hydrolase of alpha-amino acid esters in general form

Inoculum, which is a cell of a recombinant producer strain of E. coli VKPM B-11246, is prepared by incubation for 16 hours at 37 ° C in LB medium containing 17 mg / ml kanamycin. The grown culture is transferred at a ratio of 1: 200 (by volume) to LB medium containing 17 mg / ml kanamycin. The cultivation process is carried out at a temperature of 37 ° C with aeration. The synthesis of the target protein is induced by adding IPTG at OD ₆₀₀ = 0.8-1.0 OE. Cell biomass is separated by centrifugation, washed with phosphate buffer and centrifuged again.

The level of synthetase activity of the hydrolase of alpha-amino acid esters in the biomass of cells is not less than 1800 IU / g wet., 9000 IU / g dry.

The inventive method allows to increase the level of hydrolase production of alpha-amino acid esters to 20 IU / mg protein, which is 20 times higher than the production of the closest analogue.

The invention is illustrated by the following figures of graphic images:

Figure 1 - chemical structure of aminobeta-lactam antibiotics: aminopenicillins (I) and aminocephalosporins (II). The arrow indicates the pharmacologically important beta-lactam bond in the nuclei of antibiotics. The dashed line shows the acylamide bond formed during the enzymatic synthesis of amino-beta-lactams by transferring the acyl radical from the acylating agent (alpha-aminocarboxylic acid ester) containing the antibiotic side chain fragment to the amino group of the key intermediate carrying the antibiotic core.

Figure 2 - scheme of plasmid DNA pET-28a. Plasmid vector pET-28a (Novagen): T7 promoter (370-386 bp); polylinker (WatHI - XhoI) (158-203); lad (773-1852); Kan (3995-4807); replication initiation sites pBR322 ori (3286), fl ori (4903-5358).

Figure 3 - scheme of recombinant plasmid DNA pAEN. The plasmid vector pAEN: T7 promoter (370-386 bp); aehR alpha-amino acid ester hydrolase gene (6-1982); lad (2456-3535); Kan (5678-6490); replication initiation sites pBR322 ori (4969), fl ori (6586-7041).

Example 1. Construction of recombinant plasmid DNA pAHE containing the aehR gene encoding the hydrolase of alpha-amino acid esters from X. rubrilineans VKPM B-9915.

As the source of the aehR gene, genomic DNA isolated from the biomass of strain X. rubrilineans VKPM B-9915 is used. All standard genetic engineering and microbiological manipulations are carried out according to known methods [30].

The culture of strain X. rubrilineans VKPM B-9915 was grown at 27 ° C for 28 hours. Genomic DNA is isolated from the obtained biomass, which is used as a template for PCR.

For amplification of the coding part of the aehR gene, primers are used that contain the NdeI and XhoI restriction sites (PrAEHjfor 5'-gcatatgcgccgcatcgctccctg-3 'and PrAEH_rev 5'-cctegagtcagtacaccggcagactgatgaaactgg-3' polymerase) (Pment). The polymerase chain reaction is carried out according to the method of the company Fermentas. The resulting PCR product includes the coding region of the aehR gene

size 1914 bp

In order to obtain a plasmid construct for the expression of a hydrolase of alpha-amino acid esters, the PCR product containing the coding region of the aehR gene is cloned into the pET-28a expression vector (Novagen). For this, the pET-28a vector and the PCR product are treated with restriction enzymes Ndel and Xhol. A 5289 bp vector fragment is isolated from the restriction mixture. and a 1921 bp PCR product fragment The selected fragments are mixed and ligated; the resulting ligase mixture transform E. coli DH5α cells. Clone selection is carried out on LB agar medium with kanamycin. The result is a recombinant E. coli strain DH5α / pAEH containing the plasmid pAEN. The accuracy of the cloning and the absence of errors in the αehR gene sequence is confirmed by sequencing of the plasmid insert. The E. coli strain DH5α / pAEH does not synthesize alpha-amino acid ester hydrolase due to the absence of E. coli strain DH5α T7 RNA polymerase in the cells.

The recombinant pAEN plasmid DNA (SEQ ID NO 1) has a size of 7210 nucleotides (bp) and contains the alpha amino acid ester hydrolase gene (αehR) from X. rubrilineans VKPM B-9915, regulated by the T7 promoter and terminator; Kan gene providing E. coli resistance to kanamycin; replication initiation sites pBR322 and fl; the coding region of the lacI gene. At the N-end of the open reading frame, including the coding portion of the αhR gene, is located the sequence encoding 6 His (Figure 3).

Example 2. Obtaining the inventive strain of E. coli VKPM B-11246

In order to obtain a recombinant E. coli strain producing a hydrolase of alpha-amino acid esters, cells of the E. coli strain BL21 (DE3) are transformed with the plasmid pAEN by chemical transformation. The selection of transformants is carried out on LB agar containing kanamycin (17 mg / l). The result is the claimed strain of E. coli VKPM B-11246, synthesizing a hydrolase of alpha-amino acid esters from X. rubrilineans VKPM B-9915 (example 3).

Example 3. The biosynthesis of hydrolases of alpha-amino acid esters using E. coli strain VKPM B-11246 and the addition of an IPTG inducer

The starting seed is a culture of E. coli VKPM B-11246 grown on Petri dishes with agar medium. Seed is grown by incubating cells at 37 ° C for 15-17 hours on LB medium containing 17 mg / ml kanamycin.

The biosynthesis process leads to 750 ml Erlenmeyer flasks containing 100 ml

LB medium with kanamycin (17 mg / L). The seed is introduced into the fermentation medium in an amount necessary to create a concentration of about 0.5 vol.%. The producer cultivation process is carried out at a temperature of 37 ° C on a circular shaker with a rotation speed of 200-220 rpm until optical OD ₆₀₀ = 0.8-1.0 OE is reached (about 3 hours of cultivation), after which IPTG is added to the culture liquid concentration of 1 mm. Fermentation is continued under the same conditions for another 3 hours.

Cell biomass is separated by centrifugation at 5000 rpm, washed with 0.1 M phosphate buffer, pH 7.5, and centrifuged again.

From 2 l of fermentation medium (20 flasks) get 7.45 g of wet biomass. The activity of enzyme preparations for the synthesis of cephalexin is determined by the initial rate of formation of the target product under the following conditions: pH 6.0-6.2, temperature (40 ± 1) ° C, substrate concentration - (0.040 ± 0.002) M 7-ADC and (0 , 08010.004) M MEFG. The cephalexin content in the reaction mixture was determined by high performance liquid chromatography (HPLC). The solids content by the gravimetric method after the preparation is dried at (105 ± 1) ° С to constant weight. The protein content in the biomass of cells is determined by the Lowry method [31], without preliminary processing of native cells with ultrasound, since we showed that for the strains used, the protein content does not depend on the degree of destruction of the cells by ultrasound.

The resulting cell biomass is characterized by the following indicators:

- solids content: 21.4%;

- synthetase activity: 2020 IU / g wet .; 9440 IU / g dry .;

- specific activity: 23.8 IU / mg protein.

Example 4. The biosynthesis of hydrolases of alpha-amino acid esters using E. coli strain VKPM B-11246 without the addition of an inducer

The method is carried out as in example 3, with the difference that the cultivation is carried out for 6 hours without the addition of an IPTG inducer.

0.87 g of wet biomass is obtained from 200 ml of culture fluid. The resulting cell biomass is characterized by the following indicators:

- solids content of -18.4%;

- synthetase activity: 160 IU / g wet .; 870 IU / g dry .;

- specific activity: 1.9 IU / mg protein.

Example 5. (Control). Alpha-amino acid ester hydrolase biosynthesis using E. coli strain BL21 (DE3)

The method is carried out as in example 3, using for fermentation a culture of E. coli strain BL21 (DE3), different from the claimed E. coli strain VKPM B-11246 by the absence of the plasmid pAEN.

From 200 ml of culture fluid, 1.05 g of wet biomass is obtained. The resulting cell biomass is characterized by the following indicators:

- solids content of -18.4%;

- synthetase activity is not detected.

The high level of synthetase activity of alpha-amino acid ester hydrolase in the biomass of the recombinant E. coli VKPM B-11246 strain after induction of expression compared with uninduced culture confirms the expression of the aehR gene from X. rubrilineans, located in E. coli under the influence of the T7 promoter. The level of synthesis of hydrolases of alpha-amino acid esters of the claimed recombinant E. coli strain is 20 times higher than that of the closest analogue of X. rubrilineans VKPM B-9915 (Table 1).

The results of AEN biosynthesis by various producer strains of this enzyme are compared in Table 1.

Table 1 Synthetic activity of alpha-amino acid ester hydrolase in cell biomass

Synthetic activity E. coli VKPM B-11246 + IPTG (according to example 3) E. coli VKPM B-11246-IPTG (according to example 4) E.coli BL21 (DE3) (as in Example 5) X. rubrilineans VKPM B-9915 [28] IU / g wet 2020 160 0 83.3 IU / g dry 9440 870 0 450 IU / mg protein 23.8 1.9 0 About 1

Thus, the possibility of obtaining genetically modified E. coli bacteria capable of biosynthesis of hydrolase of alpha-amino acid esters from X. rubrilineans with a high level of biosynthesis of the target product was demonstrated.

The inventive method allows to synthesize intracellular hydrolase of alpha-amino acid esters in significant quantities and creates opportunities for the development of simple methods for the production of highly effective technological biocatalysts for the synthesis of aminopenicillins and aminocephalosporins.

The inventive recombinant strain-producing hydrolase of alpha-amino acid esters is classified as E. coli, the natural representatives of which are not able to synthesize this enzyme. The introduction of a new type of bacteria among the highly effective producers of hydrolases of alpha-amino acid esters provides additional opportunities for the development of effective methods for its biosynthesis.

Information sources

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Claims (2)

1. Recombinant strain of bacteria Escherichia coli VKPM B-11246 - producer of alpha-amino acid ester hydrolase from Xanthomonas rubrilineans VKPM B-9915 obtained by transforming the recipient strain Escherichia coli BL21 (DE3) plasmid DNA corresponding to the nucleotide sequence of SEQ ID NO 1 containing the coding region of the aehR gene for alpha-amino acid ester hydrolase from Xanthomonas rubrilineans VKPM B-9915 under the control of the T7 promoter, as well as the Kanamycin resistance gene. 2. The method of microbiological synthesis of a hydrolase of alpha-amino acid esters from Xanthomonas rubrilineans VKPM B-9915, comprising culturing bacteria containing the aehR gene from Xanthomonas rubrilineans VKPM B-9915, in a suitable nutrient medium, characterized in that the recombinant strain according to claim 1 is used as a producer .1, kanamycin is included in the medium, and the target product is expressed by the addition of isopropyl-β-D-thiogalactoside as an inducer.

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