CC BY
3DOI https://doi.org/10.47612/1999-9127-2022-33-76-82 UDC 579.66:577.113.3:577.15
M. A. Vinter, I. S. Kazlouski, A. I. Zinchenko
CONSTRUCTION OF A BACTERIAL STRAIN FORMING INCLUSION BODIES, EXHIBITING DIADENYLATE CYCLASE ACTIVITY
State Scientific Institution "Institute of Microbiology of the National Academy of Sciences of Belarus" 2 Kuprevich St., 220141 Minsk, Republic of Belarus e-mail: rita.vinter.abc@gmail.com
Using Recombinant DNA Technology, the novel bacterial recombinant strain Escherichia coli DAC-22, a source of diadenylate cyclase that catalyzes the transformation of adenosine-5'-triphosphate into cyclic 3',5'-diadenylate (cyclo-di-AMP), was developed. The strain was derived by the transformation of E. coli Rosetta (DE3) pLysS cells with the recombinant plasmid pET42a+ wherein the disA gene responsible for the synthesis of the diadenylate cyclase of Bacillus thuringiensis was inserted. The producing capacity of the new strain with respect to diadenylate cyclase localized in catalytically active inclusion bodies equaled 720 units per liter of liquid culture. The newly engineered strain is destined for use in the technology related to the production of pharmaceutically promising cyclo-di-AMP.
Keywords: Escherichia coli, genetic engineering, recombinant strain, inclusion bodies, diadenylate cyclase, cyclo-di-AMP.
Introduction
Diadenylate cyclase (EC 2.7.7.85; DAC) catalyzes a reaction carrying out transformation of adenosine-5'-triphosphate (ATP) into cyclic 3',5'-diadenylate (cyclo-di-AMP) represented by the structural formula in figure 1. The aforementioned compound was discovered in 2008 as a component of gram-positive bacteria and archaea [1]. Cyclo-di-AMP plays a role of pathogen-associated molecular patterns triggering the synthesis of interferon and other antiinflammatory cytokines upon their introduction into the body of humans and vertebrata. Such remarkable properties open attractive medical
prospects for cyclo-di-AMP both as a therapeutic agent [2] and a vaccine adjuvant [3, 4].
Nowadays cyclo-di-AMP is mainly produced by multistage eco-hostile chemical synthesis [5]. An alternative biocatalytic approach to the production of cyclo-di-AMP is grounded on the monophase condensation of two ATP molecules under the impact of a bacterial enzyme — recombinant DAC.
It is evident that the application of cyclo-di-AMP in vaccines or for the induction of interferons will necessitate scaling the process up to an industrial level. The problem could be solved using genetically engineered bacterial
Fig. 1. Structural formula of cyclo-di-AMP
cultures capable of the hyperproduction of DAC. However, despite a solid investigation record and commercial advantages of E. coli strains, heterologous gene expression in these bacterial cells is often accompanied by an aggregation of superproduced target proteins into water-insoluble complexes named "inclusion bodies" [6, 7].
The enzymes integrated into inclusion bodies usually require a laborious solubilization procedure to recover their activities. However, rare cases are described in literature, where the procedure for refolding of inclusion bodies is not required, since precipitated enzymes do not lose their activity [8, 9].
It should be noted that DAC-producing strains are not obtained by trivial selection and genetic methods.
Experiments, resulting in recombinant strains able to produce elevated enzyme amounts following the induction of cloned DAC gene expression, have been reported on. For instance, the recombinant bacterial strain Mycobacterium tuberculosis BCG-disA-OE containing the DAC disA gene fused with the strong mycobacterial promoter hsp60 in the pSD5vector was engineered. In comparison with a wild-type bacterial strain, the recombinant culture was distinguished by 300-times increased gene disA expression and 15-fold enhanced level of cyclo-di-AMP synthesis [10].
Regretfully, the expression of the gene encoding DAC was recorded by the authors only by measuring the amount of mRNA using real-time PCR. Data on the recombinant DAC activity measured in units per liter of liquid culture are not available and they can not be computed.
Three strain-DAC producers based on E. coli BL21(DE3) were engineered by the transformation of their cells with plasmids pGP 1973, pGP1974 and pGP1975 carrying the cloned genes of three different isoforms (DisA, CdA and CdaS) of DAC from bacteria Bacillus subtilis [11]. The findings allowing the estimation of the DAC-producing ability of bacterial strains in the units of activity per liter of liquid culture were not reported.
The previously known recombinant strain E. coli pBtdac resulted from the transformation of the recipient strain E. coli BL21(DE3) with the plasmid pET42a(+) wherein the gene, encoding DAC from B. thuringiensis, was inserted. The strain productivity with respect to DAC (localized in inclusion bodies) is relatively low constituting
330.75 units/L of liquid culture [12].
The aim of our work was to engineer the recombinant E. coli strain, exceeding in DAC biosynthetic potential the analogs described in its literature.
Materials and methods
The chromosomal DNA of the bacterial strain B. thuringiensis BT 407 (Novagen, USA) served as a source of the structural gene disA (GenelD: 67464740) encoding the DAC amino acid sequence. DNA was isolated by phenol-chloroform method with complementary cetavlon purification [13]. The disA gene was synthesized by the PCR technique using Flash polymerase (ArtBioTech, Belarus) and synthetic oligonucleotide primers: DisA_2-F (5'-GTGGTGGTCCACAACATGGA AGAAAATAAGCAACGTG-3') and DisA_2-R (5 '-GTGGTGGTGGTGCTCATTGTGTCTACTC ATATATAGATGCTCT-3'). Nucleotide sequences (underlined) complementary to the plasmid pET42a+ (Novagen, USA) were inserted into 5'-ends of the primers.
Amplification products were separated by electrophoresis in 1% agarose gel. The product, corresponding to the disA gene, was recovered and inserted into the vector pET42a+ prelinearized by the PCR technique, using primers 42Int_R (5'-GTTGTGGACCACCACCATATGTATATC TCCTTCTT-3') and pET42lin_2-F (5'-GAGCAT CACCATCACCACCACCACCACTAATTG-3') primers. The assembly of DNA fragments (the linearized vector and the DAC encoding gene) was conducted by prolonged overlap extension PCR method [14].
The resulting mixture was used to transform the competent cells of E. coli Rosetta (DE3) plysS (Novagen, USA) produced by the standard calcium method [13] with subsequent inoculation on the solid nutrient LB medium (1% tripton, 0.5% yeast extract, 1% NaCl, 1% glucose, and 2% agar-agar) comprising kanamycin in 50 |ig/ ml concentration. Grown separate colonies were analyzed for the presence of the disA gene insert by the PCR technique using the primer to the T7-promoter 5'-TAATACGACTCACTATAGGG-3', incorporated into the plasmid pET24a+ and the disA primer — DisA_R.
Amplification products were subjected to electrophoresis in 1% agarose gel. Bacterial cells comprising the plasmid pET24a+ with the disA
gene insertion were selected. As a result, the recombinant bacterial strain of E. coli capable to produce the DAC of B. thuringiensis and designated as E. coli DAC-22 was derived.
10 ml of 24-hour cell culture of E. coli DAC-22 was inoculated into 2 Erlenmeyer flasks of 2 L volume, each containing 500 ml of the LB medium with kanamycin (50 |ig/ml, pH 7.0) and was grown on the shaker at 37 °C and at an agitation rate of 200 rpm. The inducer — isopropyl-ß-D-1-thiogalactopyranoside (IPTG; CarlRoth, Germany) in the final concentration of 0.5 mM was supplied into the medium reaching optical density 0.6 (X600 nm) and the culture continued for 2 h. Upon growth termination, the cells were precipitated by centrifuging (8 000 g, 5 min), resuspended in 50 mM phosphate buffer (pH 8.0) containing 300 mM NaCl and 10 mM imidazole.
Cells were disrupted by ultrasound in the Sonifier-450 instrument (Branson, USA). The insoluble fraction of the lysate was precipitated by centrifugation at 21 000 g for 20 min, then the precipitate containing inclusion bodies was washed with the 1 M urea solution, where Triton X-100 was added to the final concentration of 2%. After centrifugation, the pellet was washed twice with 50 mM Tris-HCI buffer (pH 8.0) containing 100 mM NaCI. The resulting 120 mg (dry weight) inclusion bodies were resuspended in 20 ml of the same buffer.
Gel electrophoresis of proteins was performed in 12% polyacrylamide gel with 4% sodium dodecyl sulfate. A protein molecular weight marker (BlueEye Prestained Protein Marker) was provided by Jena Bioscience (Germany). Quantification of the target protein was performed using the ChemiDoc Gel Documentation System (BioRad, USA) and the ImageLab software (BioRad, USA).
For the synthesis of cyclo-di-AMP, the obtained inclusion bodies were added to the reaction mixture (final volume 10 ml) containing (mM): MgCl2 — 10, Tris-HCl-buffer (pH 8.0) — 10, ATP — 5, and incubated at 55 °C for 3 h. The course of the reaction was controlled by thin layer chromatography on the plates of Silica gel 60 F254 (Merck, Germany) in the system of solvents dioxane-water-25% ammonia (4:3:0.25). The reaction products were eluted from the plates with water and the concentration of substances in the eluates was determined spectrophotometrically
using the known for cyclo-di-AMP molar extinction coefficient of 27 000 (X„_ ).
^ 259 nm'
Cyclo-di-AMP was isolated from the reaction mixture by chromatography on the DEAE-Toyopearl 650 M resin column (Toyo soda, Japan) (Cl--form) using a linear NaCl gradient (0-500 mM). The eluate was concentrated 200 times with a rotary evaporator at 55 °C. The resulting solution was applied to the Sephadex G-10 column (Serva, Germany) and the end product was eluted with water. The precipitate was washed with cooled alcohol and dried in vacuo.
DAC activity was assayed as described earlier [15]. The amount of enzyme producing 1 |imole of cyclo-di-AMP in 1 min of the reaction process was defined as one unit of activity.
The experimental data presented in the work reflect the confidence interval of the arithmetic mean for 95% probability level.
Results and discussion
The shortcoming of the only found in literature strain-DAC producer with characterized activity is in its relatively low producing capacity with respect to the studied enzyme. This might be due to the inappropriate efficiency of heterologous gene expression in the recipient strain used to engineer of the recombinant variant.
The proposed strategy to create of a novel microbial source of DAC envisaged an idea of using E. coli Rosetta (DE3) plysS as the strain-recipient transformed by the plasmid pET42a(+) carrying the disA gene. The developed recombinant strain E. coli DAC-22 is distinguished by superior target protein productivity over the strain E. coli pBtdac [12].
Fig. 2 presents data evaluating the enzyme production level in the course of submerged cultivation.
It can be seen from the electrophoregram demonstrated in Figure 2 that the amount of protein with the molecular weight of 47 kDa corresponding to the target enzyme was significantly increased in the cultural fluid of the recombinant strain after induction with IPTG. According to calculations made by the gel documentation system, DAC accounted for about 70% of the total cell lysate protein, with approximately 90% of the enzyme localized in inclusion bodies.
The resulting inclusion bodies (1 ml) were
M
55 kDa
43 kDa 34 kDa
24 kDa
18 kDa
Fig. 2. Electrophoregram of the protein composition of E. coli DAC-22 Tracks: 1 — cell lysate before the induction; 2 — cell lysate after the induction; 3 — cell lysate supernatant; 4 — inclusion
bodies; M — protein molecular weight markers
Yield of cyclo-di-AMP, mol.%
Reaction time, mill
Fig. 3. Dynamics of cyclo-di-AMP accumulation in the reaction catalyzed by the inclusion bodies with recombinant DAC
added to the reaction mixture (with the final volume of 10 ml), comprising, mM: MgCl2 — 10, Tris-HCl buffer (pH 8.0) — 10, ATP — 5 (50 |imol) and incubated at 55 °C for 2 h. Under these conditions (Fig. 3), the reaction yield reached more than 90% of theoretical maximum.
Based on the results obtained, the estimated production capacity of the strain expressed in the units of activity was calculated as 720 U/L of liquid culture.
Cyclo-di-AMP was recovered from the reaction mixture as described in the section "Materials and methods". 13 mg of chromatographically pure cyclo-di-AMP was produced. As a result, the yield
of the isolated end product calculated per initial ATP substrate constituted 75% of the maximum theoretical value.
Motility of the sample in the course of thin-layer chromatography on Silica gel 60 F254 plates (Merck, Germany) in the system of solvents dioxan-water-25% ammonia solvents (4:3:0.25), as well as UV spectral parameters of the product, totally matched the characteristics of the cyclo-di-AMP standard sample purchased from Jena Bioscience (Germany).
Thus, we demonstrated the possibility of applying of inclusion bodies for efficient cyclo-di-AMP synthesis. Such an approach to the production
and utilization of DAC enzyme preparations enables to raise the considerably productive potential of a microbial strain and it provides an opportunity of the repeated use of inclusion bodies in the relatively inexpensive technology of the production of cyclic dinucleotides and their marketing as a commercially attractive pharmaceuticals.
It should be noted that heterologous protein expression plays a key role in modern biotechnology. Formation of inclusion bodies during heterologous protein expression, especially the expression of eukaryotic proteins in prokaryote cell hosts, like E. coli, poses one of the most
intricate challenges for researchers and experts in this area. With a view of minimizing the appearance of inclusion bodies, and consequently, increasing the yield of soluble proteins, numerous strategies were initiated, including genetic methods, e.g. reducing the gene dosage, physical factors — a decline in fermentation temperature, physiological techniques — mixed culture with chaperons or nutrient limitation [16]. The outcome of applying of the above-mentioned procedures is unpredictable, so that it is not surprising that they failed to develop a generally recognized process protocol accepted with regard to all the proteins aggregating into inclusion bodies.
Residual activity ofDAC %
100 so so 70 60 so 40 30 20 10 0
—- t-—
l r
)12345678i 10 1 Cycle numbe
Fig. 4. Change in the DAC activity of inclusion body preparation during its repeated use in cyclo-di-AMP synthesis
On the other part, inclusion bodies represent relatively stable protein formations. They are readily isolated following cell disintegration in the course of simple physical manipulations and may act further as effective enzyme preparations, exemplified by several literature reports.
In order to verify the statement, we conducted experimental testing of the repeated use of inclusion bodies for cyclo-di-AMP biocatalysis. It follows from Figure 4 that inclusion bodies may be recycled at least 10 times for this purpose.
Conclusion
Relying on the genetic engineering experimental technique, the novel highly efficient recombinant bacterial strain producing an enzyme (DAC in particular) in the form of catalytically active inclusion bodies was constructed. The producing capacity of the new recombinant strain with respect to the DAC contained in catalytically active inclusion bodies was 720 units/L of liquid
culture, which is more than twice of the known strain-analog.
For the first time, it was demonstrated that inclusion bodies may be used as enzyme preparations for the repeated synthesis of pharmacologically promising cyclo-di-AMP.
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М. А. Винтер, И. С. Казловский, А. И. Зинченко
СОЗДАНИЕ ШТАММА БАКТЕРИЙ, ОБРАЗУЮЩИХ ТЕЛЬЦА ВКЛЮЧЕНИЯ, ПРОЯВЛЯЮЩИХ ДИАДЕНИЛАТЦИКЛАЗНУЮ
АКТИВНОСТЬ
Государственное научное учреждение «Институт микробиологии Национальной академии наук Беларуси» Республика Беларусь, 220141, г. Минск, ул. Купревича, 2 e-mail: rita.vinter.abc@gmail.com
С помощью техники рекомбинантной ДНК создан новый бактериальный рекомбинантный штамм Escherichia coli ДАЦ-22 — продуцент диаденилатциклазы, катализирующей реакцию трансформации аденозин-5'-трифос-фата в циклический 3',5'-диаденилат (цикло-ди-АМФ). Штамм получен трансформацией клеток E. coli Rosetta (DE3) pLysS рекомбинантной плазмидой pET42a+, в которую встроен ген disA, ответственный за синтез диаденилатциклазы Bacillus thuringiensis. Продуцирующая способность нового штамма в отношении диаденилатциклазы, находящейся в составе каталитически-активных телец включения, составила 720 ед/л культуральной жидкости. Созданный штамм предназначен для использования в технологии получения фармакологически перспективного цикло-ди-АМФ.
Ключевые слова: Escherichia coli, генная инженерия, рекомбинантный штамм, тельца включения, диаде-нилатциклаза, цикло-ди-АМФ.
