danДоклады Национальной академии наук БеларусиDoklady of the National Academy of Sciences of Belarus1561-83232524-2431The Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"10.29235/1561-8323-2022-66-5-509-516dan-1090Research ArticleБИОЛОГИЯBIOLOGYГетерологичная экспрессия диаденилатциклазы в виде телец включения, обладающих ферментативной активностьюHeterologous expression of diadenylate cyclase in the form of inclusion bodies with enzymatic activityВинтерМ. А.VinterM. A.

Винтер Маргарита Андреевна – мл. науч. сотрудник

ул. Купревича, 2, 220141, Минск

Vinter Margarita A. – Junior Researcher

2, Kuprevich Str., 220141, Minsk

rita.vinter.abc@gmail.comКазловскийИ. С.KazlouskiI. S.

Казловский Илья Сергеевич – канд. биол. наук, науч.сотрудник

ул. Купревича, 2, 220141, Минск

Kazlouski Illia S. – Ph. D. (Biology), Researcher

2, Kuprevich Str., 220141, Minsk

kazlouski.illia@gmail.comhttps://orcid.org/0000-0003-2401-2586ЗинченкоА. И.ZinchenkoA. I.

Зинченко Анатолий Иванович – член-корреспондент, д-р биол. наук, профессор, заведующий лабораторией

ул. Купревича, 2, 220141, Минск

Zinchenko Anatoliy I. – Corresponding Member, D. Sc. (Biology), Professor, Head of the Laboratory

2, Kuprevich Str., 220141, Minsk

zinch@mbio.bas-net.byИнститут микробиологии Национальной академии наук БеларусиInstitute of Microbiology of the National Academy of Sciences of Belarus202203112022665509516Copyright © Винтер М.А., Казловский И.С., Зинченко А.И., 20222022Винтер М.А., Казловский И.С., Зинченко А.И.Vinter M.A., Kazlouski I.S., Zinchenko A.I.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://doklady.belnauka.by/jour/article/view/1090

С помощью техники рекомбинантной ДНК создан новый бактериальный штамм Escherichia coli ДАЦ-22, клетки которого способны осуществлять гетерологичную экспрессию диаденилатциклазы Bacillus thuringiensis – фермента, катализирующего реакцию трансформации аденозин-5′-трифосфата в циклический 3′,5′-диаденилат (цикло-ди-АМФ). Для получения этого штамма в качестве клеток-реципиентов плазмиды pET42a+ со встроенным геном disA, кодирующим диаденилатциклазу B. thuringiensis, впервые были использованы клетки E. coli «Rosetta (DE3) pLysS». Клетки нового штамма способны продуцировать гетерологичную диаденилатциклазу, около 90 % которой локализовано во фракции каталитически активных телец включения. Продуцирующая способность полученного штамма в отношении диаденилатциклазы, находящейся в составе каталитически активных телец включения, составила 720 ед/л культуральной жидкости. Образуемые этим штаммом тельца включения могут быть использованы в технологии получения фармакологически перспективного цикло-ди-АМФ.

Using the DNA recombination technique, a new bacterial strain Escherichia coli DAC-22 was derived, whose cells are able to carry out the heterologous expression of Bacillus thuringiensis diadenylate cyclase – the enzyme catalyzing the reaction of adenosine-5′-triphosphate (ATP) transformation into cyclic 3′,5′-diadenylate (cyclo-di-AMP). To derive the strain, E. coli “Rosetta (DE3) pLysS” cells were originally used as recipients of plasmid pET42a+ with the inserted gene disA encoding diadenylate cyclase of B. thuringiensis. The cells of the recombinant strain are able to produce heterologous diadenylate cyclase localized predominantly (by 90 %) in the fraction of the catalytically active inclusion bodies. The productivity of the new strain with respect to diadenylate cyclase structurally arranged as the inclusion bodies was 720 units/l of cultural fluid. The inclusion bodies formed by the newly engineered strain are intended for use in the technology of producing pharmacologically promising cyclo-di-AMP.

Escherichia coliгенная инженериярекомбинантный штаммBacillus thuringiensisгетерологичная экспрессия генательца включениядиаденилатциклазацикло-ди-АМФEscherichia coligenetic engineeringrecombinant strainBacillus thuringiensisheterologous gene expressioninclusion bodiesdiadenylate cyclasecyclo-di-AMPReferencesA decade of research on the second messenger c-di-AMP / W. Yin [et al.] // FEMS Microbiol. Rev. – 2020. – Vol. 44, N 6. – P. 701–724. https://doi.org/10.1093/femsre/fuaa019Yin W., Cai X., Ma H., Zhu L., Zhang Y., Chou S.-H., Galperin M. Y., He J. A decade of research on the second messenger c-di-AMP. FEMS Microbiology Reviews, 2020, vol. 44, no. 6, pp. 701–724. https://doi.org/10.1093/femsre/ fuaa019Intranasal delivery of influenza rNP adjuvanted with c-di-AMP induces strong humoral and cellular immune responses and provides protection against virus challenge / M. V. Sanchez [et al.] // PLoS ONE. – 2014. – Vol. 9, N 8. – Art. e104824. https://doi.org/10.1371/journal.pone.0104824Sanchez M. V., Ebensen T., Schulze K., Cargnelutti D., Blazejewska P., Scodeller E. A., Guzmán C. A. Intranasal delivery of influenza rNP adjuvanted with c-di-AMP induces strong humoral and cellular immune responses and provides protection against virus challenge. PLoS ONE, 2014, vol. 9, no. 8, art. e104824. https://doi.org/10.1371/journal. pone.0104824Cyclic di-adenosine monophosphate: a promising adjuvant candidate for the development of neonatal vaccines / D. Lirussi [et al.] // Pharmaceutics. – 2021. – Vol. 13, N 2. – Art. 188. https://doi.org/10.3390/ pharmaceutics13020188Lirussi D., Weissmann S. F., Ebensen T., Nitsche-Gloy U., Franz H. B. G., Guzman C. A. Cyclic di-adenosine monophosphate: a promising adjuvant candidate for the development of neonatal vaccines. Pharmaceutics, 2021, vol. 13, no. 2, art. 188. https://doi.org/10.3390/pharmaceutics13020188Yan, H. The Promise and challenges of cyclic dinucleotides as molecular adjuvants for vaccine development / H. Yan, W. Chen // Vaccines. – 2021. – Vol. 9, N 8. – Art. 917. https://doi.org/10.3390/vaccines9080917Yan H., Chen W. The promise and challenges of cyclic dinucleotides as molecular adjuvants for vaccine development. Vaccines, 2021, vol, 9, no. 8, art. 917. https://doi.org/10.3390/vaccines9080917Chemical synthesis, purification, and characterization of 3′-5′-linked canonical cyclic dinucleotides (CDNs) / C. Wang [et al.] // Meth. Enzymol. – 2019. – Vol. 625. – P. 41−59. https://doi.org/10.1016/bs.mie.2019.04.022Wang C., Hao M., Qi Q., Chen Y., Hartig J. S. Chemical synthesis, purification, and characterization of 3′-5′-linked canonical cyclic dinucleotides (CDNs). Methods in Enzymology, 2019, vol. 625, pp. 41−59. https://doi.org/10.1016/bs. mie.2019.04.022Villaverde, A. Protein aggregation in recombinant bacteria: Biological role of inclusion bodies / A. Villaverde, M. M. Carrio // Biotechnol. Lett. – 2003. – Vol. 25, N 17. – P. 1385–1395. https://doi.org/10.1023/a:1025024104862Villaverde A., Carrio M. M. Protein aggregation in recombinant bacteria: Biological role of inclusion bodies. Biotechnology Letters, 2003, vol, 25, no. 17, pp. 1385–1395. https://doi.org/10.1023/a:1025024104862Schramm, F. D. Protein aggregation in bacteria / F. D. Schramm, K. Schroeder, K. Jonas // FEMS Microbiol. Rev. – 2020. – Vol. 44, N 1. – P. 54–72. https://doi.org/10.1093/femsre/fuz026Schramm F. D., Schroeder K., Jonas K. Protein aggregation in bacteria. FEMS Microbiology Reviews, 2020, vol. 44, no. 1, pp. 54–72. https://doi.org/10.1093/femsre/fuz026Enzymatic synthesis of 2′-ara and 2′-deoxy analogues of c-di-GMP / A. S. Shchokolova [et al.] // Nucleos. Nucleot. Nucl. Acids. – 2015. – Vol. 34, N 6. – P. 416–423. https://doi.org/10.1080/15257770.2015.1006775Shchokolova A. S., Rymko A. N., Kvach S. V., Shabunya P. S., Fatykhava S. A., Zinchenko A. I. Enzymatic synthesis of 2′-ara and 2′-deoxy analogues of c-di-GMP. Nucleosides, Nucleotides and Nucleic Acids, 2015, vol. 34, no. 6, pp. 416–423. https://doi.org/10.1080/15257770.2015.1006775Thermostable adenosine 5′-monophosphate phosphorylase from Thermococcus kodakarensis forms catalytically active inclusion bodies / S. Kamel [et al.] // Sci. Rep. – 2021. – Vol. 11, N 1. – Art. 16880. https://doi.org/10.1038/s41598- 021-96073-5Kamel S., Walczak M. C., Kaspar F., Westarp S., Neubauer P., Kurreck A. Thermostable adenosine 5′-monophosphate phosphorylase from Thermococcus kodakarensis forms catalytically active inclusion bodies. Scientific Reports, 2021, vol. 11, no. 1, art. 16880. https://doi.org/10.1038/s41598-021-96073-5Re-engineered BCG overexpressing cyclic di-AMP augments trained immunity and exhibits improved efficacy against bladder cancer / A. K. Singh [et al.] // Nat. Commun. – 2022. – Vol. 13, N 1. – Art. 878. https://doi.org/10.1038/s41467- 022-28509-zSingh A. K., Praharaj M., Lombardo K. A., Yoshida T., Matoso A., Baras A. S., Zhao L., Srikrishna G., Huang J., Prasad P., Powell J. D., Kates M., McConkey D., Pardoll D. M., Bishai W. R., Bivalacqua T. J. Re-engineered BCG overexpressing cyclic di-AMP augments trained immunity and exhibits improved efficacy against bladder cancer. Nature Communications, 2022, vol. 13, no. 1, art. 878. https://doi.org/10.1038/s41467-022-28509-zCyclic di-AMP homeostasis in Bacillus subtilis: both lack and high level accumulation of the nucleotide are detrimental for cell growth / F. M. Mehne [et al.] // J. Biol. Chem. – 2013. – Vol. 288, N 3. – P. 2004–2017. https://doi.org/10.1074/jbc. m112.395491Mehne F. M., Gunka K., Eilers H., Herzberg C., Kaever V., Stülke J. Cyclic di-AMP homeostasis in Bacillus subtilis: both lack and high level accumulation of the nucleotide are detrimental for cell growth. Journal of Biological Chemistry, 2013, vol. 288, no. 3, pp. 2004−2017. https://doi.org/10.1074/jbc.m112.395491Создание рекомбинантного штамма Escherichia coli – продуцента диаденилатциклазы и ее использование для синтеза цикло-ди-АМФ / И. С. Казловский [и др.] // Вес. Нац. акад. навук Беларусi. Сер. бiял. навук. – 2015. – № 4. – С. 51–55.Kazlovskij I. S., Radevich D. S., Rymko A. N., Shchokolova А. S., Kvach S. V., Zinchenko A. I. Construction of Escherichia coli strain, producing di-adenylate cyclase and its application for cyclic di-AMP synthesis. Vestsi Natsyyanal’nai akademii navuk Belarusi. Seryya biyalagichnykh navuk = Proceedings of the National Academy of Sciences of Belarus. Biological series, 2015, no. 4, рр. 51–55 (in Russian).Green, M. R. Molecular cloning. A laboratory manual. 4th ed. / M. R. Green, J. Sambrook. – New York, 2012. – 630 p.Green M. R., Sambrook J. Molecular cloning: a laboratory manual, fourth ed. New York, 2012. 630 p.Quan, J. Circular polymerase extension cloning of complex gene libraries and pathways / J. Quan, J. Tian // PLoS ONE. – 2009. – Vol. 4, N 7. – Art. e6441. https://doi.org/10.1371/journal.pone.0006441Quan J., Tian J. Circular polymerase extension cloning of complex gene libraries and pathways. PLoS ONE, 2009, vol. 4, no. 7, art. e6441. https://doi.org/10.1371/journal.pone.0006441Challenges associated with the formation of recombinant protein inclusion bodies in Escherichia coli and strategies to address them for industrial applications / A. Bhatwa [et al.] // Front. Bioeng. Biotechnol. – 2021. – Vol. 9. – Art. 630551. https:// doi.org/10.3389/fbioe.2021.630551Bhatwa A., Wang W., Hassan Y. I., Abraham N., Li X.-Z., Zhou T. Challenges associated with the formation of recombinant protein inclusion bodies in Escherichia coli and strategies to address them for industrial applications. Frontiers in Bioengineering and Biotechnology, 2021, vol. 9, art. 630551. https://doi.org/10.3389/fbioe.2021.630551

The authors declare that there are no conflicts of interest present.