vguitВестник Воронежского государственного университета инженерных технологийProceedings of the Voronezh State University of Engineering Technologies2226-910X2310-1202VSUET10.20914/2310-1202-2021-1-263-269vguit-2696Research ArticleПищевая биотехнологияFood biotechnologyСравнительная экспрессия рекомбинантной фосфолипазы А2 в Komagataella phaffii в зависимости от модификации сигнального пептида альфа-фактораComparative expression of recombinant phospholipase A2 in Komagataella phaffii depending on the modification of the alpha-factor signaling peptidehttps://orcid.org/0000-0002-4281-2758БытякД. С.BytyakD. S.

магистр, кафедра биохимии и биотехнологии, пр-т Революции, 19, г. Воронеж, 394036, Россия

master student, biochemistry and biotechnology department, Revolution Av., 19 Voronezh, 394036, Russia

bytyak.denis@mail.ruhttps://orcid.org/0000-0002-5895-9463ГладченкоЮ. А.GladchenkoY. A.

генный инженер, Центр биотехнологий, Белгородская область, Красногвардейский р-н, с. Малобыково, ул. Белая Вежа, д. 1, 309927, Россия

genetic engineer, Center for Biotechnology, Belgorod Region, Krasnogvardeysky district, Malobykovo village, Belaya Vezha str., 1, 309927, Russia

dngfv5@gmail.comhttps://orcid.org/0000-0002-7784-9892РяполоваА. В.RyapolovaA. V.

студент, кафедра медицинской биохимии и микробиологии, пр-т Революции, 19, г. Воронеж, 394036

student, medical biochemistry and microbiology department, Revolution Av., 19 Voronezh, 394036, Russia

anac142009@yandex.ruhttps://orcid.org/0000-0002-2863-0771КорнееваО. С.KorneevaO. S.

д.б.н., профессор, кафедра биохимии и биотехнологии, пр-т Революции, 19, г. Воронеж, 394036, Россия

Dr. Sci. (Biol.), professor, biochemistry and biotechnology department, Revolution Av., 19 Voronezh, 394036, Russia

korneeva-olgas@yandex.ruhttps://orcid.org/0000-0002-3433-2754МотинаЕ. А.MotinaE. A.

к.т.н., доцент, кафедра биохимии и биотехнологии, пр-т Революции, 19, г. Воронеж, 394036, Россия

Сand. Sci. (Engin.), biochemistry and biotechnology department, Revolution Av., 19, Voronezh, 394036, Russia

emotina18@mail.ruВоронежский государственный университет инженерных технологийРоссияVoronezh State University of Engineering TechnologiesRussian FederationООО «Инновационный центр «Бирюч-Новые Технологии»Russian FederationInnovation Center Biryuch-New TechnologiesRussian Federation202110052021831263269Copyright © Бытяк Д.С., Гладченко Ю.А., Ряполова А.В., Корнеева О.С., Мотина Е.А., 20212021Бытяк Д.С., Гладченко Ю.А., Ряполова А.В., Корнеева О.С., Мотина Е.А.Bytyak D.S., Gladchenko Y.A., Ryapolova A.V., Korneeva O.S., Motina E.A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.vestnik-vsuet.ru/vguit/article/view/2696

В настоящее время Российский рынок ферментных препаратов фосфолипазы А2 представлен коммерческими препаратами иностранных производителей: Nagase (Япония) и Maxapal (Нидерланды). Однако растущий спрос и необходимость снижения себестоимости производства фосфолипазы А2 требуют разработки новых суперпродуцентов фосфолипазы А2. В связи с чем целью работы является сравнительная экспрессия рекомбинантной фосфолипазы А2 в Komagataella phaffii в зависимости от модификации сигнального пептида альфа-фактора. Объектом исследования является дрожжевой штамм-реципиент Komagataella phaffii X-33. Исследования проводились в соответствии с общепринятыми нормами и подходами. Для работы использованы гены фосфолипазы А2 из Streptomyces violaceoruber. Целевые последовательности синтезированы в компании «Евроген» (Россия) и заклонированы в составе TE-вектора pUC57. В ходе выполнения работы проведена сборка генетических конструкций pPICZaA-Pla2 и PPICZmf4iA-Pla2, содержащих ген фосфолипазы А2 Streptomyces violaceoruber под нативным сигналом a-MF и его модификацией mf4i. Также проведена трансформация дрожжей Komagataella phaffii X-33 полученными генетическими конструкциями. В результате проведенных исследований показано, что в среднем достоверных отличий в уровне экспрессии и удельной активности рекомбинантной фосфолипазы А2 метилотрофными дрожжами K. Phaffii X-33 при использовании нативного сигнала секреции a-MF и его модифицированного варианта mf4i не обнаружено. Однако использование фактора секреции mf4i позволяет получить более высокую продукцию фосфолипазы А2 у отдельных клонов (трансформантов). Полученные данные указывают на перспективность использования фактора секреции mf4i для создания сверхпродуцентов ферментов на основе дрожжей K. Phaffii X-33.

Currently, the Russian market of phospholipase A2 enzyme preparations is represented by commercial preparations of foreign manufacturers: Nagase (Japan) and Maxapal (the Netherlands). However, the growing demand and the need to reduce the cost of production of phospholipase A2 require the development of new super-producers of phospholipase A2. In this connection, the aim of the work is to compare the expression of recombinant phospholipase A2 in Komagataella phaffii depending on the modification of the alpha-factor signaling peptide. The object of the study is the recipient yeast strain Komagataella phaffii X-33. The studies were conducted in accordance with generally accepted norms and approaches. Phospholipase A2 genes from Streptomyces violaceoruber were used for this worK. The target sequences were synthesized in the company "Eurogen" (Russia) and cloned as part of the TE vector pUC57. In the course of the work, the genetic constructs pPICZaA-Pla2 and PPICZmf4iA-Pla2 containing the Streptomyces violaceoruber phospholipase A2 gene were assembled under the native signal a-MF and its modification mf4i. The transformation of the yeast Komagataella phaffii X-33 with the obtained genetic constructs was also carried out. As a result of the conducted studies, it was shown that on average, there were no significant differences in the level of expression and specific activity of recombinant phospholipase A2 in methylotrophic yeast K. Phaffii X-33 when using the native a-MF secretion signal and its modified version mf4i. However, the use of the secretion factor mf4i allows for higher production of phospholipase A2 in individual clones. The obtained data indicate the prospects of using the secretion factor mf4i to create super-producers of enzymes based on yeast K. Phaffii X-33.

Komagataella phaffiiфосфолипаза А2Streptomyces violaceoruberгенная инженериябиотехнология ферментовэкспрессияAOX промоторсигнальный пептидKomagataella phaffiiphospholipase A2Streptomyces violaceorubergenetic engineeringenzyme biotechnologyexpressionAOX promotersignal peptideReferencesDennis E.A., Cao J., Hsu V.I.I., Magrioti V. et al. Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention // Chem Rev. 2011. V. 111. P. 6130–6185. doi: 10.1021/cr200085wDennis E.A., Cao J., Hsu V.I.I., Magrioti V. et al. Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention. Chem Rev. 2011. vol. 111. pp. 6130–6185. doi: 10.1021/cr200085wSamy R.P., Gopalakrishnakone P., Stiles B.G., Girish K.S. et al. Snake venom phospholipases A(2): a novel tool against bacterial diseases // Curr Med Chem. 2012. V. 19. P. 6150–6162. doi: 10.2174/092986712804485791Samy R.P., Gopalakrishnakone P., Stiles B.G., Girish K.S. et al. Snake venom phospholipases A(2): a novel tool against bacterial diseases. Curr Med Chem. 2012. vol. 19. pp. 6150–6162. doi: 10.2174/092986712804485791Muller V.D., Russo R.R., Cintra A.C., Sartim M.A. et al. Crotoxin and phospholipases A2 from Crotalus durissus terrificus showed antiviral activity against dengue and yellow fever viruses // Toxicon. 2012. V. 59. P. 507–515. doi: 10.1016/j.toxicon.2011.05.021Muller V.D., Russo R.R., Cintra A.C., Sartim M.A. et al. Crotoxin and phospholipases A2 from Crotalus durissus terrificus showed antiviral activity against dengue and yellow fever viruses. Toxicon. 2012. vol. 59. pp. 507–515. doi: 10.1016/j.toxicon.2011.05.021Muller V.D., Soares R.O., dos Santos N.N., Trabuco A.C. et al. Phospholipase A2 isolated from the venom of Crotalus durissus terrificus inactivates dengue virus and other enveloped viruses by disrupting the viral envelope // PLoS One. 2014. V. 9. №. 11. P. e112351. doi: 10.1371/journal.pone.0112351Muller V.D., Soares R.O., dos Santos N.N., Trabuco A.C. et al. Phospholipase A2 isolated from the venom of Crotalus durissus terrificus inactivates dengue virus and other enveloped viruses by disrupting the viral envelope. PLoS One. 2014. vol. 9. no. 11. pp. e112351. doi: 10.1371/journal.pone.0112351Russo R.R., M?ller V.D.M., Cintra A.C.O., Figueiredo L.T.M. et al. Phospholipase A2 crotoxin B isolated from the venom of Crotalus durissus terrificus exert antiviral effect against dengue virus and yellow fever virus through its catalytic activity // J Virol Antivir Res. 2014. V. 3. P. 1. doi: 10.1016/j.toxicon.2011.05.021Russo R.R., M?ller V.D.M., Cintra A.C.O., Figueiredo L.T.M. et al. Phospholipase A2 crotoxin B isolated from the venom of Crotalus durissus terrificus exert antiviral effect against dengue virus and yellow fever virus through its catalytic activity. J Virol Antivir Res. 2014. vol. 3. pp. 1. doi: 10.1016/j.toxicon.2011.05.021Castillo J.C., Vargas L.J., Segura C., Guti?rrez J.M. et al. In vitro antiplasmodial activity of phospholipases A2 and a phospholipase homologue isolated from the venom of the snake Bothrops asper // Toxins (Basel). 2012. V. 4. P. 1500–1516. doi: 10.3390/toxins4121500Castillo J.C., Vargas L.J., Segura C., Guti?rrez J.M. et al. In vitro antiplasmodial activity of phospholipases A2 and a phospholipase homologue isolated from the venom of the snake Bothrops asper. Toxins (Basel). 2012. vol. 4. pp. 1500–1516. doi: 10.3390/toxins4121500Nunes D.C., Figueira M.M., Lopes D.S., De Souza D.L. et al. BnSP7 toxin, a basic phospholipase A2 from Bothrops pauloensis snake venom, interferes with proliferation, ultrastructure and infectivity of Leishmania (Leishmania) amazonensis // Parasitology. 2013. V. 140. P. 844–854. doi:10.1017/S0031182013000012Nunes D.C., Figueira M.M., Lopes D.S., De Souza D.L. et al. BnSP 7 toxin, a basic phospholipase A2 from Bothrops pauloensis snake venom, interferes with proliferation, ultrastructure and infectivity of Leishmania (Leishmania) amazonensis. Parasitology. 2013. vol. 140. pp. 844–854. doi:10.1017/S0031182013000012Silveira L.B., Marchi-Salvador D.P., Santos-Filho N.A., Silva F.P. et al. Isolation and expression of a hypotensive and anti-platelet acidic phospholipase A2 from Bothrops moojeni snake venom // J Pharm Biomed Anal. 2013. V. 73. P. 35–43. doi: 10.1016/j.jpba.2012.04.008Silveira L.B., Marchi-Salvador D.P., Santos-Filho N.A., Silva F.P. et al. Isolation and expression of a hypotensive and anti-platelet acidic phospholipase A2 from Bothrops moojeni snake venom. J Pharm Biomed Anal. 2013. vol. 73. pp. 35–43. doi: 10.1016/j.jpba.2012.04.008Rodrigues R.S., Izidoro L.F., de Oliveira R.J., Sampaio S.V. et al. Snake venom phospholipases A2: a new class of antitumor agents // Protein Pept Lett. 2009. V. 16. P. 894–898. doi: 10.2174/092986609788923266Rodrigues R.S., Izidoro L.F., de Oliveira R.J., Sampaio S.V. et al. Snake venom phospholipases A2: a new class of antitumor agents. Protein Pept Lett. 2009. vol. 16. pp. 894–898. doi: 10.2174/092986609788923266Borrelli G.M., Trono D. Recombinant lipases and phospholipases and their use as biocatalysts for industrial applications // Int. J.N. Sci. 2015. V. 16. P. 20774–20840. doi: 10.3390/ijms160920774Borrelli G.M., Trono D. Recombinant lipases and phospholipases and their use as biocatalysts for industrial applications. Int. J.N. Sci. 2015. vol. 16. pp. 20774–20840. doi: 10.3390/ijms160920774Murakami M., Sato H., Miki Y., Yamamoto K. et al. A new era of secreted phospholipase A2 (sPLA2) // J Lipid Res. 2015. V. 56. P. 1248–1261. doi: 10.1194/jlr.R058123Murakami M., Sato H., Miki Y., Yamamoto K. et al. A new era of secreted phospholipase A2 (sPLA2). J Lipid Res. 2015. vol. 56. pp. 1248–1261. doi: 10.1194/jlr.R058123Quach N.D., Arnold R.D., Cummings B.S. Secretory phospholipase A2 enzymes as pharmacological targets for treatment of disease. // Biochem Pharmacol. 2014. V. 90. P. 338–348. doi: 10.1016/j.bcp.2014.05.022Quach N.D., Arnold R.D., Cummings B.S. Secretory phospholipase A2 enzymes as pharmacological targets for treatment of disease. Biochem Pharmacol. 2014. vol. 90. pp. 338–348. doi: 10.1016/j.bcp.2014.05.022Ahmad M., Hirtz M., Pitcher H., Schwab H. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous production // Appl Microbiol Biotechnol. 2014. V. 98. P. 5301–5317. doi: 10.1007/s00253-014-5732-5Ahmad M., Hirtz M., Pitcher H., Schwab H. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous production. Appl Microbiol Biotechnol. 2014. vol. 98. pp. 5301–5317. doi: 10.1007/s00253-014-5732-5Kang Z., Huang H., Zhang Y., Du G. et al. Recent advances of molecular toolbox construction expand Pichia pastoris in synthetic biology applications // World J Microbiol Biotechnol. 2017. V. 33. №. 1. P. 1-8. doi: 10.1007/s11274-016-2185-2Kang Z., Huang H., Zhang Y., Du G. et al. Recent advances of molecular toolbox construction expand Pichia pastoris in synthetic biology applications. World J Microbiol Biotechnol. 2017. vol. 33. no. 1. pp. 1-8. doi: 10.1007/s11274-016-2185-2Liu A., Yu X.-W., Sha C., Xu Y. Streptomyces violaceoruber Phospholipase A2: expression in Pichia pastoris, properties, and application in oil degumming // Appl Biochem Biotechnol. 2015. V. 175. №. 6. P. 3195-3206. doi: 10.1007/s12010-015-1492-7Liu A., Yu X.-W., Sha C., Xu Y. Streptomyces violaceoruber Phospholipase A2: expression in Pichia pastoris, properties, and application in oil degumming. Appl Biochem Biotechnol. 2015. vol. 175. no. 6. pp. 3195-3206. doi: 10.1007/s12010-015-1492-7Takemori D., Yoshino K., Eba C., Nakano H. et al. Extracellular production of phospholipase A 2 from Streptomyces violaceoruber by recombinant Escherichia coli // Protein Expression and Purification. 2012. V. 81. №. 2. P. 145-150. doi: 10.1016/j.pep.2011.10.002Takemori D., Yoshino K., Eba C., Nakano H. et al. Extracellular production of phospholipase A 2 from Streptomyces violaceoruber by recombinant Escherichia coli. Protein Expression and Purification. 2012. vol. 81. no. 2. pp. 145-150. doi: 10.1016/j.pep.2011.10.002Yu X.W., Sun W.H., Wang Y.Z., Xu Y. Identification of novel factors enhancing recombinant protein production in multi-copy Komagataella phaffii based on transcriptomic analysis of overexpression effects // Sci. Rep. 2017. V. 7. №. 1. P. 1-12. doi: 10.1038/s41598-017-16577-xYu X.W., Sun W.H., Wang Y.Z., Xu Y. Identification of novel factors enhancing recombinant protein production in multi-copy Komagataella phaffii based on transcriptomic analysis of overexpression effects. Sci. Rep. 2017. vol. 7. no. 1. pp. 1-12. doi: 10.1038/s41598-017-16577-xCodon Usage Database. URL: http://www.kazusa.or.jp/codon/index.html.Codon Usage Database. URL: http://www.kazusa.or.jp/codon/index.html.EasySelect Pichia Expression Kit. For Expression of Recombinant Proteins Using pPICZ and pPICZ? in Pichia pastoris. User manual // Invitrogen. 2010. 86 p.EasySelect Pichia Expression Kit. For Expression of Recombinant Proteins Using pPICZ and pPICZ? in Pichia pastoris. User manual. Invitrogen. 2010. 86 p.Valli M., Tatto N.E., Peymann A., Gruber C. et al. Curation of the genome annotation of Pichia pastoris (Komagataella phaffii) CBS7435 from gene level to protein function // FEMS yeast research. 2016. V. 16. №. 6. doi: 10.1093/femsyr/fow051Valli M., Tatto N.E., Peymann A., Gruber C. et al. Curation of the genome annotation of Pichia pastoris (Komagataella phaffii) CBS7435 from gene level to protein function. FEMS yeast research. 2016. vol. 16. no. 6. doi: 10.1093/femsyr/fow051

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