vestibИзвестия Национальной академии наук Беларуси. Серия биологических наукProceedings of the National Academy of Sciences of Belarus, Biological Series1029-89402524-230XThe Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"10.29235/1029-8940-2022-67-1-66-74vestib-789Research ArticleСтатьиКаротиногенез в клетках Haematococcus pluvialis при совместном действии избытка NаCl в среде культивирования и света высокой интенсивностиCarotenogenesis in Haematococcus pluvialis cells under the combined action of excess of NaCl in the culture medium and high light intensityСамовичТ. В.SamovichT. V.

Самович Татьяна Викторовна - кандидат биологических наук, старший научный сотрудник.

ул. Академическая, 27, 220072, Минск.

Tatsiana V. Samovich - Ph. D. (Biol.), Senior Researcher, Institute of Biophysics and Cell Engineering of the National Academy of Sciences of Belarus.

27, Akademicheskaya Str., 220072, Minsk.

samovich77@gmail.comВязовЕ. В.ViazauYa. V.

Вязов Евгений Викторович - кандидат биологических наук, старший научный сотрудник.

ул. Академическая, 27, 220072, Минск.

Yauhen V. Viazau - Ph. D. (Biol.), Senior Researcher, Institute of Biophysics and Cell Engineering of the National Academy of Sciences of Belarus.

27, Akademicheskaya Str., 220072, Minsk.

yauhen.viazau@gmail.comГончарикР. Г.GoncharikR. G.

Гончарик Руслан Геннадьевич - научный сотрудник.

ул. Академическая, 27, 220072, Минск.

Ruslan G. Goncharik – Researcher, Institute of Biophysics and Cell Engineering of the National Academy of Sciences of Belarus.

27, Akademicheskaya Str., 220072, Minsk.

rusgon@mail.ruПечёнкинаЕ. И.PechenkinaE. I.

Печёнкина Елизавета Ивановна - младший научный сотрудник.

ул. Академическая, 27, 220072, Минск.

Elizaveta I. Pechenkina - Junior Researcher, Institute of Biophysics and Cell Engineering of the National Academy of Sciences of Belarus.

27, Akademicheskaya Str., 220072, Minsk.

eliza9214504@mail.ruКозелН. В.KozelN. V.

Козел Николай Владимирович - кандидат биологических наук, заведующий лабораторией.

ул. Академическая, 27, 220072, Минск.

Nikolai V. Kozel - Ph. D. (Biol.), Head of the Laboratory, Institute of Biophysics and Cell Engineering of the National Academy of Sciences of Belarus.

27, Akademicheskaya Str., 220072, Minsk.

kozel.mikalai@gmail.comИнститут биофизики и клеточной инженерии НАН БеларусиInstitute of Biophysics and Cell Engineering of the National Academy of Sciences of Belarus2022020220226716674Copyright © Самович Т.В., Вязов Е.В., Гончарик Р.Г., Печёнкина Е.И., Козел Н.В., 20222022Самович Т.В., Вязов Е.В., Гончарик Р.Г., Печёнкина Е.И., Козел Н.В.Samovich T.V., Viazau Y.V., Goncharik R.G., Pechenkina E.I., Kozel N.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestibio.belnauka.by/jour/article/view/789

Исследованы параметры роста и развития микроводоросли Haematococcus pluvialis, а также процесс каротиногенеза, включая пигментный состав и экспрессию ключевых генов биосинтеза астаксантина, в клетках водоросли в условиях совместного действия света высокой интенсивности и избытка NaCl в среде культивирования. Установлено, что сочетание избытка NaCl в среде культивирования Haematococcus pluvialis, инициированного на стадии покоя микроводоросли, и действия света высокой интенсивности не является эффективным способом индукции накопления астаксантина в клетках Haematococcus pluvialis. Выявлено снижение уровня экспрессии одного из ключевых генов каротиногенеза, кодирующего фитоинсинтазу, в таких условиях. В то же время установлено, что совместное действие света высокой интенсивности и избытка NaCl приводит к повышению доли более ценных с точки зрения биотехнологии цисизомеров астаксантина в общем пуле пигмента.

The parameters of growth and development of the microalga Haematococcus pluvialis, as well as the process of carotenogenesis, including the pigment composition and expression of the key genes of astaxanthin biosynthesis, were studied in algal cells under the combined action of excess of NaCl in the culture medium and high light intensity. It was found that the combination of excess of NaCl in the culture medium for Haematococcus pluvialis, initiated at the resting stage of the microalga, and high light intensity is not an effective method for induction of the accumulation of astaxanthin in the cells of Haematococcus pluvialis. A decrease in the level of expression of one of the key genes of carotenogenesis - phytoene synthase gene, was revealed under such conditions. At the same time, it was found that the combined action of high light intensity and excess of NaCl led to an increase in the ratio of astaxanthin cis-isomers, which are more biotechnologically valuable, in the total pigment pool.

Haematococcus pluvialisсолевой стресссвет высокой интенсивностикаротиногенезастаксантинHaematococcus pluvialissalt stresshigh light intensitycarotenogenesisastaxanthinРабота финансирована в рамках Государственной программы научных исследований «Биотехнологии» (задание 1.42, 2019-2020 гг.).This work was supported by the State Program of Scientific Research “Biotechnologies” (grant 1.42, 2019-2020).ReferencesAstaxanthin, a carotenoid with potential human health and nutrition / G. Hussein [et al.] // J. Nat. Prod. - 2006. -Vol. 69, N 3. - P. 443-449. https://doi.org/10.1021/np050354+Hussein G., Sankawa U., Goto H., Matsumoto K., Watanabe H. Astaxanthin, a carotenoid with potential in human health and nutrition. Journal of Natural Products, 2008, vol. 69. no. 3, pp. 443-449. https://doi.org/10.1021/np050354+Lorenz, R. T. Commercial potential for Haematococcus pluvialis as a natural source of astaxanthin / R. T. Lorenz, G. R. Cysewski // Trends Biotechnol. - 2000. - Vol. 18, N 4. - P. 160-167. https://doi.org/10.1016/s0167-7799(00)01433-5Lorenz R. T., Cysewski G. R. Commercial potential for Haematococcus pluvialis as a natural source of astaxanthin. Trends in Biotechnology, 2000, vol. 18, no. 4, pp. 160-167. https://doi.org/10.1016/s0167-7799(00)01433-5Naguib, Y. M. A. Antioxidant activities of astaxanthin and related carotenoids / Y. M. A. Naguib // J. Agr. Food Chem. -2000. - Vol. 48, N 4. - P. 1150-1154. https://doi.org/10.1021/jf991106kNaguib Y. M. A. Antioxidant activities of astaxanthin and related carotenoids. Journal of Agricultural and Food Chemistry, 2000, vol. 48, no. 4, pp. 1150-1154. https://doi.org/10.1021/jf991106kdel Campo, J. A. Outdoor cultivation of microalgae for carotenoid production: current state and perspectives / J. A. del Campo, M. Garda-Gonzalez, M. G. Guerrero // Appl. Microbiol. Biotechnol. - 2007. - Vol. 74, N 6. - P 1163-1174. https://doi.org/10.1007/s00253-007-0844-9del Campo J. A., Garda-Gonzalez M., Guerrero M. G. Outdoor cultivation of microalgae for carotenoid production: current state and perspectives. Applied Microbiology and Biotechnology, 2007, vol. 74, no. 6, pp. 1163-1174. https://doi.org/10.1007/s00253-007-0844-9Spiller, G.A. Safety of an astaxanthin-rich Haematococcus pluvialis algal extract: a randomized clinical trial / G. A. Spiller, A. Dewell // J. Med. Food. - 2003. - Vol. 6, N 1. - P. 51-56. https://doi.org/10.1089/109662003765184741Spiller G. A., Dewell A. Safety of an astaxanthin-rich Haematococcus pluvialis algal extract: a randomized clinical trial. Journal of Medicinal Food, 2003, vol. 6, no. 1, pp. 51-56. https://doi.org/10.1089/109662003765184741Our history [Electronic resource] // AstaReal. - Mode of access: https://www.astareal.com/en/history/. - Date of access: 20.04.2021.Our history (2021). Available at: https://www.astareal.com/en/history/ (accessed 20.04.2021).Cyanotech - Whole Health through Hawaiian microalgae [Electronic resource] // Cyanotech. - Mode of access: www.cyanotech.com. - Date of access: 20.04.2021.Cyanotech - Whole Health through Hawaiian microalgae (2021). Available at: http://www.cyanotech.com (accessed 20.04.2021).Одноклеточные водоросли как возобновляемый биологический ресурс / Г. С. Минюк [и др.] // Мор. экол. журн. -2008. - Т. 7, № 2. - С. 5-23.Minyuk G. S., Drobetskaya I. V., Chubchikova I. N., Terent'eva N. V. Unicellular algae as a renewable resource. Morskoi ekologicheskii zhurnal [Marine ecological journal], 2008, vol. 7, no. 2, pp. 5-23 (in Russian).Micro- and macro-algae: utility for industrial applications. Outputs from EPOBIO project / A. S. Carlsson [et al.]. -Chippenham : CPL Press, 2007. - 86 p.Carlsson A. S., van Beilen J. B., Moller R., Clayton D. Micro- and macro-algae: utility for industrial applications. outputs from epobio project. Chippenham, CPL Press, 2007. 86 p.Commercial applications of microalgae / P. Spolaore [et al.] // J. Biosci. Bioeng. - 2006. - Vol. 101, N 2. - P. 87-96. https://doi.org/10.1263/jbb.101.87Spolaore P., Joannis-Cassan C., Duran E., Isambert A. Commercial applications of microalgae. Journal of Bioscience and Bioengineering, 2006, vol. 101, no. 2, pp. 87-96. https://doi.org/10.1263/jbb.101.87Влияние NaCl на продуктивность водоросли Haematococcus pluvialis и содержание в ее клетках фотосинтетических пигментов, активных форм кислорода и астаксантина / Н. Г Аверина [и др.] // Вес. Нац. акад. навук Беларуст Сер. бiял. навук. - 2018. - Т. 63, № 3. - С. 263-275.Averina N. G., Kozel N. V., Shcherbakov R. A., Radyuk M. S., Manankina E. E., Goncharik R. G., Shalygo N. V. Effect of NaCl on Haematococcus pluvialis productivity and content in its cells of photosynthetic pigments, active oxygen forms and astaxanthin. Vestsi Natsyyanal'nai akademii navuk Belarusi. Seryya biyalagichnykh navuk = Proceedings of the National Academy of Sciences of Belarus. Biological series, 2018, vol. 63, no. 3, pp. 263-275 (in Russian).Печенкина, Е. И. Влияние фотосенсибилизатора бенгальского розового на продуктивность и пигментный состав Haematococcus pluvialis / Е. И. Печенкина, Т. В. Самович, Н. В. Козел // Журн. Белорус. гос. ун-та. Биология. -2021. - № 1. - С. 58-69.Pechenkina E. I., Samovich T. V., Kozel N. V. The influence of Rose Bengal photosensitizer on the productivity and pigment composition of Haematococcus pluvialis. Zhurnal Belorusskogo gosudarstvennogo universiteta. Biologiya [Journal of the Belarusian State University. Biology], 2021, no. 1, pp. 58-69 (in Russian).Каталог генетического фонда хозяйственно полезных видов водорослей / С. С. Мельников [и др.]. - Минск : Беларус. навука, 2011. - 101 с.13 Melnikov S. S., Manankina E. E., Budakova E. A., Shalygo N. V. Catalogue of the genetic fund of economically useful algae species. Minsk, Belaruskaya navuka Publ., 2011. 101 p. (in Russian).Rudic, V. Process for cultivation of green alga Haeamatococcus pluvialis (Flotow) / V. Rudic, T. Dudnicenco. - 2000. -MD Patent Nr. a 2000 0154.Rudic V., Dudnicenco T. Process for cultivation of green alga Haeamatococcus pluvialis (Flotow). 2000. MD Patent Nr. a 2000 0154.Rodriguez-Amaya, D. B. HarvestPlus handbook for carotenoid analysis / D. B. Rodriguez-Amaya, M. Kimura. -Washington : HarvestPlus, 2004. - 63 p.Rodriguez-Amaya D. B., Kimura M. HarvestPlus handbook for carotenoid analysis. Washington, HarvestPlus, 2004. 63 p.The identification of chlorophyll and its derivatives in the pigment mixtures: HPLC-chromatography, visible and mass spectroscopy studies / S. V. Milenkovic [et al.] // Adv. Technol. - 2012. - Vol. 1. - P 16-24.Milenkovic S. V., Zvezdanovic J. B., Andelkovic T. D., Markovic D. Z. The identification of chlorophyll and its derivatives in the pigment mixtures: HPLC-chromatography, visible and mass spectroscopy studies. Advanced Technologies, 2012, vol. 1, pp. 16-24.Forni, E. HPLC separation and fluorimetric estimation of chlorophylls and pheophytins in fresh and frozen peas / E. Forni, M. Ghezzi, A. Polesello // Chromatography. - 2012. - Vol. 26. - P. 120-124. https://doi.org/10.1007/BF02268135Forni E., Ghezzi M., Polesello A. HPLC separation and fluorimetric estimation of chlorophylls and pheophytins in fresh and frozen peas. Chromatography, 2012, vol. 26, pp. 120-124. https://doi.org/10.1007/BF02268135Yuan, J.-P. Hydrolysis kinetics of astaxanthin esters and stability of astaxanthin of Haematococcus pluvialis during saponification / J.-P. Yuan, F. Chen // J. Agr. Food Chem. - 1999. - Vol. 47, N 1. - P. 31-35. https://doi.org/10.1021/jf980465xYuan J.-P., Chen F. Hydrolysis kinetics of astaxanthin esters and stability of astaxanthin of Haematococcus pluvialis during saponification. Journal of Agricultural and Food Chemistry, 1999, vol. 47, no. 1, pp. 31-35. https://doi.org/10.1021/jf980465xLivak, K. J. Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) method / K. J. Livak, T. D. Schmittgen // Methods. - 2001. - Vol. 25, N 4. - P. 402-408. https://doi.org/10.1006/meth.2001.1262Livak K. J., Schmittgen T. D. Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) method. Methods, 2001, vol. 25, no. 4, pp. 402-408. https://doi.org/10.1006/meth.2001.1262Kakizono, T. Effect of carbon/nitrogen ratio on encystment accompanied with astaxantin formation in a green alga, Haematococcus pluvialis / T. Kakizono, M. Kobayashi, S. Nagai // J. Ferment. Bioeng. - 1992. - Vol. 74, N 6. - P. 403-405. https://doi.org/10.1016/0922-338X(92)90041-RKakizono T., Kobayashi M., Nagai S. Effect of carbon/nitrogen ratio on encystment accompanied with astaxantin formation in a green alga, Haematococcus pluvialis. Journal of Bioscience and Bioengineering, 1992, vol. 74, no. 6, pp. 403405. https://doi.org/10.1016/0922-338X(92)90041-RLerfall, J. Effect of high pressure processing on astaxanthin stability / J. Lerfall, S. Birkeland // Int. J. Food Sci. Tech. -2014. - Vol. 49, N 1. - P. 294-297. https://doi.org/10.1111/ijfs.12271Lerfall J., Birkeland S. Effect of high pressure processing on astaxanthin stability. International Journal of Food Science and Technology, 2014, vol. 49, no. 1, pp. 294-297. https://doi.org/10.1111/ijfs.12271Efficient and environmentally friendly method for carotenoid extraction from Paracoccus carotinifaciens utilizing naturally occurring Z-isomerization-accelerating catalysts / M. Honda [et al.] // Process Biochem. - 2019. - Vol. 89. -P. 146-154. https://doi.org/10.1016/j.procbio.2019.10.005Honda M., Kageyama H., Hibino T., Sowa T., Kawashima Y. Efficient and environmentally friendly method for carotenoid extraction from Paracoccus carotinifaciens utilizing naturally occurring Z-isomerization-accelerating catalysts. Process Biochemistry, 2019, vol. 89, pp. 146-154. https://doi.org/10.1016/j.procbio.2019.10.005Rapid and efficient conversion of all-E-astaxanthin to 9Z- and 13Z-isomers and assessment of their stability and antioxidant activities / C. Yang [et al.] // J. Agr. Food Chem. - 2017. - Vol. 65, N 4. - P. 818-826. https://doi.org/10.1021/acs.jafc.6b04962Yang C., Zhang L., Zhang H., Sun Q., Liu R., Li J., Wu L., Tsao R. Rapid and efficient conversion of all-E-astaxanthin to 9Z- and 13Z-isomers and assessment of their stability and antioxidant activities. Journal of Agricultural and Food Chemistry, 2017, vol. 65, no. 4, pp. 818-826. https://doi.org/10.1021/acs.jafc.6b04962Anti-inflammatory effects of different astaxanthin isomers and the roles of lipid transporters in the cellular transport of astaxanthin isomers in Caco-2 cell monolayers / C. Yang [et al.] // Agr. Food Chem. - 2019. - Vol. 67, N 22. - P. 6222-623 1 . https://doi.org/10.1021/acs.jafc.9b02102Yang C., Hassan Y. I., Liu R., Zhang H., Chen Y., Zhang L., Tsao R. Anti-Inflammatory Effects of different astaxanthin isomers and the roles of lipid transporters in the cellular transport of astaxanthin isomers in Caco-2 cell monolayers. Agricultural and Food Chemistry, 2019, vol. 67, no. 22, pp. 6222-6231. https://doi.org/10.1021/acs.jafc.9b02102Improved carotenoid processing with sustainable solvents utilizing z-isomerization-induced alteration in physicochemical properties: a review and future directions / M. Honda [et al.] // Molecules. - 2019. - Vol. 24, N 11. - P. 2149. https://doi.org/10.3390/molecules24112149Honda M., Kageyama H., Hibino T., Zhang Y., Diono W., Kanda H., Yamaguchi R., Takemura R., Fukaya T., Goto M. Improved carotenoid processing with sustainable solvents utilizing Z-isomerization-induced alteration in physicochemical properties: a review and future directions. Molecules, 2019, vol. 24, no. 11, p. 2149. https://doi.org/10.3390/molecules24112149Consumption of oxygen by astaxanthin biosynthesis: a protective mechanism against oxidative stress in Haematococcus pluvialis (Chlorophyceae) / Y. Li [et al.] // J. Plant Physiol. - 2008. - Vol. 165, N 17. - P. 1783-1797. https://doi.org/10.1016/j.jplph.2007.12.007Li Y., Sommerfeld M., Chen F., Hu Q. Consumption of oxygen by astaxanthin biosynthesis: a protective mechanism against oxidative stress in Haematococcus pluvialis (Chlorophyceae). Journal of Plant Physiology, 2008, vol. 165, no. 17, pp. 1783-1797. https://doi.org/10.1016/j.jplph.2007.12.007Steinbrenner, J. Regulation of two carotenoid biosynthesis genes coding for phytoene synthase and carotenoid hydroxylase during stress-induced astaxanthin formation in the green alga Haematococcus pluvialis / J. Steinbrenner, H. Linden // Plant Physiol. - 2001. - Vol. 125, N 2. - P. 810-817. https://doi.org/10.1104/pp.125.2.810Steinbrenner J., Linden H. Regulation of two carotenoid biosynthesis genes coding for phytoene synthase and carotenoid hydroxylase during stress-induced astaxanthin formation in the green alga Haematococcus pluvialis. Plant Physiology, 2001, vol. 125, no. 2, pp. 810-817. https://doi.org/10.1104/pp.125.2.810

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