vguitВестник Воронежского государственного университета инженерных технологийProceedings of the Voronezh State University of Engineering Technologies2226-910X2310-1202VSUET10.20914/2310-1202-2026-3-167-171vguit-3892Research ArticleПищевые системыFood systemsМетоды направленной метаболической инженерии Saccharomyces cerevisiae для программирования органолептического профиля пиваMethods of directed metabolic engineering of Saccharomyces cerevisiae for programming the organoleptic profile of beerhttps://orcid.org/0009-0007-3133-4467КулигинД. Р.KuliginD. R.kuligin470@gmail.comhttps://orcid.org/0009-0004-9485-664XДыгайД. В.DygaiD. V.loli_top@inbox.ruhttps://orcid.org/0000-0002-5989-0752МуравьевА. С.MuravevA. S.hntrun@mail.ruВоронежский государственный университет инженерных технологийРоссияVoronezh State University of Engineering TechnologiesRussian Federation202601102026883166170Copyright © Кулигин Д.Р., Дыгай Д.В., Муравьев А.С., 20262026Кулигин Д.Р., Дыгай Д.В., Муравьев А.С.Kuligin D.R., Dygai D.V., Muravev A.S.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/3892

Развитие технологий синтетической биологии и точного геномного редактирования открывает новые перспективы в пищевой биотехнологии. В пивоварении ключевым производственным микроорганизмом выступают штаммы дрожжей Saccharomyces cerevisiae, метаболизм которых напрямую определяет сенсорные характеристики конечного продукта — букет эфиров, высших спиртов, фенольных соединений и органических кислот. Традиционная селекция штаммов ограничена в возможности целевой модуляции сложных признаков. Целью работы являлась экспериментальная проверка возможности направленного изменения органолептического профиля пива методами редактирования генома пивоваренных дрожжей. На основе штамма S. cerevisiae WLP001 эля типа с использованием системы CRISPR/Cas9 получены два модифицированных штамма: с интеграцией в локус HO кассеты сверхэкспрессии генов ATF1 и MTT1 под контролем конститутивного промотора PGK1p и с полным удалением открытой рамки считывания гена POF1. Модельное брожение проводили в лабораторных ферментерах вместимостью 2 дм³ на охмелённом сусле с массовой долей экстракта 12 % при температуре 20 °C в трёх биологических повторностях; полученные образцы анализировали методом ГХ-МС и оценивали по методологии EBC. У штамма со сверхэкспрессией концентрация ацетата изоамила возросла с 1,8 до 5,67 мг/дм³, ацетата этила — с 12,5 до 35,0 мг/дм³ (p < 0,01) при неизменном уровне изоамилового спирта, что свидетельствует о перераспределении потока метаболитов в сторону этерификации. У штамма с делецией POF1 4-винилгваякол не детектирован при содержании 0,35 мг/дм³ в контроле. Сенсорная оценка подтвердила аналитические данные. Модификации не повлияли на степень сбраживания (76–78 %), флокуляционную способность и жизнеспособность клеток после брожения (свыше 95 %), что указывает на возможность изолированного изменения ароматобразующего потенциала без ущерба для технологических свойств штамма.

Advances in synthetic biology and precise genome editing open new prospects for food biotechnology. In brewing, the key production microorganism is Saccharomyces cerevisiae, whose metabolism directly determines the sensory characteristics of the final product — the bouquet of esters, higher alcohols, phenolic compounds and organic acids. Conventional strain selection offers limited scope for the targeted modulation of complex traits. The aim of this work was to experimentally verify the possibility of directed modification of the organoleptic profile of beer by genome editing of brewing yeast. Using the CRISPR/Cas9 system, two modified strains were obtained from the ale-type strain S. cerevisiae WLP001: one carrying an overexpression cassette of the ATF1 and MTT1 genes under the constitutive PGK1p promoter integrated into the HO locus, and one with complete deletion of the POF1 open reading frame. Model fermentation was carried out in 2 dm³ laboratory fermenters on hopped wort with an extract content of 12 % at 20 °C in three biological replicates; the resulting samples were analysed by GC-MS and evaluated according to the EBC methodology. In the overexpression strain, the isoamyl acetate concentration increased from 1.8 to 5.67 mg/dm³ and ethyl acetate from 12.5 to 35.0 mg/dm³ (p < 0.01), while the isoamyl alcohol level remained unchanged, indicating a redistribution of metabolic flux towards esterification. In the POF1 deletion strain, 4-vinylguaiacol was not detected, against 0.35 mg/dm³ in the control. Sensory evaluation confirmed the analytical data. The modifications did not affect the degree of attenuation (76–78 %), flocculation ability or post-fermentation cell viability (above 95 %), indicating that the aroma-forming potential can be altered in isolation without compromising the technological properties of the strain.

Saccharomyces cerevisiaeметаболическая инженерияCRISPR/Cas9ароматические соединенияпивоварениесенсорный профильSaccharomyces cerevisiaemetabolic engineeringCRISPR/Cas9aromatic compoundsbrewingsensory profileReferencesDiCarlo J.E., Norville J.E., Mali P. et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Research. 2013. vol. 41. no. 7. pp. 4336–4343. doi: 10.1093/nar/gkt135.DiCarlo J.E., Norville J.E., Mali P. et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Research. 2013. vol. 41. no. 7. pp. 4336–4343. doi: 10.1093/nar/gkt135.Gallone B., Steensels J., Prahl T. et al. Domestication and divergence of Saccharomyces cerevisiae beer yeasts. Cell. 2016. vol. 166. no. 6. pp. 1397–1410. doi: 10.1016/j.cell.2016.08.020.Gallone B., Steensels J., Prahl T. et al. Domestication and divergence of Saccharomyces cerevisiae beer yeasts. Cell. 2016. vol. 166. no. 6. pp. 1397–1410. doi: 10.1016/j.cell.2016.08.020.Krogerus K., Magalhães F., Vidgren V., Gibson B.R. Novel brewing yeast hybrids: creation and application. Applied Microbiology and Biotechnology. 2017. vol. 101. no. 1. pp. 65–78. doi: 10.1007/s00253-016-8007-5.Krogerus K., Magalhães F., Vidgren V., Gibson B.R. Novel brewing yeast hybrids: creation and application. Applied Microbiology and Biotechnology. 2017. vol. 101. no. 1. pp. 65–78. doi: 10.1007/s00253-016-8007-5.Lian J., Bao Z., Hu S., Zhao H. Engineered CRISPR/Cas9 system for multiplex genome editing in Saccharomyces cerevisiae. Metabolic Engineering. 2015. vol. 29. pp. 286–296. doi: 10.1016/j.ymben.2015.04.004.Lian J., Bao Z., Hu S., Zhao H. Engineered CRISPR/Cas9 system for multiplex genome editing in Saccharomyces cerevisiae. Metabolic Engineering. 2015. vol. 29. pp. 286–296. doi: 10.1016/j.ymben.2015.04.004.Mans R., et al. CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. FEMS Yeast Research. 2015. vol. 15. no. 2. doi: 10.1093/femsyr/fov004.Mans R., et al. CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. FEMS Yeast Research. 2015. vol. 15. no. 2. doi: 10.1093/femsyr/fov004.Nielsen J., Keasling J.D. Engineering cellular metabolism. Cell. 2016. vol. 164. no. 6. pp. 1185–1197. doi: 10.1016/j.cell.2016.02.004.Nielsen J., Keasling J.D. Engineering cellular metabolism. Cell. 2016. vol. 164. no. 6. pp. 1185–1197. doi: 10.1016/j.cell.2016.02.004.Saerens S.M.G., Delvaux F., Verstrepen K.J., Thevelein J.M. Production and biological function of volatile esters in Saccharomyces cerevisiae. Microbial Biotechnology. 2010. vol. 3. no. 2. pp. 165–177. doi: 10.1111/j.1751-7915.2009.00106.x.Saerens S.M.G., Delvaux F., Verstrepen K.J., Thevelein J.M. Production and biological function of volatile esters in Saccharomyces cerevisiae. Microbial Biotechnology. 2010. vol. 3. no. 2. pp. 165–177. doi: 10.1111/j.1751-7915.2009.00106.x.Saerens S.M.G., et al. The Saccharomyces cerevisiae EHT1 and EEB1 genes encode novel enzymes with medium-chain fatty acid ethyl ester synthesis and hydrolysis capacity. Journal of Biological Chemistry. 2006. vol. 281. no. 7. pp. 4446–4456. doi: 10.1074/jbc.M512028200.Saerens S.M.G., et al. The Saccharomyces cerevisiae EHT1 and EEB1 genes encode novel enzymes with medium-chain fatty acid ethyl ester synthesis and hydrolysis capacity. Journal of Biological Chemistry. 2006. vol. 281. no. 7. pp. 4446–4456. doi: 10.1074/jbc.M512028200.Steensels J., Verstrepen K.J. Taming wild yeast: potential of conventional and nonconventional yeasts in industrial fermentations. Annual Review of Microbiology. 2014. vol. 68. pp. 61–80. doi: 10.1146/annurev-micro-091213-113025.Steensels J., Verstrepen K.J. Taming wild yeast: potential of conventional and nonconventional yeasts in industrial fermentations. Annual Review of Microbiology. 2014. vol. 68. pp. 61–80. doi: 10.1146/annurev-micro-091213-113025.Verstrepen K.J., Derdelinckx G., Dufour J.P. et al. Flavor-active esters: adding fruitiness to beer. Journal of Bioscience and Bioengineering. 2003. vol. 96. no. 2. pp. 110–118. doi: 10.1016/S1389-1723(03)90112-5.Verstrepen K.J., Derdelinckx G., Dufour J.P. et al. Flavor-active esters: adding fruitiness to beer. Journal of Bioscience and Bioengineering. 2003. vol. 96. no. 2. pp. 110–118. doi: 10.1016/S1389-1723(03)90112-5.Fujii T., Yoshimoto H., Tamai Y. Acetate esters in beer and their control during fermentation. Journal of Bioscience and Bioengineering. 1997. vol. 84. no. 2. pp. 150–155.Fujii T., Yoshimoto H., Tamai Y. Acetate esters in beer and their control during fermentation. Journal of Bioscience and Bioengineering. 1997. vol. 84. no. 2. pp. 150–155.Yoshimoto H. et al. Genetic and physiological factors affecting formation of ethyl acetate in Saccharomyces cerevisiae. J. of Bioscience and Bioengineering. 2002. vol. 94. no. 4. pp. 347–351. doi: 10.1016/S1389-1723(02)80179-5.Yoshimoto H. et al. Genetic and physiological factors affecting formation of ethyl acetate in Saccharomyces cerevisiae. J. of Bioscience and Bioengineering. 2002. vol. 94. no. 4. pp. 347–351. doi: 10.1016/S1389-1723(02)80179-5.Jessop-Fabre M.M. et al. EasyClone-MarkerFree: a vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9. Biotechnology Journal. 2016. vol. 11. no. 8. pp. 1110–1117.Jessop-Fabre M.M. et al. EasyClone-MarkerFree: a vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9. Biotechnology Journal. 2016. vol. 11. no. 8. pp. 1110–1117.Gietz R.D., Schiestl R.H. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature Protocols. 2007. vol. 2. no. 1. pp. 31–34. doi: 10.1038/nprot.2007.13.Gietz R.D., Schiestl R.H. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature Protocols. 2007. vol. 2. no. 1. pp. 31–34. doi: 10.1038/nprot.2007.13.Bordbar A., Monk J.M., King Z.A., Palsson B.O. Constraint-based models predict metabolic and associated cellular functions. Nature Reviews Genetics. 2014. vol. 15. no. 2. pp. 107–120. doi: 10.1038/nrg3643.Bordbar A., Monk J.M., King Z.A., Palsson B.O. Constraint-based models predict metabolic and associated cellular functions. Nature Reviews Genetics. 2014. vol. 15. no. 2. pp. 107–120. doi: 10.1038/nrg3643.Han S., Jang H.W., Park S. et al. Unlocking the flavor potential of brewing yeast with CRISPR/Cas9 genome editing. LWT – Food Science and Technology. 2025. vol. 230. article 118254. doi: 10.1016/j.lwt.2025.118254.Han S., Jang H.W., Park S. et al. Unlocking the flavor potential of brewing yeast with CRISPR/Cas9 genome editing. LWT – Food Science and Technology. 2025. vol. 230. article 118254. doi: 10.1016/j.lwt.2025.118254.Yeast bioflavoring in beer: complexity decoded and built up again. Fermentation. 2024. vol. 10. no. 4. article 183. doi: 10.3390/fermentation10040183.Yeast bioflavoring in beer: complexity decoded and built up again. Fermentation. 2024. vol. 10. no. 4. article 183. doi: 10.3390/fermentation10040183.Dank A., Smid E.J., Notebaart R.A. CRISPR-Cas genome engineering of esterase activity in Saccharomyces cerevisiae steers aroma formation. BMC Research Notes. 2018. vol. 11. article 682. doi: 10.1186/s13104-018-3788-5.Dank A., Smid E.J., Notebaart R.A. CRISPR-Cas genome engineering of esterase activity in Saccharomyces cerevisiae steers aroma formation. BMC Research Notes. 2018. vol. 11. article 682. doi: 10.1186/s13104-018-3788-5.Mertens S. et al. Reducing phenolic off-flavors through CRISPR-based gene editing of the FDC1 gene in Saccharomyces cerevisiae × Saccharomyces eubayanus hybrid lager beer yeasts. PLOS ONE. 2019. vol. 14. no. 10.Mertens S. et al. Reducing phenolic off-flavors through CRISPR-based gene editing of the FDC1 gene in Saccharomyces cerevisiae × Saccharomyces eubayanus hybrid lager beer yeasts. PLOS ONE. 2019. vol. 14. no. 10.Krogerus K., Gibson B. Polygenic analysis in absence of major effector ATF1 unveils novel components in yeast flavor ester biosynthesis. mBio. 2018. vol. 9. no. 5. article e01279-18. doi: 10.1128/mBio.01279-18.Krogerus K., Gibson B. Polygenic analysis in absence of major effector ATF1 unveils novel components in yeast flavor ester biosynthesis. mBio. 2018. vol. 9. no. 5. article e01279-18. doi: 10.1128/mBio.01279-18.

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