The project aims to study the sporogenesis of non-pathogenic bacteria of the genus Bacillus. The project will study the factors that activate or inhibit the process of spore formation, determine the mechanisms of sporulation and germination of bacilli endospores. The main problem that the project aims to solve is to identify and study the aspects that affect the sporulation of Bacillus and conduct targeted genomic editing of two promising strains of Bacillus licheniformis and Bacillus paralicheniformis, producing amylolytic and proteolytic enzymes, respectively, using the CRISPR-Cas9 system to obtain asporogenic variants of producer strains.

Abstract

Endospores form inside a bacterial cell and are a highly specialized type of cell designed to survive harsh conditions. Endospores are highly resistant to starvation, high temperatures, ionizing radiation, mechanical damage, chemical solvents, detergents, hydrolytic enzymes, desiccation, extreme pH, and antimicrobial agents. Spore-forming bacteria are considered potential hazards in the food chain, and control of spore-forming bacteria requires an understanding of the characteristics of endospores. Non-pathogenic sporeforms cause significant economic damage to food producers. The most frequently isolated spores were Bacillus, accounting for approximately 68.9% of all isolates. Selective prevention of sporulation by Bacillus species is the task of scientists and technologists, which will increase the production capacity of fermented strains. To ensure the safe production of food and shelf-stable products, there is a clear need for additional knowledge about spore-forming bacteria, and the use of CRISPR/Cas9 will allow the production of asporogenic producer strains.

Project goal

The goal of the project is to investigate the mechanism of sporulation and germination of Bacillus endospores and to edit the genomes of Bacillus licheniformis and Bacillus paralicheniformis to produce amylolytic and proteolytic asporogenic strains.

Expected results

• Physicochemical factors affecting spore formation will be studied: temperature, irradiation, pressure, sonication, influence of chemical agents, enzymes, component composition of medium, fermentation conditions.
• Adhesiveness and hydrophobicity/hydrophilicity of endospores will be studied; effects of temperature, humidity, pressure, ultraviolet and microwave irradiation, exposure to ultrasound, treatment with acids, alkali, detergents, mutagens, enzymes, antibiotics on spore viability will be determined.
• Conditions for spore activation and germination will be studied: temperature conditions, humidity, effects of chemicals: metal salts, amino acids, secondary metabolites, medium.
• Data on genes involved in sporogenesis of Bacillus bacteria will be studied, nucleotide sequences of B.licheniformis and B.paralicheniformis genomes will be analyzed for gene knockout and editing.
• Genomic editing of amylolytic B.licheniformis and proteolytic B.paralicheniformis strains will be performed and their asporogenic modifications will be obtained.

Project leader

Khassenov Bekbolat, h-index 10, Head of the Laboratory of Genetics and Biochemistry for Microorganisms, Candidate of Chemical Sciences, Full Professor in Biological Sciences. Prof. Khassenov B. is the author of more than 120 publications and the author of 22 patents of the Republic of Kazakhstan. ResearcherID: AAM-8657-2020, ORCID: 0000-0003-4572-948X, Scopus Author ID: 36096620800

Research team members

Akishev Zhiger, PhD, h-index 7, specializes in genetic research and enzyme biochemistry, senior researcher, author of 33 publications and 5 patents of Republic of Kazakhstan. ResearcherID: N-6206-2017, ORCID: 0000-0001-9943-1625, Scopus Author ID: 56674741700

Aktayeva Saniya, PhD, h-index 3, specializes in the study of spore-forming proteolytic and keratinolytic microorganisms, senior researcher, author of 15 publications and 2 patents of Republic of Kazakhstan. ResearcherID: AAR-5133-2020, ORCID: 0000-0001-6346-5866, Scopus Author ID: 57439359000

Mussakhmetov Arman, Master, finished PhD program, h-index 4, specializes in enzyme biochemistry and biotechnology, senior researcher, 33 years old, author of 21 publications and 3 patents of the Republic of Kazakhstan. ResearcherID: AAQ-9945-2020, ORCID: 0000-0002-6182-3487, Scopus Author ID: 57203751227

Popova Victoria, 2nd year Master’s student of the Department of Biology and General Genetics of the Eurasian National University named after L.N. Gumilyov, specialization – genetics of microorganisms, laboratory assistant, author of 3 publications. ResearcherID: JLN-1946-2023, ORCID: 0009-0001-1211-7244

Publications and protection documents

1. Aktayeva S., Baltin K., Kiribayeva A., Akishev Zh., Silayev D., Ramankulov Ye., Khassenov B. Isolation of Bacillus sp. A5.3 Strain with Keratinolytic Activity // Biology (MDPI). 2022, Vol 11, Issue 2, e244. https://doi.org/10.3390/biology11020244  (Q2, IF 4.2, CiteScore 4.0, Percentile 61).
2. Akishev Zh., Aktayeva S., Abdullayeva A., Baltin K., Kiribayeva A, Mussakhmetov A, Tursunbekova A., Ramankulov Ye., Khassenov B. Obtaining of recombinant camel chymosin and testing its milk-clotting activity on cow’s, goat’s, ewes’, camel’s and mare’s milk Biology (MDPI). 2022, Vol 11, Issue 11, e1545. https://doi.org/10.3390/biology11111545  (Q2, IF 4.2, CiteScore 4.0, Percentile 61).
3. Kiribayeva A., Mukanov B., Silayev D., Akishev Zh., Ramankulov Ye., Khassenov B. Cloning, expression, and characterization of a recombinant xylanase from Bacillus sp. T6 // PLoS One. 2022. Vol.17(3). e0202232. https://doi.org/10.1371/journal.pone.0202232 (Q2, IF 3.7, CiteScore 6.0, Percentile 87).
4. Akishev Zh., Kiribayeva A, Mussakhmetov A, Baltin K., Ramankulov Ye., Khassenov B. Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastoris // Heliyon. 2021. Volume 7, Issue 5, e07137 https://doi.org/10.1016/j.heliyon.2021.e07137  (CiteScore 2.7, Percentile 75).
5. Matta E., Kiribayeva A., Khassenov B., Matkarimov B., Ishchenko A. Insight into DNA substrate specificity of PARP1-catalysed DNA poly(ADP-ribosyl)ation // Scientific Reports. 2020. Vol.10(1), 3699. pp.1-11. https://doi.org/10.1038/s41598-020-60631-0  (Q1, IF 4.38, CiteScore 7.1, Percentile 93).
6. Turgimbayeva A, Abeldenov S, Zharkov D.O, Ishchenko A.A, Ramankulov Y, Saparbaev M, Khassenov B. Characterization of biochemical properties of an apurinic/apyrimidinic endonuclease from Helicobacter pylori // PLoS One. 2018. Vol.13(8). e0202232. https://doi.org/10.1371/journal.pone.0202232 (Q2, IF 3.24, CiteScore 5.3, Percentile 92).
7. Kalendar R., Khassenov B., Ramankulov Ye., Samuilova O., Ivanov K. Fast PCR: An in silico tool for fast primer and probe design and advanced sequence analysis // Genomics. 2017. Vol.109. pp. 312-319. https://doi.org/10.1016/j.ygeno.2017.05.005 (Q1, IF 5.736, CiteScore 4.0, Percentile 48).
8. Abeldenov S., Talhaoui I., Zharkov D.O., Ishchenko A.A., Ramanculov E., Saparbaev M., Khassenov B. Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from Mycobacterium tuberculosis // DNA Repair. 2015, Vol. 33. pp. 1-16 https://doi.org/10.1016/j.dnarep.2015.05.007 (Q1, IF 4.913, CiteScore 5.9, Percentile 68).
9. Mussakhmetov A., Shumilin I.A., Nugmanova R., Shabalin I.G., Baizhumanov T., Toibazar D., Khassenov B., Minor W., Utepbergenov D. A transient post-translational modification of active site cysteine alters binding properties of the parkinsonism protein DJ-1 // Biochemical and Biophysical Research communications. 2018. Vol.504(1). Pp. 328-333. https://doi.org/10.1016/j.bbrc.2018.08.190 (Q2, IF 3.572, CiteScore 5.5, Percentile=62).
10. Aktayeva S., Baltin K., Ramankulov Ye., Khassenov B. Isolation and identification of the keratin degrading bacteria // Journal of Biotechnology. 2019. Vol. 305S. P.S30. (Q1, IF 4.913, CiteScore 5.9, Percentile 68).
11. Sarsen A., Saginova M., Akishev Zh., Aktayeva S., Manabayeva Sh., Khassenov B. Molecular phylogenetic analysis of Tulipa (Liliaceae) from Aksu-Zhabagly Nature Reserve // Plant Science Today. 2023 Vol 10, Issue 2, P.302–309. https://doi.org/10.14719/pst.2153 (Q4, Impact Factor 0.9, CiteScore 1.1, Percentile 28).
12. Aktayeva S., Sarsen A., Mussakhmetov A., Kiribayeva A., Tursunbekova A., Khassenov B. Development of microbiological diffusion inhibition test for the determination of antibiotic residues in the milk // Eurasian Journal of Applied Biotechnology, 2023. No. 2. P.44-5. https://doi.org/10.11134/btp.2.2023.6 (RSCI).
13. Aktayeva S., Kiribayeva A., Makasheva D., Astrakhanov M., Tursunbekova A., Baltin K., Khassenov B. Isolation, identification and usage of Bacillus strains in microbial inhibition test in milk Eurasian Journal of Applied Biotechnology, 2022. No. 4. P.49-57 https://doi.org/10.11134/btp.4.2022.6 (RSCI).
14. Aktayeva S.A., Baltin K.K., Kiribayeva A.K., Ramankulov E.M., Khassenov B.B. Bacterial strain Bacillus licheniformis T7 – producer of proteases and keratinases. Patent of the Republic of Kazakhstan №35357 from 12.11.2021.
15. Barshevskaya L.V., Sotnikov D.V., Zherdev A.V., Khassenov B.B., Baltin K.K., Eskendirova S.Z., Mukanov K.K., Mukantayev K.K., Dzantiev B.B. Triple Immunochromatographic System for Simultaneous Serodiagnosis of Bovine Brucellosis, Tuberculosis, and Leukemia // Biosensors (Basel). 2019 Vol.9 (4), 115. pp. 1-10. https://doi.org/10.3390/bios9040115  (Q2, IF 5.972, Cite Score 5.6, Percentile 89).

Achieved results

2024

The effect of physicochemical factors, biochemical factors on the process of sporulation of the bacterium Bacillus paralicheniformis T7 was studied. It was found that the addition of magnesium sulfate, iron sulfate, potassium chloride, manganese or calcium to the medium stimulates the formation of endospores of the strain. Adhesion, hydrophobicity/hydrophilicity of Bacillus paralicheniformis T7 endospores were studied, their resistance to abiotic and biotic stress factors, physical and chemical effects, antibiotics, and lysing agents was determined. It was found that endospores have high hydrophobicity in aqueous solutions, are resistant to ultrasound, retain the ability to germinate after heating at 90 °C for 60 min, withstand heat treatment at 121 °C for 30 min, and are resistant to 3 M phosphoric acid and 200 mM NaOH. Spores of Bacillus paralicheniformis T7 are resistant to lincosamides, ansamycins, quinolones, fluoroquinolones, macrolides, tetracyclines, aminoglycosides, glycopeptides at a concentration of 10 μg/mL and withstand treatment with lysozyme at a concentration of 100 mg/mL.

Publication for 2024

1. Aktayeva S., Khassenov B. High keratinase and other types of hydrolase activity of the new strain of Bacillus paralicheniformis // PLoS One. 2024. Vol.19(10). e0312679. https://doi.org/10.1371/journal.pone.0312679
2. [In Rus] Popova V.S., Kiribaeva A.K., Silayev D.V., Khassenov B.B. Study of thermostable amylase from the Bacillus paralicheniformis strain // Eurasian Journal of Applied Biotechnology. No.3S, 2024 Special issue “International Scientific Conference “Astana Biotech 2024”. 2024. Astana, p.133. https://doi.org/10.11134/btp.3S.2024.121