The ability to utilize a number of polyhydric alcohols as a sole carbon source has been studied in the Yarrowia lipolytica yeast. The efficiency of the promoter of the Y. lipolytica native AraDH2 gene encoding the enzyme D-mannitol/D-arabitol dehydrogenase was assessed during yeast growth on a minimal medium with different carbon sources. For this purpose, the promoter region of the AraDH2 gene was transcriptionally fused with the green fluorescent protein hrGFP gene, and the construct was used to transform Y. lipolytica . A β-carotene producing strain of Y. lipolytica was created using the described promoter; the strain carried the Mucor circinelloides CarRP and CarB genes encoding the bifunctional enzyme phytoene synthase/lycopene β-cyclase (CarRP) and phytoene dehydrogenase (CarB) as well as the GGPPSs7 gene Synechococcus sp . geranylgeranyl pyrophosphate synthase. The nucleotide sequence encoding the fused CarRP and GGPPSs7 under the regulation of the pAraDH2 promoter and the CarB gene under the control of the pTEF promoter were introduced into the yeast genome. As a result, a transformant was obtained capable of producing 66.3, 121.2, and 148.9 mg/L of β-carotene after 5 days of cultivation in test tubes on media containing glycerol, sucrose, and glucose, respectively. The obtained results testify to the high potential of the pAraDH2 promoter in the field of genetic engineering.

In this article, strong promoters of thermotolerant methylotrophic yeast Ogataea haglerorum have been characterized. Promoters play a key role in the regulation of gene expression; therefore, they are the important element of expression vectors. Strong and strictly regulated promoters are a powerful tool for creating highly productive strains — producers of recombinant proteins. To expand the potential of the O. haglerorum expression system natural methanol-induced promoters of the OhMOX and OhFMD genes and the constitutive promoter of the OhGAP gene were studied in comparison with the promoter of the MOX gene from O. polymorpha yeast. A gene encoding recombinant β-mannanase was used as a reporter gene. It has been shown that in O. haglerorum yeast cells, the expression level (strength) of the pOhMOX promoter is about 1.4–1.9 times higher relative to the pOpMOX promoter from O. polymorpha yeast. The obtained data on the strength of promoters from yeast O. haglerorum can be useful in designing producers of recombinant proteins and optimizing metabolic pathways in methylotrophic yeast O. haglerorum.

Successful assembly of bacteriophage MS2 virus-like particles (VLPs) from a secreted recombinant capsid protein in the culture medium of the Komagataella phaffii methylotrophic yeast has been shown. The yield of VLPs was 5 g/L and reached 30

In this work, it was shown for the first time that inactivation of the L-lysine uptake transport system from the external environment leads to an increase in the level of L-threonine synthesis by the Escherichia coli strain that produces it, while the addition of lysine to the culture medium reduces the productivity of the unmodified strain, which indicates the influence of the intracellular pool of lysine on the efficiency of threonine biosynthesis. A study of the possible mechanisms of this influence showed that neither the previously known inhibition of aspartate kinase III (LysC) activity, nor the repression of the synthesis of aspartate semialdehyde dehydrogenase (Asd) and glutamate dehydrogenase (GdhA) individually are reasons for the decrease in culture productivity. The results we obtained may indicate both the presence of a previously unknown mechanism for the regulation of threonine metabolism by lysine and the fact that several enzymes in the threonine biosynthesis pathway are simultaneously affected by this amino acid. At the same time, changing the permeability of the membrane to lysine, or reducing its intracellular concentration as a result of changing the efficiency of its biosynthesis, is a promising approach for improving threonine-producing strains.

The expression potential of various strains from the Collection of the National Bio-Resource Center (BRC VKPM) belonging to the species Komagataella kurtzmanii, K. phaffii, and K. mondaviorum has been assessed by the level of production of the heterologous enzyme, Citrobacter freundii phytase. Heterologous expression in the K. mondaviorum strains was observed for the first time. We identified the strains of K. phaffii Y-4288, K. mondaviorum Y-4331 and K. phaffii Y-4287 species with a high level production of the heterologous enzyme, a high growth rate, the ability to accumulate a large amount of biomass, and moderate thermotolerance. It has been shown that the average productivity of the transformants based on K. phaffii Y-4288, K. mondaviorum Y-4331, and K. phaffii Y-4287 strains exceeds that of the commercial industrial recipient strain K. phaffii GS115 Y-2837 by more than 3, 5, and 6 times, respectively. The K. phaffii Y-4287 and K. mondaviorum Y-4331 strains exhibited moderate thermotolerance and the ability to accumulate a heterologous product at 37°C. The high expression potential of the identified strains opens up the possibility of creating recipient strains on their basis for high-level production of heterologous proteins.

The expression potential of various strains from the Collection of the National Bio-Resource Center (BRC VKPM) collection belonging to the species Komagataella kurtzmanii, K. phaffii, K. mondaviorum has been assessed by the level of production of the heterologous enzyme Citrobacter freundii phytase. Heterologous expression in the K. mondaviorum strains was observed for the first time. The strains of K. phaffii Y-4288, K. mondaviorum Y-4331 and K. phaffii Y-4287 were identified with a high level production of the heterologous enzyme, a high growth rate, the ability to accumulate a large amount of biomass and moderate thermotolerance. It was shown that the average productivity of the transformants based on K. phaffii Y-4288, K. mondaviorum Y-4331, and K. phaffii Y-4287 strains exceeds that of the commercial industrial recipient strain K. phaffii GS115 Y-2837 by more than 3, 5 and 6 times, respectively. The K. phaffii Y-4287 and K. mondaviorum Y-4331 strains exhibited moderate thermotolerance and the ability to accumulate a heterologous product at 37° C. The high expression potential of the identified strains opens up the possibility of creating recipient strains on their basis for high-level production of heterologous proteins. Key words: Komagataella, Pichia pastoris, thermotolerance, expression of heterologous proteins Funding - This work was supported by the Ministry of Science and Higher Education of the Russian Federation (grant no. 075-15-2019-1658 dated October 31, 2019) and was carried out using the resources of the Unique Scientific Facility of the "All-Russian Collection of Industrial Microorganisms" National Bio-Resource Center, NRC «Kurchatov Institute»---GOSNIIGENETIKA.

Recombinant Yarrowia lipolytica yeast has been used as a host strain for the expression of the Saccharomyces cerevisiae ScSUC2 gene and Y. lipolytica YlHXK1 gene encoding invertase and hexakinase, respectively. The expression was carried out on the background of the enhanced pathway of the synthesis of neutral lipids. This allowed the yeast to efficiently utilize glucose as a single carbon source. The engineered strain accumulated neutral lipids in amount of 50.7% of biomass dry weight, when cultured in tubes in minimal medium with nitrogen limitation and high sucrose content. The next metabolic engineering step was the use of the CRISPR-Cas9 editing system to introduce a heterologous β-carotene synthesis pathway by the expression of the Mucor circinelloides genes CarRP and CarB, encoding the bifunctional enzyme phytoene synthase/licopene-β-cyclase (carRP) and phytoene dehydrogenase (carB), as well as an increase in the expression level of the Y. lipolytica YlGGS1 gene, encoding geranyl diphosphate synthase enzyme. The β-carotene production on a sucrose-containing medium was shown for the first time; it amounted for 24.0 mg/g dry biomass, or 406.9 mg/L, on the 5th day of cultivation in tubes in a rich medium with a sucrose portioned supply (50 g/L). The corresponding values for a rich medium with a higher sucrose concentration (90 g/L) were 21.9 mg/g dry biomass and 625.8 mg/L. Yarrowia lipolytica, β-carotene, sucrose, CRISPR-Cas9 The plasmids pdKu70Yl-URA3, pLTet-SP-CAT, pARS-Cre-reverse, pMW-att-Cm and pGPD1Yl were kindly provided by Ph.D. I.A. Laptev ("Kurchatov Institute"-GOSNIIGENETIKA NRC). The work was carried out using the equipment of the Multipurpose Scientific Facility of the "Russian State Collection of Industrial Microorganisms" National Bio-Resource Center, NRC «Kurchatov Institute»-GOSNIIGENETIKA. The work was supported by state assignment no. 14 of 25.07.2012 AAAA-A20-120093090016-9. The quantitative analysis of lipids and β-carotene was carried out by V.A. Zolottsev within the framework of the "Long-Term Program of Fundamental Scientific Research in the Russian Federation (2021-2030)".

Screening of glycerol-producing yeasts resistant to high osmotic pressure of substrates has been carried out among collection strains and strains isolated from natural sources associated with bee habitat. In total, more than 170 strains of osmotolerant yeasts were investigated which belong to 9 genera and the following13 species: Candida apicola, C. magnolia, Debaryomyces hansenii, D. marama, D. polymorphus, Hansenula ciferri, Kluyveromyces lactis, K. marxianus, Pichia farinose, Saccharomyces cerevisiae, Starmerella bombi, Schizosaccharomyces pombe, and Zhygosaccharomyces rouxii. The Kluyveromyces lactis VKPM Y-4429 strain producing 77.4 g/L of glycerol for 24 h of cultivation on media with a high glucose (300 g/L) and NaCl (5-6%) content was selected. These conditions cause osmotic shock, and as a consequence, a higher glucose conversion to glycerol. glycerol biosynthesis, fermentation, osmotolerant yeast, screening The work was supported by the State Assignment № AAAA-A20-120093090016-9 and the work was carried out using the Unique Scientific Facility of the "All-Russian Collection of Industrial Microorganisms" National Bio-Resource Center, NRC «Kurchatov Institute»---GOSNIIGENETIKA (NBC VKPM).

In this study, Y1ACL2 was inactivated by two methods: traditional approach based on homologous recombination and uracil marker and markerless system using CRISPR/Cas9. The efficiency of YIACL2 inactivation using traditional approach was 4% (one ΔYlacl2 strain out of 24 tested transformants) whereas knockout efficiency using CRISPR/Cas9 system was 75% (18 ΔYlacl2 strains out of 24 tested transformants). YIACL2 null mutant strains were not able to utilize citrate as a single carbon source. Growth kinetics was investigated in the media with glucose and acetate as a single carbon source. The fact that ΔYlacl2 is able to grow in the minimal medium with glucose as a single carbon source provides evidence that there is an alternative source of acetyl-CoA on carbohydrate substrates in Y. lipolytica. Yarrowia lipolytica, CRISPR/Cas9 system, ATP citrate lyase, YIACL2. The work was carried out using the equipment of the Unique Scientific Facility of BRC VKPM with technical support of the Centre for Collective Use of NRC «Kurchatov Institute» -GosNIIgenetika and with financial support of Russian Federation (state task No. 595-00003-19 PR) as well as partially supported by grant No. MK-2241.2019.7.

An E. coli strain in which all known pathways of threonine catabolism were inactivated (Δtdh, ΔltaE, ΔAilvA, ΔtdcB, AyiaY) has been constructed. The possibility of an alternative pathway for the isoleucine synthesis by expressing heterologous citramalate synthase from Leptospira interrogans in an E. coli strain carrying the ΔilvA deletion was demonstrated. It was observed that the cimA overexpression has a negative effect on the threonine production. We developed a system for regulated gene expression based on the inducible promoter PLtetO and TetR repressor of the tetracycline operon. A threonine producing strain B-1201 in which the cimA gene is expressed under the control of the regulated promoter was constructed. A correlation of the threonine productivity and the expression level of the cimA gene was shown by culturing the B-1201 strain in fermenter. The optimal inductor content for the maximum threonine accumulation was also determined. Escherichia coli, strain, threonine, citramalate synthase This work was supported by the Ministry of Education and Science of the Russian Federation (project code RFMEFI61017X0011), and it was carried out using the equipment of the National Bio-Resource Center «All-Russian Collection of Industrial Microorganisms», NRC «Kurchatov Institute» - GOSNIIGENETIKA. The authors are grateful to Dr. I. V. Manukhov for providing the MG1655Z1strain and pZE21-lux plasmid, and for teaching techniques of bioluminescence measuring.

The heterologous expression and characteristics of a new xylanase from Pyromyces finnis have been described. The endo-l,4-β-xylanase XylP (EC 3.2.1.8) consists of 223 amino acids and 19 residues of a putative signal peptide in the N-terminal region. The amino acid sequence of the mature protein has the greatest homology with the sequence of the native catalytic N-terminal domain of Neocallimastix patriciarum endo-l,4-β-xylanase (84%). A synthetic nucleotide sequence encoding a mature XylP protein was expressed in Pichia pastoris. The purified recombinant enzyme showed activity with birch xylan and arabinoxylan. When using birch xylan as a substrate, the optimum pH for the enzyme was 5.0, and the optimum temperature was 50 °C. The specific activity of the xylanase was 4700 U/mg protein, and Km and Vmax were equal to 0.51 mg/mL and 7395.3 umol/(min∙mg), respectively. The recombinant XylP protein showed moderate thermal stability and high pH stability, resistance to digestive enzymes and protein inhibitors of grain xylanases. It was also shown that the Mg2+, Co2+ and Li+ ions have a positive effect on the enzyme activity. xylanase, xylan, feed enzyme, Pichia pastoris, Pyromyces finnis The work was performed with the financial support of the Ministry of Education and Science of Russia (Unique Project Identifier RFMEFI60717X0180) using the Unique Scientific Installation -National Bioresource Center «All-Russian Collection of Industrial Microorganisms», NRC «Kurchatov Institute» - GOSNIIGENETIKA

In recent years, mycelial fungi have faced competition from recombinant yeast as producers of feed enzymes. An intensive study on genetic diversity identified the yeast genes encoding feed enzymes, the specific activity of which is much higher than that in mycelial fungi. In addition, these genes were expressed in yeast much more efficiently than in mycelial fungi. The use of yeast recombinant producers allowed the expansion of the production of a line of industrial enzymes with a significant reduction in their cost. The advantages of yeast producers of recombinant enzymes include the ability to obtain monoenzymes, which are part of various enzyme complexes used for different purposes. Pichia pastoris methylotrophic yeast is the most attractive subject for the creation of recombinant protein-producing strains.

A Pichia pastoris yeast strain producing endo-l,4-β-xylanase from Paenibacillus brasilensis with an activity of 54,400 U/mL after 140 h of fermentation in a laboratory fermenter has been obtained. A number of approaches were used to increase the level of the xylanase production in this strain: optimization of the target gene codon composition, multiple integration of the expression cassette into the recipient strain chromosome using the Cre-lox recombination system, and also improving the heterologous protein folding via the overexpression of the HAC1i gene from Pichia pastoris. xylanase, xylan, Cre-lox system, HAC1p transcriptional activator, multicopy strain, Paenibacillus brasilensis, Pichia pastoris The work was performed with the financial support of the Ministry of Education and Science of Russia (Unique Project Identifier RFMEFI60717X0180) using the Multipurpose Scientific Installation of «All-Russian Collection of Industrial Microorganisms» National Bio-Resource Center, NRC «Kurchatov Institute» - GosNIIgenetika.

The co-expression of genes encoding β-glucanases from Paenibacillus jamilae and Rhizomucor miehei in the Komagataella kurtzmanii methylotrophic yeast system has been carried out. The recombinant strains that simultaneously produced both enzymes with a total activity of 1132 and 1464 units for 96 h of cultivation in flasks were obtained. The combination of enzyme properties made it possible to achieve a substantial extension of the pH range of β-glucanase activity, as well as to bring the temperature and pH optima of the enzymes closer to those optimal for use in feed production. A vector has been developed that allows the efficient simultaneous expression of genes due to the sequential integration of expression cassettes into the recipient strain chromosome. It was also shown that a change in the ratio of the number of genes integrated into the chromosome of the host cell allows to directionally influence the total properties of recombinant enzymes. co-expression, β-glucanase, β-glucan, Paenibacillus jamilae, Rhizomucor miehei, Komagataella kurtzmanii, Pichia pastoris The work was financially supported by the Ministry of Science and Education (Project Unique Identifier RFMEFI60717X0179) using the Multipurpose Scientific Installation of National Bio-Resource Center «All-Russian Collection of Industrial Microorganisms», NRC «Kurchatov Institute» - GOSNIIGENETIKA.

Effect of overexpression of the HAC1 genes from Pichia pastoris and Saccharomyces cerevisiae on the production of heterologous enzymes, Escherichia coli phytase and Paenibacillus brasilensis xylanase, in P. pastoris cells has been studied. Codon composition of the phytase and xylanase encoding genes was optimized, and the genes were expressed in P. pastoris under the control of AOX1 promoter. The obtained multi-copy strains produced in vitro 927 U/mL phytase and 1,401 U/ml xylanase activity. Overexpression of the HAC1 gene from P. pastoris was shown to increase the phytase and xylanase production by 46% and 41%, respectively. Overexpression of HAC1 from S. cerevisiae increased the phytase production by 28% and xylanase by 20%. Data obtained could be helpful in the construction of industrial enzyme-producing strains based on P. pastoris. phytase, xylanase, Нас1р transcription activator, UPR, Pichia pastoris, Saccharomyces cerevisiae The work was carried out using Multipurpose Scientific Installation All-Russian Collection of Industrial Microorganisms National Bioresource Center, NRC «Kurchatov Institute» -GosNIIgenetika. The authors are grateful to A.V. Nikulin (Sintol LLC, Russia) for the assistance in the real-time PCR experiments. The work was financially supported by the Ministry of Education and Science of the Russian Federation (Unique Identifiers of the Projects are RFMEFI57917X0145 and RFMEFI60717X0180).

The genes for bacterial phytases from Citrobacter freundii and Yersinia intermedia were expressed for the first time in a thermotolarant yeast Ogataea polymorpha. A comparative analysis of the properties of recombinant phytases produced by Ogataea polymorpha and Pichia pastoris yeasts was carried out. It was shown that the stability, pH and temperature profiles of the enzyme activities are the same regardless of the host strain. It was proved that O. polymorpha yeast can be used to create producers of feed enzymes and to develop a technology for their cultivation at temperatures above 37 °C. The prospects of using the O. polymorpha yeast for these purposes were evaluated. Ogataea (Hansenula) polymorpha, Pichia pastoris, methylotrophic yeast, thermal tolerance, producer, recombinant phytase The work was financially supported by the Ministry of Science and Higher Education of RF (Project Unique Identifier RFMEFI57917X0145) using the Multipurpose Scientific Installation of All-Russian National Collection of Industrial Microorganisms National Bioresource Center, NRC «Kurchatov Institute»-GosNIIgenetika.

The isolation, heterologous gene expression, and characteristics of a new β-glucanase from Bacillus pumilus is described. The bgl1 gene from the Bacillus pumilus Bg57 VKPM В-13195 strain, which consists of 729 nucleotides, encodes a secreted endo-β-l,3(4)-D-glucanase (EC 3.2.1.6) containing 214 amino acids and 28 residues of the putative signal peptide in the N-terminal area. The nucleotide sequence of bgl1 and amino acid sequence of the mature Bgl protein have the highest homology (89 and 95%, respectively) with the sequences for β-l,3-l,4-glucanase from Bacillus licheniformis. A fragment of the gene encoding the mature protein was expressed in Pichia pastoris. The purified recombinant enzyme of Bgl was active towards barley β-glucan and lichenin. With barley β-glucan, the optimal pH and temperature were shown to be 6.0 and 50°C, respectively; Km and Vmax were 2.2 mg/mL and 3036.4 μmol/(min mg), respectively. The recombinant protein Bgl showed a high specific activity, thermal stability, and resistance to digestive enzymes. Other characteristics of the recombinant protein, including pH stability and sensitivity to metal ions and chemical reagents, were also determined.

The expression of four genes of bacterial endo-β-l,3(4)-D-glucanases from B. pumilus Bg57 (VKPM В-13195), Paenibacillus polymyxa Bg23 (VKPM В-4056), Bacillus subtilis Bg11 (VKPM B-13178), and Bacillus amyloliquefaciens Bg76 (VKPM B-13184), which are members of the family of GH16 glycohydrolases, in the expression system of Pichia pastoris are compared. Technologically valuable characteristics of the recombinant β-glucanases, such as the specific activity, pH and thermal stability, temperature and pH optima, resistance to digestive enzymes, and substrate specificity, were studied. It was shown that the enzymes from B. pumilus and P. polymyxa have the highest biotechnological potential for the creation of β‑glucanase producers based on recombinant P. pastoris yeast strains for further use in fodder production.

Efficient expression of the man1 gene for Aspergillus aculeatus β-1,4-mannanase was demonstrated in Pichia pastoris yeast for the first time, and industrially important characteristics of the synthesized enzyme were confirmed. β-Mannanases are industrial enzymes that can be used, in particular, in the feed industry. The most important requirements for feed enzymes are a broad pH range, thermal stability, and high specific activity. The obtained data indicate that the man1 gene from A. aculeatus is potentially useful for the construction of industrial mannanase producers based on Pichia pastoris yeast.