RU2722563C1 - Yeast komagataella kurtzmanii transformant producing beta-glucanase - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and represents a transformant of yeast Komagataella kurtzmanii, producing endo-β-1,3(4)-D-gluconase from Bacillus pumilus or enzyme, amino acid sequence of which is homologous to it by at least 96 %, namely endo-β-1,3(4)-D-glucanase from Bacillus safensis, or Bacillus stratosphericus, or Bacillus altitudinis, or Bacillus aerius, or Bacillus cellulasensis, or Bacillus australimaris.

EFFECT: invention enables to obtain endo-β-1,3(4)-D-glucanase with high degree of effectiveness.

1 cl, 2 dwg, 9 ex

Description

The invention relates to microbiology and biotechnology and relates to the production of a yeast strain Komagataella kurtzmanii capable of producing beta-glucanase (β-glucanase).

β-glucans are a family of polysaccharides consisting of D-glucose monomers linked via β-glycosidic bonds and are a natural component of the cell walls of bacteria, fungi, yeast and cereals, such as oats and barley. β-glucans of various origins differ in structure, branching level and molecular weight, as well as physicochemical properties.

β-glucanase is a group of enzymes that catalyze the cleavage of β-glucans with β-1,2-, β-1,3-, β-1,4- and β-1,6-bonds.

Important among the enzymes belonging to this group are β-1,3-1,4-glucanases, which are widely used, in particular, in brewing and in the production of food additives [FEBS Lett., 1975, 52, 202-207] .

The use of β-glucanases in beer fermentation leads to an increase in the extraction of barley seeds and a decrease in the amount of wort, a decrease in the formation of excess viscosity and a decrease in the appearance of sediment in beer.

β-1,3-1,4-glucanases are also used as an additive to the feed of farm animals with a single-chamber stomach. In the poultry and pig industries, the water-soluble non-starch β-glucan polysaccharide acts as an anti-nutrient. Pet food mixed with β-1,3-1,4-glucanase and xylanase enzymes enhances weight gain, feed intake and digestibility of nitrogen (+ 5.6%) and lipids (+ 6.2%), as well as reduces the formation of sticky droppings, which significantly reduces sanitary problems [Trends in Biotechnology, 1993, 11 (10), 424-430].

The natural sources of β-glucanases are various microorganisms: bacteria, fungi, yeast and actinomycetes

Most feed enzyme preparations, which include β-glucanases, are of fungal origin. However, from a scientific and technological point of view, it is of great interest to develop recombinant yeast-based enzyme producers, since they are more convenient for genetic engineering work and they accumulate the target enzyme faster than mushroom producers. A significant advantage of yeast producers is also that it is much easier to create mono-enzyme producers on their basis, while mushroom producers usually synthesize complexes of cellulitic enzymes. The industrial production of monozymes allows one to more efficiently solve the problem of compiling the optimal composition of enzymes using various substrates.

Currently, the most promising is the creation of producers based on recombinant strains of methylotrophic yeast of the genus Pichia, Komagataella or Hansenula [J Ind Microbiol Biotechnol 2009, 36: 1435-1438],

The use of methylotrophic yeast allows one to achieve high expression rates of heterologous proteins with a high cell density, as well as a high level and quality of N-glycosylation of proteins.

Especially often, methylotrophic yeast Pichia pastoris [FEMS Microbiol. Rev., 2000, 24, 45-66].

Shown [J. Ind. Microbiol. Biotechnol., 2012 39 (6), 869-876] that the bgl16C1 gene from Penicillium pinophilum encoding endo-1,3 (4) -β-D-glucanase is efficiently expressed in Pichia pastoris yeast cells, with recombinant β activity -glucanase in the culture fluid when cultured in a 15-liter fermenter is 28721 U / ml.

Also known are recombinant strains of Pichia pastoris producing thermostable β-1,3-1,4-glucanase from Bacillus amyloliquefaciens. [CN 101899458]

In [Biotechnology, 2018, T. 34, No. 5, pp. 12-22], the bgl gene from Bacillus pumilus, encoding β-glucanase, which belongs to the class of endo-β-1,3 (4) -D-glucanases ( E.S. 3.2.1.6).

Since β-glucanase genes of various origin are expressed in methylotrophic yeast with different efficiencies [Biotechnology, 2018, 34 (4), 26-36], an important task for constructing effective producers is the choice of genes encoding β-glucanase and efficiently working in yeast.

The search for new highly active β-glucanases possessing the properties necessary for their industrial use and capable of efficiently expressing themselves in yeast, as well as the creation of industrially significant producers based on them, is an urgent task.

Komagataella kurtzmanii is a new species of methylotrophic yeast isolated from Pichia pastoris [Antonie van Leeuwenhoek, 2013, 104 (3), Published online, DOI 10.1007 / s10482-013-9956-7].

In the work [Tyurin O.V. Development of a gene expression system based on methylotrophic Komagataella kurtzmanii yeast: Ph.D. biol. sciences. GosNIIgenetika, Moscow, 2014.] the expression system for such yeast is described and its efficiency for the production of heterologous proteins is shown on model strains.

The task of the invention is to expand the arsenal of recombinant microorganisms producing β-glucanase.

The problem was solved by obtaining a Komagataella kurtzmanii yeast transformant producing β-glucanase containing the bgl gene encoding endo-β-1,3 (4) -D-glucanase from Bacillus pumilus or an enzyme whose amino acid sequence is homologous to it by at least 96 %

Enzymes whose amino acid sequence is homologous to endo-β-l, 3 (4) -D glucanase from Bacillus pumilus [NCBI: MH553379] are no less than 96%, for example, endo-β-1,3 (4) - D-glucanase from Bacillus safensis [NCBI: WP_034622736.1], or endo-β-1,3 (4) -D-glucanase from Bacillus stratosphericus [NCBI: WP_103132685.1], or endo-β-1,3 (4 ) -D-glucanase from Bacillus altitudinis [NCBI: WP_073416011.1], or endo-β-1,3 (4) -D-glucanase from Bacillus aerius [NCBI: PYH23823.1], or endo-β-1,3 (4) -D-glucanase from Bacillus cellulasensis [NCBI: KIL24001.1], or endo-β-1,3 (4) -D-glucanase from Bacillus australimaris [NCBI: WP_060698046.]

Obtaining the claimed transformants involves introducing the bgl gene from Bacillus pumilus into Komagataella kurtzmanii yeast cells using an expression cassette that includes the bgl gene, a promoter suitable for working in Komagataella kurtzmanii yeast, a signal peptide for the secretion of the enzyme into the culture fluid, terminator, marker a gene and, preferably, a site for homologous integration into the chromosome. Integration can be carried out by both homologous and non-homologous recombination. The transformation of the expression cassette into Komagataella kurtzmanii yeast cells can be carried out by any suitable method, for example, by electroporation [Tyurin O.V. Development of a gene expression system based on methylotrophic Komagataella kurtzmanii yeast: Ph.D. biol. sciences. State Research Institute of Genetics, Moscow, 2014.]

The construction of expression cassettes is carried out by standard methods of genetic engineering [Rybchin V.N. Fundamentals of Genetic Engineering. - SPb .: SPbSTU, 1999. Sambrook J., Maniatis T., Fritsch E. Molecular cloning: a laboratory manual. - N.Y. Cold Spring Harbor Laboratory, 1989.] using genetic elements suitable for work with Komagataella kurtzmanii yeast [O. Tyurin Development of a gene expression system based on methylotrophic Komagataella kurtzmanii yeast: Ph.D. biol. sciences. State Research Institute of Genetics, Moscow, 2014.]

The invention is illustrated by the following figures:

FIG. 1 Expression cassette AOX1-artHH-Bgl1ng.

FIG. 2 Electrophoresis of the bgl Bacillus pumilus gene

The invention is confirmed by the following examples.

Example 1. Obtaining the plasmid pUC19mx-Bgl1.

The bgl structural gene is amplified using PCR. The chromosomal DNA of the strain Bacillus pumilus Bg57 VKPM B-13195 is used as a template for PCR [Biotechnology, 2018, V. 34, No. 5, P. 12-22].

PCR primers are N1344m (5'-catccatggaaaagagatcccaaacgggcgggtcgttttat) and N1345m (5'-atactcgagttatctttttgtgtaacgcacccag).

The sticky ends in the amplified DNA fragment are opened using restriction enzymes NcoI and XhoI, and the obtained NcoI / XhoI DNA fragment is cloned in the laboratory vector pUC19mx, a derivative of the standard vector pUC19 containing the modified polylinker recognition sites of restriction enzymes NcoI and XhoI. Cloning yields the plasmid pUC19mx-Bgl1, in which the bgl gene has a direction opposite to the lacZ 'gene, and which is subsequently used as a source of the bgl DNA encoding fragment.

Example 2. Modification of the structural gene bgl of Bacillus pumilus

The nucleotide sequence of the bgl structural gene of the Bacillus pumilus Bg57 strain VKPM B-13195 cloned in the pUC19mx-Bgll vector is determined.

The DNA sequence of the cloned gene is modified to inactivate the unique BamHI site and the middle of the three potential N-glycosylation sites. To carry out the modification, PCR-mediated site-directed mutagenesis is used.

The template for PCR is the plasmid DNA pUC19mx-Bgl1. PCR amplification is carried out in 2 stages.

At the first stage, the mutant gene sequence is obtained in the form of 3 DNA fragments amplified by PCR and having pairwise overlapping ends:

Fragment 1 was prepared using primers N1344m (5'-catccatggaaaagagatcccaaacgggcgggtcgttttat) and N1426 (5'-atttgcacgccacgtacagtt)

Fragment 2 was prepared using primers N1425 (5'-aactgtacgtggcgtgcaaataacgtggctatgacctcat) and N1430 (5'-aacaccgttgtaagatccgagcca)

Fragment 3 was prepared using primers N1431 (5'-tggctcggatcttacaacggtgtt) and N1345m (5'-atactcgagttatctttttgtgtaacgcacccag)

Amplified DNA fragments are purified using the Qiagen kit (Qiagen, cat. No. 28706).

The next step is PCR-mediated ligation of 3 purified DNA fragments. For this purpose, PCR is carried out using fragments 1, 2 and 3 as a template mixture. The primers for PCR amplification are N1344m and N1345m. The amplified DNA fragment was eluted from the agarose gel and, after opening the ends using restriction enzymes NcoI and XhoI, were cloned into the plasmid pUC19mx (see Example 1), which was digested at the same sites. As a result of cloning, plasmid pUC19mx-Bgl1ng is obtained, in which the nucleotide sequence of the cloned gene is confirmed by sequencing.

Thus, when mutating the bgl sequence, the BamHI restriction enzyme recognition site is inactivated using a nucleotide substitution that does not lead to an amino acid residue substitution, and the N-glycosylation site is inactivated by a nucleotide substitution leading to the replacement of the asparagine residue with a glutamine residue. A modified gene containing both mutations is called Bgl1ng.

The resulting plasmid pUC19mx-Bgl1ng contains an NcoI / XhoI DNA fragment containing the modified Bgl1ng gene.

Example 3. Construction of a plasmid for the integration of the Bgl1ng gene in the genome of the yeast K.kurtzmanii

The plasmid for integration is constructed on the basis of the vector pPH93-AOX1 _Y727 -HSA [Tyurin O.V. Development of a gene expression system based on methylotrophic yeast Komagataella kurtzmanii: Ph.D. thesis. biol. sciences. State Research Institute of Genetics, Moscow, 2014.].

This vector contains the genetic elements of the strain K.kurtzmanii_ VKPM Y-727 [Tyurin O.V. Development of a gene expression system based on methylotrophic yeast Komagataella kurtzmanii: Ph.D. thesis. biol. sciences. State Research Institute of Genetics, Moscow, 2014]. These include His4 marker marker gene, as well as a DNA fragment enclosing the promoter region of the AOX1 gene. In this case, the AOX1 promoter region instead of the natural unique SacI site contains an artificial nucleotide sequence encoding the MluI and BamHI sites, moreover, the sequence of the known bacterial vector pUC18 containing the origin of replication and the antibiotic resistance gene ampicillin was cloned into the MluI site. The 3'-terminal region of the AOX1 promoter as part of the pPH93-AOX1 _Y727 -HSA vector is fused to the DNA sequence encoding the signal art peptide. The target integrative plasmid pRH727-Bgl1ng for transformation of yeast cells is constructed by ligation of three DNA fragments, namely:

- XhoI / PstI DNA fragment of the vector pPH93-AOX1 _Y727 -HSA, comprising all the vector elements and the DNA sequence encoding the signal art peptide;

- PstI / NcoI DNA fragment of the plasmid pUC18x-GAL1matHH-GH encoding part of the leader polypeptide containing the doubled amino acid sequence of the pro-region of the S. cerevisiae yeast protein HSP150 [RU 2460795];

- NcoI / XhoI DNA fragment of the plasmid pUC19mx-Bgl1ng containing the modified structural gene Bgl1ng β-glucanase Bacillus pumilus.

As a result of ligation, the target integrative plasmid pPH727-Bgl1ng containing the integrative MluI / MluI DNA fragment containing 1 copy of the target expression cassette AOX1-artHH-Bgl1ng is obtained. In plasmid yeast, this plasmid mediates the expression of the Bacillus β-glucanase methanolus β-glucanase structural gene under the control of pacilus pumilus pumilus -induced promoter AOX1, and β-glucanase secretion is directed by the artHH leader polypeptide comprising the art signal peptide and the doubled pro-region of S. cerevisiae yeast protein HSP150.

The MluI / MluI integrative DNA fragment of plasmid pPH727-Bgl1ng is used to transform yeast cells. This DNA fragment does not contain E. coli DNA.

Example 4. Construction of an auxiliary plasmid A3d-Bgl1ng

An auxiliary plasmid is obtained on the basis of a modified bacterial vector pUC18, the EcoRI site of which is inactivated using the BglII linker (5'-gagatctc). The modified vector is called pUC18b.

PCR amplification of the DNA fragment of the genomic DNA of the VKPM strain Y-727 of the K.kurtzmanii yeast is carried out, which includes the region of transcription termination of the AOX1 gene. Amplification is carried out using primers AOX1T-dir (5'-taactcgagaaaagagaggctgaagctgg) and AOX1T-rev (5'-ttgagatctcgtacgagaagaaaaaatgac). As a result of amplification, a DNA fragment of about 220 nucleotide residues in size is obtained. The sticky ends of the amplified DNA fragment are opened using restriction enzymes XhoI and BglII.

The auxiliary plasmid A3d-Bgl1ng is constructed by targeted ligation of the following three DNA fragments:

- AatII / XhoI DNA fragment of the plasmid pPH727-Bgl1ng, comprising the AOX1 promoter fused to the artHH-Bgl1ng structural gene, mediating the expression and secretion of Bacillus pumilus β-glucanase in yeast cells;

- XhoI / BglII DNA fragment obtained as a result of amplification of the genomic DNA of the yeast strain K.kurtzmanii VKPM Y-727, enclosing the transcription termination region of the AOX1 gene;

- BglII / AatII DNA fragment of the modified vector pUC18b, comprising the origin of replication and the gene for resistance to ampicillin of the bacterial vector pUC18.

The resulting auxiliary plasmid A3d-Bgl1ng contains a BamHI / BglII DNA fragment encoding the AOX1-artHH-Bgl1ng-aox1T target expression cassette, including the AOX1 promoter, the artHH leader region, the Bacillus pumilus β-glucanase structural gene, and aox transcription terminator. The indicated cassette is surrounded by unique restriction sites AatII, BamHI and BglII, the location of which allows genetic engineering manipulations leading to doubling and tripling of the cassette on this auxiliary vector.

Example 5. Construction of a plasmid for multi-copy integration of the Bgl1ng gene in the K.kurtzmanii yeast genome

At the first stage, using the auxiliary plasmid A3d-Bgl1ng, double the target expression cassette AOX1-artHH-Bgl1ng-aox1T. For this purpose, the AatII / BglII DNA fragment of the auxiliary plasmid A3d-Bgl1ng is cloned into the same plasmid cleaved at the AatII / BamHI sites. As a result of this genetic engineering manipulation, the plasmid A3d- (Bgl1ng) x2 containing the AatII / BglII DNA fragment containing 2 tandem copies of the target expression cassette AOX1-artHH-Bgl1ng-aox1T is obtained.

In the next step, 2 copies of the target copy of the target expression cassette AOX1-artHH-Bgl1ng-aox1T from the auxiliary plasmid are transferred to the integrative plasmid pPH727-Bgl1ng containing another 1 copy of the expression cassette. For this purpose, the AatIIl / BglII DNA fragment of the auxiliary plasmid A3d- (Bgl1ng) x2 is cloned into the plasmid pPH727-Bgl1ng, split at the AatII / BamHI sites.

Cloning yields the plasmid pPH727- (Bgl1ng) x3 containing an integrative MluI / MluI DNA fragment containing 3 tandem copies of the target expression cassette AOX1-artHH-Bgl1ng, including (Fig. 1):

1. The bgl Bacillus pumilus gene, integrated into a single reading frame with the nucleotide sequence of the artHH signal peptide, under the control of the AOX1 _Y727 promoter

2. Aox1T transcription terminator

3. Yeast selective marker His4

The MluI / MluI integrative DNA fragment of plasmid pPH727- (Bgl1ng) x3 is used to transform yeast cells. This DNA fragment does not contain E. coli DNA.

Example 6. Obtaining transformants of Komagataella kurtzmanii yeast carrying the bgl gene from Bacillus pumilus

The MluI / MluI DNA fragment of the constructed plasmid pPH727- (Bgl1ng) x3 (Example 5) was used to transform cells of the laboratory recipient strain K. kurtzmanii 727ΔHis4 VKPM Y-4462 obtained from the K.kurtzmanii VKPM Y-727 strain by deletion of the His4 gene.

The transformation procedure is carried out by electroporation, as described in [Tyurin O.V. Development of a gene expression system based on methylotrophic Komagataella kurtzmanii yeast: Ph.D. biol. sciences. State Research Institute of Genetics, Moscow, 2014.].

The integrative MluI / MluI fragment contains, at the open ends, DNA sequences homologous to the promoter region of AOX1, allowing its integration into the promoter region of AOX1 of the yeast genome.

As a result of this procedure, Y727-Bgl1 transformants are obtained, in the genome of each of which a different number of copies of the target expression cassette is integrated, including the Bacillus pumilus β-glucanase gene.

Example 7. The cultivation of transformed strains.

Yeast transformants K. kurtzmanii are cultured in flasks at a temperature of 29 ° C on a rotary shaker (250 rpm). Cultivation is carried out in a liquid medium YPgM of the following composition (wt.%): Peptone - 2, yeast extract - 1, glycerin - 0.5, methanol - 0.5, water - the rest. Transformants are seeded in the initial titer of 5 × 10 ⁵ -5 × 10 ⁶ ml ^-1 . Cultures are grown for 69 hours, 24 and 48 hours after the start of cultivation, a solution of 50% methanol in an amount of 1/100 of the volume of medium is introduced into the flasks. After 69 hours of growth, the cell biomass is separated from the culture medium by centrifugation at 16,000 g for 2 minutes using 1.5 ml tubes, after which the clarified culture liquid is transferred to clean tubes and stored in a refrigerator at + 4 ° C.

The selection of transformants characterized by a high concentration of β-glucanase Bacillus pumilus in the culture fluid is carried out by electrophoretic analysis.

Samples for electrophoretic analysis of the level of production are obtained by concentrating proteins of the culture fluid. The analysis is carried out on a 15% polyacrylamide gel under denaturing reducing conditions according to a standard procedure, for example, using the Mini-PROTEAN Tetra Cell system (# 165-8000). Buffers, sample preparation, application and electrophoresis are done according to the "BIO-RAD" instructions supplied with the system. A mixture of pre-stained proteins with a known molecular weight in the range of 10 to 250 kDa (Thermo Scientific ™, PageRuler ™ Plus Prestained Protein Ladder, # 26620) is used as markers. Gel staining with Coomassie solution was performed using a Thermo Scientific ™ kit (Pierce ™ Mini Gel Power Staining Kit, # 22840) according to the attached instructions.

As a result, transformant No. 89 was selected from the set of obtained transformants, characterized by the highest concentration of the target β-glucanase protein Bacillus pumilus in the culture fluid.

The amount of β-glucanase enzyme in the culture fluid is determined using the DNS method [Anal. Chem., 1959, 31 (3), 426-428].

When cultured under the described conditions, the selected transformant No. 89 provides the production of β-glucanase Bacillus pumilus in the amount of 3000 units in 1 ml of culture fluid.

To confirm the presence in the chromosome of the selected transformant of the bgl Bacillus pumilus gene insert by the polymerase chain reaction (PCR) method, chromosomal DNA isolated from the cells of the selected transformant and specific primers BglPum-f and BglPum -r are used

BglPum -f 5'-ggttatgtgttctgggaacctc-3 ',

BglPum-r 5'-ttatctttttgtgtaacgca-3 '

PCR reaction mode:

95 ° C - 3 min - 1 cycle

30 cycles:

95 ° C - 30 sec.

60 ° С - 30 sec.

72 ° C - 60 sec.

72 ° C - 5 min. - 1 cycle

To control the magnitude of the amplified DNA fragment during electrophoresis, the molecular marker GeneRuler 1 kb DNA Ladder (Fermentas) was used (line 2, Fig. 2, the size of the fragments from the bottom up 10000, 8000, 6000, 5000, 4000, 3500, 3000, 2500, 2000, 1500 , 1000, 750, 500, 250 bp). The production of a DNA fragment of 642 bp (line 1 of Fig. 2) indicates the presence of a bacillus pumilus bgl gene insertion strain on the chromosome

Example 8. Obtaining transformants of Komagataella kurtzmanii yeast carrying the bgl gene from Bacillus safensis

The plasmid vector pPIC-bglSaf containing the integrative expression cassette is obtained. The total genomic DNA of the natural strain of Bacillus safensis VKPM B-13331 is used as a source of the bgl gene. The bgl gene DNA is synthesized by PCR using bgl-saf-1 and bgl-saf-2 primers containing restriction sites at the 5'-ends for cloning EcoRI and NotI, respectively:

bgl-saf-1 5'-aagaattccaaacgggcggttcgttatatga-3 '

bgl-saf-1 5'-aagcggccgcttatctaattgtgtaaggca-3 '

The resulting DNA fragment is treated with restriction enzymes EcoRI and NotI and cloned into the expression vector pPIC9

(htttp: //www.thermofisher.com/order/catalog/product/V17520).

To obtain an integrative expression cassette, pPIC-bglPum plasmid is treated with BglII restriction endonuclease.

The integrative expression cassette consists of the following elements:

1. The bgl_Bacillus safensis gene, integrated into a single reading frame with the nucleotide sequence of the α-factor signal peptide, under the control of the AOX1 promoter

2. Transcriptional terminator TTAOX1

3. Yeast selective marker His4

4. The area of integration is the nucleotide sequence of the AOX1 gene

The transformation of the indicated integrative expression cassette into Komagataella kurtzmanii 727ΔHis4 yeast VKPM Y-4462 yeast, the selection of the most active transformants and determination of β-glucanase activity in the culture fluid are carried out as described in examples 6 and 7. The presence of the selected transformant of the bgl gene insert from Bacillus safensis on the chromosome is confirmed by the method polymerase chain reaction (PCR).

According to the fermentation results, the most productive transformant No. 154 is selected, which, upon cultivation, synthesizes β-glucanase in the amount of 1134 units / ml of culture fluid.

Example 9. Obtaining transformants of the yeast Pichia pastoris carrying the bgl gene from Bacillus altitudinis.

The plasmid vector pPIC-bglAlt containing the integrative expression cassette is obtained. The total genomic DNA of the Bacillus altitudinis strain VKPM B-11231 is used as a source of the bgl gene. The bgl gene DNA is synthesized by PCR using bgl-saf-1 and bgl-saf-2 primers containing restriction sites for cloning at the 5'-ends - EcoRI and NotI, respectively:

bgl-alt-1 5'-aagaattccaaacgggcggttcgttatatga-3 '

bgl-alt-1 5'-aagcggccgcttatctaattgtgtaaggca-3 '

The resulting DNA fragment is treated with restriction enzymes EcoRI and NotI and cloned into the expression vector pPIC9

(http://www.thermofisher.com/order/catalog/product/V17520).

To obtain an integrative expression cassette, pPIC-bglPum plasmid is treated with BglII restriction endonuclease.

The integrative expression cassette consists of the following elements:

1. The bgl_Bacillus altitudinis gene, integrated into a single reading frame with the nucleotide sequence of the α-factor signal peptide, under the control of the AOX1 promoter

2. Transcriptional terminator TTAOX1

3. Yeast selective marker His4

4. The area of integration is the nucleotide sequence of the AOX1 gene

The transformation of the indicated integrative expression cassette into Komagataella kurtzmanii 727ΔHis4 yeast VKPM Y-4462 yeast, the selection of the most active transformants and determination of β-glucanase activity in the culture fluid are carried out as described in examples 6 and 7. The presence of the selected bgl gene insert transformant from Bacillus altitudinis on the chromosome is confirmed by the method polymerase chain reaction (PCR)

According to the fermentation results, the most productive transformant No. 27 was selected, which, when cultivated, synthesizes β-glucanase in the amount of 998 units / ml of culture fluid.

Claims (2)

1. The transformant yeast Komagataella kurtzmanii producing β-glucanase containing the bgl gene encoding endo-β-l, 3 (4) -D-glucanase from Bacillus pumilus or an enzyme whose amino acid sequence is homologous to endo-β-l, 3 (4 ) -D-glucanase from Bacillus pumilus not less than 96%. 2. The transformant according to claim 1, in which endo-β-l, 3 is used as an enzyme whose amino acid sequence is homologous to endo-β-l, 3 (4) -D-glucanase from Bacillus pumilus, at least 96% (4) -D-glucanase from Bacillus safensis, or endo-β-l, 3 (4) -D-glucanase from Bacillus stratosphericus, or endo-β-l, 3 (4) -D-glucanase from Bacillus altitudinis, or endo-β-l, 3 (4) -D-glucanase from Bacillus aerius, or endo-β-l, 3 (4) -D-glucanase from Bacillus cellulasens, or endo-β-l, 3 (4) -D Glucanase from Bacillus australimaris.

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