RU2605629C1 - RECOMBINANT STRAIN OF YEAST Pichia pastoris - PRODUCER OF THE SECRETED THERMOSTABLE XYLOGLUCANASE CODED BY SYNTHETIC GENE, AND METHOD FOR MICROBIOLOGICAL SYNTHESIS OF SECRETED THERMOSTABLE XYLOGLUCANASE BASED ON THIS STRAIN - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to biochemistry, genetic engineering and biotechnology, in particular, to strain Pichia pastoris VKPM Y-4269. This is a producer of secreted thermostable xyloglucanase of GH74 class. Said xyloglucanase encoded by synthetic gene corresponding to SEQ ID NO 1. Present invention also relates to a method for microbiological synthesis of xyloglucanase. Above method involves culturing a recombinant microorganisms, containing gene xyloglucanase, in aerobic conditions in a nutrient medium to maximum accumulation of the end product. This nutritional medium includes sources of carbon, nitrogen and mineral additives. Above method is characterised by that is as a producer is used a recombinant strain Pichia pastoris VKPM Y-4269.

EFFECT: present invention enables to obtain secretable thermostable xyloglucanase of class GH74 with high output and purity.

2 cl, 2 dwg, 3 ex

Description

The group of inventions relates to biotechnology, in particular to xyloglucanase biosynthesis, and is a recombinant strain of Pichia pastoris yeast containing a synthetic xyloglucanase gene in the genome and capable of synthesizing secreted thermostable xyloglucanase, as well as a method of microbiological synthesis of thermostable xyloglucanase based.

Plant biomass is a cheap renewable source of sugars for biotechnology needs. Enzymatic hydrolysis is an effective and environmentally friendly way to convert plant biomass to sugar. A variety of plant materials necessitates the use of a wide range of enzymes for hydrolysis.

Xyloglucan (KG) is one of the main polysaccharides of the primary cell wall of dicotyledons and many monocotyledonous plants. The KG molecule consists of 1,4-β-D-glucotetrose monomers, in which three of the four glucose residues are linked by α-1,6 bonds with D-xylose residues. Some of the glucose residues, in turn, may be linked by β-1,2 bonds to D-galactose or L-arabinose residues. D-galactose residues may be linked by α-1,2 bonds to L-fucose residues. The modification of xylose residues is species-specific, which determines many types of CG in plants. In the cell wall, KG performs a structural function, forming a matrix into which cellulose microfibrils are immersed, therefore, complete hydrolysis of cellulose is impossible without preliminary hydrolysis of KG.

Enzymes that specifically cleave β-1,4 bonds of the KG molecule are called xyloglucanases. Endo-xyloglucanases (xyloglucan-specific endo-β-1,4-glucanases, EC 3.2.1.151) randomly cleave KG, resulting in the formation of oligoxyloglucans of various lengths. Exo-xyloglucanases (EC 3.2.1.155 and EC 3.2.1.150) processively hydrolyze the KG molecule from the ends of the chain. Complete hydrolysis of KG is carried out as a result of the combined action of both types of enzymes. According to the CAZy base classification, KG hydrolyzing enzymes belong to the GH74, GH5, GH9, GH12, GH16, GH26, GH44 families of glycosyl hydrolases. KG hydrolyzing enzymes can also hydrolyze cellulose.

Oligosaccharides obtained from KG can be used in food and feed industries, and can also be used in medicine, pharmaceuticals. It is known that some plant oligosaccharides have immunomodulating / stimulating, prebiotic, antibiotic, antiviral, antitumor, antiparasitic, anticoagulation, antineurodegenerative properties, and can also lower lipids and cholesterol. The possibility of obtaining oligosaccharides with such properties makes KG interesting objects for study and subsequent use.

The information sources contain information about microorganisms - natural producers of xyloglucanases: Geotrichum sp. (Yaoi and Mitsuishi, 2004), Aspergillus japonicus, Chrysosporium lucknowense, Trichoderma reesei (Grishutin et al., 2004; Markov et al., 2005; Qi et al., 2013), Phanerochaete chrysosporium (Ishida et al., 2007). By methods of selection and radiation mutagenesis, strains synthesizing increased amounts of xyloglucans were obtained: Penicillium verruculosum (RU 2361918), Penicillium funiculosum (RU 2323254), Aspergillus aculeatus (RU 2303057).

Genetic engineering strains of Escherichia coli, producers of recombinant xyloglucanases from Geotrichum sp. M128 (US 2004038367; Yaoi and Mitsuishi, 2002), Fusarium graminearum (Habrylo et al., 2012), Rhizomucor miehei (Song et al., 2013). Various strains of fungi are used as recipients for the heterologous expression of fungal xyloglucanases: xyloglucanases from Malbranchea cinnamomea (US 6,500,658) are expressed in Aspergillus oryzae, xyloglucanase Penicillium xenoglucenicum Penicillium isenus (Tr. 2358756), in Aspergillus niger, xyloglucanase from Thielavia australiensis and proprietary xyloglucanase (WO 2014138983; Hasper et al., 2002).

Sometimes, bacterial recipients other than E. coli are used for heterologous expression of fungal xyloglucanases, for example, Acidothermus cellulolyticus is used for heterologous expression of xyloglucanases from Acremonium sp., Cladorrhinum foecundissimum, Humicola insolens, Thielavia tergus agerus gerus infusius tergis, trichoderus agerus regius thermophila (Vlasenko et al., 2010).

Genetic engineering strains of Pichia pastoris yeast, producers of recombinant xyloglucanases from Phanerochaete chrysosporium (Ishida et al., 2007), are also described.

The heterologous expression systems used in the production of a recombinant enzyme in bacteria and microscopic fungi have their technological and economic advantages and disadvantages. Methods using bacterial expression systems, such as E. coli, have such limitations as the inability to reproduce all post-translational modifications characteristic of eukaryotes (glycosylation, phosphorylation, the formation of disulfide bonds between cysteine residues) and the formation of insoluble inclusion bodies. The disadvantages of producer strains based on microscopic fungi of the genera Aspergillus and Penicillium include a low level of synthesis of recombinant enzymes compared to E. coli and difficulties with genetic manipulation of the strains due to non-standardized and underdeveloped genetic tools (Demain and Vaishnav, 2009).

Interest in the production of recombinant proteins using methylotrophic yeast, in particular of the genus Pichia, Komagataella or Hansenula, is based on a number of advantages of these microorganisms both from a scientific and a technological point of view. Unlike E. coli, methylotrophic yeasts are capable of all post-translational modifications characteristic of eukaryotic cells, including the secretion of proteins into the culture medium. The low amount of endogenous secreted yeast proteins significantly facilitates the isolation and purification of the target recombinant product (Macauley-Patrick and Fazenda, 2005). A culture of methylotrophic yeast can be grown in fermenters to higher densities compared to the traditionally used Saccharomyces cerevisiae yeast, which allows a higher level of production of the target protein (Gellissen, 2000). Methylotrophic yeast has some of the most powerful promoters in nature. They are also distinguished by an alternative mechanism of protein glycosylation, which allows the synthesis of a low immunogenic target protein.

As the closest analogue of the claimed strain, let us consider a strain of the yeast Pichia pastoris containing the gene XGH74B from the Phanerochaete chrysosporium plasmid pPICZα-A under the control of the AOX1 promoter and expressing the glycosyl hydrolase of class GH74, specifically hydrolyzing xyloglucan (Ishida et al., 2007). This strain was created in order to obtain a recombinant enzyme and to study its structure and properties and has not been characterized by the level of synthesis of XGH74B. According to the results of a comparative analysis (http://blast.ncbi.nlm.nih.gov), the homology level of the synthetic mt74sin gene encoding xyloglucanase of the present invention and the XGH74B gene from Phanerochaete chrysosporium is 55%.

The closest analogue of the proposed method of microbiological synthesis of xyloglucanase is a method for producing an enzyme preparation with xyloglucanase activity in the fermentation of Penicillium canescens strains multicopy of the homologous xegA gene (RU 2358756). The xyloglucanase activity at the end of fermentation in the culture fluid of P. canescens XG9 strains (VKM No. F-3939D) was at least 60 units / ml.

The task of the claimed group of inventions is to develop a method of microbiological synthesis of xyloglucanase based on a recombinant strain of Pichia pastoris, combining a high level of production of a heterologous enzyme with its thermal stability.

The problem is solved by:

- designing a recombinant strain of the yeast Pichia pastoris VKPM Y-4269 - producer of secreted thermostable xyloglucanase GH74 encoded by a synthetic gene corresponding to SEQ ID NO 1,

- development of a method of microbiological synthesis of xyloglucanase based on a recombinant strain of the yeast Pichia pastoris VKPM Y-4269

The process of constructing the inventive strain consists of the following steps:

- designing the synthetic mt74sin gene (SEQ ID NO 1) based on the nucleotide sequence of the mt74 gene encoding xyloglucanase of the GH74 family from Myceliophthora thermophila VKPM F-244.

- designing an integrative plasmid DNA (plasmid) pPIC-mt74sin containing the synthetic xyloglucanase gene mt74sin.

- designing a recombinant strain of Pichia pastoris / mt74sin yeast capable of synthesizing thermostable xyloglucanase of the GH74 family.

- development of a method for microbiological synthesis of xyloglucanase based on a recombinant strain of the yeast Pichia pastoris / mt74sin, combining a high level of synthesis of heterologous xyloglucanase of the GH74 family with its thermal stability.

Step 1. Construction of a synthetic mt74sin gene based on the nucleotide sequence of the mt74 gene encoding xyloglucanase of the GH74 family from strain Myceliophthora thermophila VKPM F-244

The design of the mt74sin nucleotide sequence is developed based on the coding region of the mt74 gene from Myceliophthora thermophila VKPM F-244, identical to the coding region of the GH74 family glycosyl hydrolase gene MYCTH_116384 (GenBank: AEO58927.1) from Myceliophthora thermophila / DSCRM646464464994 BCMCR4644642 ) The mt74 gene from Myceliophthora thermophila VKPM F-244 encodes xyloglucanase, which is confirmed by analysis of the proteins secreted by the VKPM F-244 strain using a combination of zymogram and MALDI-TOF methods.

The mt74 gene sequence is analyzed, introns are deleted, and, guided by information on the composition of the codons of actively expressed Pichia pastoris genes, nucleotide triplets are replaced so that the content of G + C matches the natural genes. The structure of the resulting mRNA is analyzed and, if necessary, made replacements to remove the extensive secondary structures and restriction sites XhoI and BglII, which then flank the sequence for subsequent insertion into the plasmid construct. The resulting sequence is used for the synthesis of mt74sin DNA. The homology of the mt74sin gene and the coding region of the MYCTH_116384 gene from Myceliophthora thermophila ATCC 42464 is 73.5%.

Step 2. Construction of an integrative plasmid construct pPIC-mt74sin

The plasmid pPIC-mt74sin was constructed by cloning the DNA fragment obtained in step 1 containing the synthetic mt74sin gene into pPIC-101 vector designed for integration into Pichia pastoris strains (his 4, mut +). The pPIC-101 vector of 7404 base pairs contains the AOX1 promoter; AOX1 terminator; the coding region of the HIS4 gene; the coding region of the bla gene, which provides resistance of E. coli strains to ampicillin; Replicon pBR-322; a sequence encoding a signal peptide α-factor yeast.

Plasmid pPIC-mt74sin, size 9285 base pairs, along with the genes of the vector pPIC-101, contains the coding region of the synthetic xtoglucanase mt74sin gene under the control of the AOX1 promoter. (Fig. 2).

Step 3. Obtaining a recombinant strain of Pichia pastoris / mt74sin

As the recipient strain, the Pichia pastoris strain (his 4, mut +) is used, which does not synthesize xyloglucanase. Competent cells of this strain are transformed with the plasmid pPIC-mt74sin. The result is a recombinant strain of Pichia pastoris / mt74sin capable of synthesizing thermostable xyloglucanase of the GH74 family.

Pichia pastoris / mt74sin is deposited in the All-Russian collection of industrial microorganisms as Pichia pastoris VKPM Y-4269;

The inventive strain of Pichia pastoris VKPM Y-4269 has the following morphological and physiological and biochemical characteristics:

Morphological features

When cultured at a temperature of 28 ° C for 48 hours on an agarized YPD medium of the following composition, wt. %:

peptone - 2,

yeast extract - 1,

glucose - 2,

agar - 2,

water is the rest.

The cells are oval, 3-4 microns in diameter; budded; true budding, many-sided; true mycelium does not form. Colonies of light beige color with an even edge, a matte surface, a lenticular profile and a pasty consistency are formed on a YPD agar medium. When growing in a YPD liquid medium at 28 ° С for 24 h of cultivation, the liquid is cloudy, the precipitate is white, coagulation is not observed, the culture does not form wall films.

Physiological and biochemical characteristics

The strain is an optional anaerobic. Growth temperature - 20-33 ° С (optimum - 28 ° С); The pH of the cultivation medium is 4.8-7.4 (optimum is 6.0). The strain can use glucose, glycerin, methanol, oleate, sorbitol, rhamnose as carbon sources. It does not utilize galactose, xylose, arabinose. As sources of nitrogen, the strain can use amino acids, ammonium sulfate, ammonium nitrate. The essential features of the strain are the lack of need for histidine. The Pichia pastoris strain VKPM Y-4269 synthesizes recombinant secreted thermostable xyloglucanase.

The method of microbiological synthesis of xyloglucanase in General

Inoculum, which is a cell of a recombinant producer strain, is prepared by incubation for 15-24 hours at a temperature of 29 ° C on YPD medium with constant aeration on a thermostatic shaker (250 rpm). Then the grown culture is transferred in a ratio of 1: 200 (by volume) to YPgM medium of the following composition, wt. %:

peptone - 2,

yeast extract - 1,

glycerin 0.5,

methanol - 0.5,

water is the rest.

The biosynthesis process leads to Erlenmeyer flasks containing 100 ml of YPgM medium for 62 hours in a rotary shaker-thermostat (250 rpm), at a temperature of 28 ° C. Every 24 hours, methanol induction is carried out by aseptically adding 50% methanol solution to the tubes to a final concentration of 0.5%. After 62 hours, the biomass is separated by centrifugation. The presence of recombinant xyloglucanase in the culture medium is determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate followed by a zymogram (Maniatis et al., 1984). A quantitative assessment of the level of activity of recombinant xyloglucanase is carried out using the DNS method (Miller, 1959).

The level of synthesis of xyloglucanase by the claimed method is at least 60 units / ml of culture fluid, which is not inferior to the closest analogue. The optimum activity of the recombinant xyloglucanase produced by the strain Pichia pastoris VKPM Y-4269 is 60 ° C, pH 5.0. The recombinant xyloglucanase produced by the Pichia pastoris strain VKPM Y-4269 is thermostable: upon incubation of the enzyme preparation for one hour at 50 ° C, pH 5.0 retains 100% of the enzymatic activity, and as a result of incubation for 60 hours, pH 5.0 retains 80 % enzymatic activity.

The invention is illustrated by the following figures of graphic images.

FIG. 1 - Scheme of plasmid DNA pPIC-101. Plasmid vector pPIC-101: AOX1 promoter; AOX1 terminator; HIS4 gene; the bla gene providing resistance to ampicillin (ApR).

FIG. 2 - Scheme of recombinant plasmid DNA pPIC-mt74sin. Plasmid vector pPIC-mt74sin: AOX1 promoter; AOX1 terminator; HIS4 gene; the bla gene providing ampicillin resistance (ApR); mt74syn.

Example 1. Construction of recombinant plasmid DNA pPIC-mt74sin containing the synthetic gene mt74sin encoding xyloglucanase family GH74

All standard genetic engineering and microbiological manipulations are carried out according to known methods (Maniatis et al., 1984).

The mt74sin synthetic gene is obtained by optimizing the nucleotide sequence of the mt74 gene for synthesis in Pichia pastoris. Using the mfold Web Server program (http://unafold.rna.albany.edu/doc/old-mfold-manual/), the structure of the resulting mRNA is checked for extensive secondary structures, and using the Vector NTI program version 9 (Invitrogen) for the presence of restriction sites XhoI and BglII. Substitutions are made to the gene sequence to remove unwanted restriction sites and secondary structures. The ends of the nucleotide sequence are modified with restriction sites XhoI and BglII for subsequent insertion of fragments into the plasmid construct. The resulting sequence is used for the synthesis of mt74sin DNA (SEQ ID NO 1).

Plasmid p-mt74sin is constructed by cloning a fragment containing the mt74sin gene into the integrative vector pPIC-101 (Fig. 1).

Hydrolysis of the plasmid pPIC-101 and the DNA fragment containing the mt74sin gene is carried out by restriction enzymes BglII and XhoI (Fermentas, Latvia). The reaction mixture is purified with a kit for DNA extraction from reaction mixtures (Biosilica, Russia) according to the manufacturer's instructions. Ligation is carried out in a 20 μl reaction mixture containing 20 ng of DNA vector, 1.5 ng of DNA fragment and 5 units T4 DNA ligase (Fermentas, Latvia), according to the manufacturer's method. Competent cells of E. coli strain XL1 blue MRF ′ (Stratagene, USA) (genotype Δ (mcrA) 183 Δ (mcrCB-hsdSMR-mrr) 173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [transform] were transformed with the obtained ligase mixture in an amount of 5 μl F ′ proAB lacIqZΔM15 Tn10 (Tetr)]). Plasmid DNA of the obtained transformants is analyzed by digestion with restriction enzymes BglII and XhoI. As a result, clones containing BglII / XhoI fragments of a size equal to the original fragment of the insert are selected. The correctness of cloning is confirmed by sequencing of each of the recombinant inserts by the Sanger method on an ABI 3500 genetic analyzer (Life technology, USA). The result is a recombinant plasmid pPIC-mt74sin with a size of 9,285 base pairs containing, along with the genes of the vector pPIC-101, also a synthetic gene mt74sin encoding secreted thermostable xyloglucanase of the GH74 family (SEQ ID NO 2) (Fig. 2).

Example 2. Obtaining the inventive strain of Pichia pastoris VKPM Y-4269 - producer of xyloglucanase family GH74

In order to obtain a recombinant strain of Pichia pastoris VKPM Y-4269, a producer of xyloglucanase, cells of the strain Pichia pastoris GS115 (his 4, mut +) (Invitrogen) are transformed with plasmid pPIC-mt74sin. The transformation is carried out with 5 mg of linearized plasmid DNA, to obtain which the original plasmid is treated with restriction endonuclease MluI.

The yeast culture of the strain Pichia pastoris GS115 (his 4, mut +) is grown on YPD medium, under aerobic conditions (250 rpm) to an optical density of 1-2 units. Biomass cells separated by centrifugation at 3000 g for 5 minutes are resuspended in buffer containing 1 mM dithiothreitol and 100 mM HEPES, then incubated on a shaker for 30 minutes and, after washing twice with cold deionized water, are resuspended in 1 ml of 1 M sorbitol. The suspension is divided into aliquots of 40 μl. Electroporation is carried out on a GenePulser Xcell (Biorad) instrument in cuvettes with a gap of 2 mm at 1500 V, 25 μF, 600 Ohms. After electroporation, 1 ml of a cold solution of 1 M sorbitol is quickly added, incubated for 1 hour at a temperature of 30 ° C and plated on a selective medium for selection of transformants. The selection of transformants is carried out on a minimal medium M9 containing, by weight. %:

KH ₂ PO ₄ - 0.1;

MgSO ₄ - 0.05;

NaCl - 0.01;

CaCl ₂ - 0.01;

(NH ₄ ) ₂ SO ₄ 0.35;

glucose - 2;

thiamine - 0.02;

riboflavin - 0.02;

nicotinic acid - 0.02;

p-aminobenzoic acid - 0.02;

calcium pantothenate - 0.02;

biotin - 0.0002;

pyridoxine - 0.02;

inositol - 1;

folic acid - 0.02;

water is the rest.

Cells are incubated at 29 ° C for 3 days. Transformants are detected by PCR screening. The result is the claimed strain of Pichia pastoris VKPM Y-4269, producing a secreted thermostable xyloglucanase.

Example 3. Microbiological synthesis of thermostable secreted xyloglucanase of the claimed strain of Pichia pastoris VKPM Y-4269

The seed material is grown by incubating cells of the Pichia pastoris strain VKPM Y-4269 for 15-24 hours at a temperature of 29 ° C on YPD medium with constant aeration (250 rpm). The YPgM medium is inoculated with prepared seed and the biosynthesis process is carried out under aerobic conditions at a temperature of 28 ° C, by induction with methanol.

The level of xyloglucanase synthesis by the claimed method is at least 60 U / ml, which is 51 times higher than the level of xyloglucanase synthesis by the natural producer Myceliophthora thermophila VKPM F-244 (1.17 units / ml of culture fluid). The optimum activity of recombinant xyloglucanase produced by the strain Pichia pastoris VKPM Y-4269 is 60 ° C, pH 5.0. When incubated for one hour at 50 ° C, pH 5.0, 100% of the enzymatic activity is preserved, and when incubated for one hour at 60 ° C, pH 5.0, 80% of the enzymatic activity is preserved.

Thus, a recombinant yeast strain Pichia pastoris VKPM Y-4269 was obtained, capable of biosynthesis of secreted thermostable xyloglucanase of the GH74 family encoded by a synthetic gene. When culturing by the claimed method, the level of synthesis of the target product of the claimed strain is comparable to that of the nearest analogue, but unlike the closest analogue, the xyloglucanase produced is thermostable.

Information sources

1. Maniatis T., Fritsch E., Sambrook J., Molecular Cloning, M .: Mir, 1984; DNA cloning. Methods Ed. D. Glover, Per. from English., Moscow, Mir, 1988.

2. Demain AL, Vaishnav P. // Production of recombinant proteins by microbes and higher organisms // Biotechnol Adv., 2009.

3. Grishutin S.G., Gusakov A.V., Markov A.V., Ustinov B.B., Semenova M.V., Sinitsyn A.P. // Specific xyloglucanases as a new class of polysaccharide-degrading enzymes. // Biochim Biophys Acta, 2004.

4. Habrylo O., Song X., Forster A., Jeltsch J.M., Phalip V. // Characterization of the four GH12 Endoxylanases from the plant pathogen Fusarium graminearum. // J. Microbiol Biotechnol., 2012.

5. Hasper A.A., Dekkers E., van Mil M., van de Vondervoort P.J., de Graaff L.H. // EglC, a new endoglucanase from Aspergillus niger with major activity towards xyloglucan. // Appl Environ Microbiol., 2002.

6. Ishida T., Yaoi K., Hiyoshi A., Igarashi K., Samejima M. // Substrate recognition by glycoside hydrolase family 74 xyloglucanase from the basidiomycete Phanerochaete chrysosporium. // FEBS J., 2007.

7. Markov A.V., Gusakov A.V., Kondratyeva E.G., Okunev O.N., Bekkarevich A.O., Sinitsyn A.P. // New effective method for analysis of the component composition of enzyme complexes from Trichoderma reesei. // Biochemistry (Mosc), 2005.

8. Miller G.L. // Use of dinitrosalicylic acid reagent for determination of reducing sugar // Analytical Chemistry. 1959.

9. Qi H., Bai F., Liu A. // Purification and characteristics of xyloglucanase and five other cellulolytic enzymes from Trichoderma reesei QM9414. // Biochemistry (Mosc), 2013.

10. Yaoi K., Mitsuishi Y. // Purification, characterization, cloning, and expression of a novel xyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase. // J Biol Chem., 2002.

11. Yaoi K., Mitsuishi Y. // Purification, characterization, cDNA cloning, and expression of a xyloglucan endoglucanase from Geotrichum sp. M128 // FEBS Lett., 2004.

12. Song S., Tang Y., Yang S., Yan Q., Zhou P., Jiang Z. // Characterization of two novel family 12 xyloglucanases from the thermophilic Rhizomucor miehei. // Appl Microbiol Biotechnol., 2013.

M., Cherry J., Xu F. // Substrate specificity of family 5, 6, 7, 9, 12, and 45 endoglucanases // Bioresour Technol., 2010.13. Vlasenko E.,

M., Cherry J., Xu F. // Substrate specificity of family 5, 6, 7, 9, 12, and 45 endoglucanases // Bioresour Technol., 2010.

14. Macauley-Patrick S, Fazenda ML (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22: 249-270.

15. Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl. Microbiol. Biotechnol. 54 (6): 741-750.

16. RU 2358756 // A method for producing an enzyme preparation for cleaving hemicellulose heteropolysaccharides of the plant cell wall and an enzyme preparation (options) // Priority date: 11.26.2007.

17. RU 2303057 // Strain of the mycelial fungus Aspergillus aculeatus - producer of a complex of carbohydrases containing xylanases, beta-glucanases, pectinases and xyloglucanases // Priority date: 11/14/2005

18. RU 2323254 // Penicillium funiculosum mycelium strain - producer of a carbohydrase complex containing cellulase, glucanase, glucosidase, xylanase and xyloglucanase, and a method for producing an enzyme preparation of a carbohydrase complex for saccharification of lignocellulosic materials // Priority date: 04.04.2006.

19. RU 2361918 // Penicillium verruculosum mycelium strain - producer of a complex of cellulases, xylanase and xyloglucanase and a method for producing an enzyme preparation of a complex of cellulases, xylanase and xyloglucanase for hydrolysis of cellulose and hemicellulose // Priority date: 26.02.2008.

20. US 6500658 // Xyloglucanase from Malbranchea // Priority date: US20000653778 20000901; WO2000DK00450 20000811; US19990149397P 19990817

21. US 2004038367 // Novel xyloglucan oligosaccharide-degrading enzyme, polynucleotide encoding the enzyme, and method of preparing the enzyme // Priority date: JP20020083433 20020325

22. US 2004067569 A1 // Polypeptides having xyloglucanase activity and nucleic acids encoding same // Priority date: US20030420191 20030418; US20020373987P20020419.

23. WO 2014138983 A1 // Novel cell wall deconstraction enzymes of Malbranchea cinnamomea, Thielavia australiensis, and Paecilomyces byssochlamydoides, and uses its // Priority date: US201361783222P 20130314; US201361783313P20130314; US201361783485P 20130314.

Claims (2)

1. Recombinant yeast strain Pichia pastoris VKPM Y-4269 - producer of secreted thermostable xyloglucanase class GH74 encoded by a synthetic gene corresponding to SEQ ID NO 1. 2. A method of microbiological synthesis of xyloglucanase, comprising cultivating recombinant microorganisms containing the xyloglucanase gene, under aerobic conditions in a nutrient medium including carbon, nitrogen and mineral additives to the maximum accumulation of the target product, characterized in that the recombinant strain according to claim 1 is used as a producer. one.

Images