RU2378372C2 - Genetic maker to ensure expression of target homologous and heterologous genes in cells of filamentous fungus penicillium verruculosum; used as host, method for making strain of fungus penicillium verruculosum and method for making enzymatic agent - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention represents the method for making an enzyme, preferentially cellulase or xynalase, decomposing cellulose or xylenes respectively by cultivation of strain of fungus Penicillium verruculosum. Said strain is derived from transformation of a cell of fungus Penicillium verruculosum with a genetic maker containing a target coding enzyme sequence functionally connected with regulatory gene elements of cellobiohydrolase I Penicillium verruculosum which represent a promotor and terminator of a cellobiohydrolase I gene, while an identification peptide herewith depends on the fact what target enzyme is.

EFFECT: invention allows producing high efficient enzymes.

6 cl, 6 dwg, 1 tbl, 4 ex

Description

The invention in the field of biotechnology relates to the microbiological industry and is a method for producing enzyme preparations with carbohydrase activity by culturing multicopy strains of mycelial fungi of the genus Penicillium verruculosum transformed with DNA fragments with cloned homologous and heterologous genes.

The energy needs of mankind at the present stage of its development require the use of renewable energy sources, primarily plant biomass. The basis of the bioconversion of plant biomass is the enzymatic hydrolysis of cellulose and hemicellulose to sugars with their subsequent processing by fermentation to ethanol or transformation into other products of microbial synthesis.

In the global process of decomposition of plant biomass, the main role is played by bacteria and mycelial fungi. Deep destruction of cellulose and hemicellulose is carried out under the influence of polyenzymatic systems of cellulases and hemicellulases (xylanase). Currently, many countries are searching for new mushroom producers of cellulases and hemicellulases. The most widespread mushroom producers, representatives of the genus Trichoderma, which have a high secretory ability. In turn, fungi of the genus Penicillium synthesize enzymatic complexes of cellulases and hemicellulases of a more balanced composition and more efficiently break down cellulose and plant materials containing it. At the same time, individual Penicillium enzymes have high catalytic activity and operational stability.

In the last decade, multicopy fungal superproducers of enzymes have become widespread, in which the amplification of genes encoding secreted enzymes has allowed a sharp increase in the productivity of microorganism strains to produce cheap industrial enzymes. Known multicopy producers of endo-β1,4-glucanases include strains based on Trichoderma longibrachiatum [Clarkson et al., 1995, US Patent 5,419,778], Tr.reesei [Saloheimo et al., 1994, WO Patent 94/28117] and Humicola insolens [Rasmussen et al., 1991, Patent WO 91/17243]. Various fungal multicopy xylanase producers are known - Aspergillus niger [van den Broek et al., 1994, US Patent 5,358,864], Tr.reesei [Nevalainen et al., 1994, US Patent 5,298,405], Thermoascus auranticus [Yu et al., 1990, US Patent 4,966,850], H.insolens [Schulein et al., 1997, US Patent 5,610,048].

Of the fungi of the genus Penicillium, Penicillium verruculosum is of great interest.

The prototype of the developed technical solution can be the patent of the Russian Federation No. 2288267 '"A method for producing a feed complex enzyme preparation (options) and a Penicillium canescens strain (options)", which describes a method for producing preparations with high endoglucanase, xylanase, phytase and pectin-lyase activity during fermentation multicopy strains of P.canescens in which the regulatory region of the included gene copies was represented by promoters of either the β-galactosidase gene or the P.canescens xylanase gene.

The technical problem solved by the group of developed technical solutions consists in developing a method for producing enzyme preparations with various types of cellulase and hemicellulase (xylanase) activity during the fermentation of multicopy strains obtained by transformation of recipient highly productive P.verruculosum strains with cloned DNA regions with homologous and heterologous genes cellulase and hemicellulase (xylanase) enzymes.

The technical result obtained by the implementation of the developed technical solutions consists in obtaining industrial enzyme preparations with high activity of individual cellulase and hemicellulase (xylanase) enzymes during the fermentation of new multicopy strains of the fungi of the genus Penicillium obtained using recombinant DNA molecule technology.

To obtain the indicated technical result, it was proposed to use the genetic construct to ensure the expression of the target homologous and heterologous genes in the cells of the P. verruculosum mycelium fungus used as the host, containing the target coding sequence functionally linked to the regulatory elements of the P. verruculosum cellobiohydrolase I gene, which are promoter, signal peptide and terminator of the cellobiohydrolase I.

Also, to obtain the indicated technical result, it is proposed to use a method for producing a P.verruculosum fungus strain, an enzyme producer encoded by a homologous or heterologous nucleotide sequence that transforms a P.verruculosum fungal cell with a genetic construct containing a target coding sequence functionally linked to regulatory elements of the cellobiohydrolase I P gene .verruculosum, which are a promoter, signal peptide and terminator of the cellobiohydrolase I.

In addition, to obtain the indicated technical result, it was proposed to use a method for producing an enzyme preparation, including cellulase and hemicellulase (xylanase) enzymes, by cultivating microorganisms, moreover, the strain P.verruculosum is obtained by the method described above, the obtained strain is cultured and the target product is isolated from the culture fluid mushroom. In the first embodiment of the indicated method for obtaining the enzyme preparation, a P.verruculosum PV-Cbh1P strain (BKM F-3973D) is obtained which is multicopy homologous to the cbhI gene of P. verruculosum cellobiohydrolase I. In a second embodiment of the method for producing an enzyme preparation, a strain P.verruculosum PV-Egl4T (BKM F-3976D) is obtained, multicopy of the heterologous eglIV gene, endoglucanase IV Tr.longibrachiatum. In a third embodiment of the method for producing an enzyme preparation, a strain P.verruculosum PV-Xyl3T (BKM F-3979D) is obtained, multicopy of the heterologous xylIII gene - xylanase III Tr.longibrachiatum.

These options do not exhaust the possibilities of the developed method.

The development scheme of a method for producing each of the enzyme preparations having one of the cellulase or xylanase activities consists of one preparatory and three typical steps.

Preparatory step: A gene encoding P. verruculosum cellobiohydrolase I is isolated and an expression vector for transformation of P.verruculosum cells is constructed in which the target protein gene is linked to the regulatory sequences (promoter and terminator) of the gene encoding P. verruculosum cellobiohydrolase I.

Step 1. By cloning in a phage vector or by PCR synthesis, a DNA fragment is obtained that encodes a gene for each of these enzymes with a full regulatory region or only the structural part of the sequence encoding a mature protein. This DNA fragment is included in the structure of a vector molecule. An expression plasmid is obtained in which the nucleotide sequence of the structural part of the target gene is functionally aligned with the nucleotide sequence encoding the promoter region and signal peptide of the cbhI gene of P. verruculosum cellobiohydrolase I (or a linear DNA fragment containing the nucleotide sequence of the gene and the above regulatory regions).

Step 2. Each expression plasmid with the incorporated gene (or a DNA fragment in linear form) is transformed with a recipient highly productive strain P.verruculosum 537 (niaD ^- ) under cotransformation conditions together with a plasmid (or a DNA fragment in linear form) carrying the niaD gene sequence, as a marker of selection, and carry out the selection of transformants that secrete the desired activity into the culture fluid. The selected transformants are fermented in rocking flasks and the most productive variant (s) of the producer strain are selected among them.

Stage 3. The fermentation process of the selected most active variant producer strain is carried out in fermenters, enzyme preparations with high activity of the target enzyme are obtained and characterized.

The invention is illustrated by the following examples.

Example 1. Isolation and sequencing of a DNA fragment carrying the cbhI gene of cellobiohydrolase P.verruculosum (cellobiohydrolase I of the 7th family of glycoside hydrolases, molecular weight 66 kDa), and obtaining a vector plasmid pUC-LIC based on the structural elements of this gene.

Based on the homology of the amino acid and nucleotide sequences of fungal cellobiohydrolases of the family 7 glucoside hydrolases, two oligonucleotide primers are synthesized:

(1) 5'-ACCAACTGCTACACCGGCAACACCTGG-3 '

(2) 5'CGGTAAGGGTTGAA (G / A) TCACA (G / A) TC (G / A) CAICCATC-3 ',

where I is inosine.

Then, using the PCR polymerase chain reaction (PCR), an internal DNA fragment of the cellobiohydrolase I gene of 660 bp is synthesized. The genomic DNA of the P. verruculosum strain isolated from mycelium is used as a template for PCR. The nucleotide sequence of the obtained fragment is determined by the Sanger method on both chains. A comparative analysis of the nucleotide sequences of the DNA of the sample and the genes of fungal cellobiohydrolases shows that the maximum homology between the sequence of the isolated DNA of the sample and the sequences of cellobiohydrolases from P. funiculosum and P.occcitanis is 90% and 89%, respectively, which allows the resulting sequence to be attributed to a fragment of cellobiohydrolase I of family 7 glucoside hydrolase.

To obtain the full-sized gene of cellobiohydrolase I P.verruculosum, the inverse PCR method is used. For this, the genomic DNA of P.verruculosum is treated with restriction enzymes EcoRI, SalI, XbaI, PstI and circulated using T4 DNA ligase, which was the template for inverse PCR. To amplify the remaining sections of the cellobiohydrolase gene, two oppositely directed oligonucleotide primers are used.

(3) 5'-AACAACTCCAACACCGGAATCGGCAACCAC-3 '

(4) 5'-AACCGGTGACGAAGTTGAGACGCAACGAGT-3 '

On a DNA matrix obtained by hydrolysis of genomic DNA using restriction enzyme EcoRI, the maximum value of the amplified PCR fragment reaches 2000 bp The nucleotide sequence of the DNA of the gene is determined by both chains by the Sanger method. The cellobiohydrolase I gene of P. verruculosum consists of 1575 bp and does not contain introns. The translated amino acid sequence of cellobiohydrolase I includes 525 amino acids.

Inverse PCR is used to determine the structure of the promoter and terminator of the cellobiohydrolase I gene. Primers (3) and (4) are used, as well as new primers (5), (6) and (7) are synthesized for DNA sequencing of the promoter part of the gene in the 5'-untranslated region:

(5) 5'-GATAATGTCTGATTCTTCAAGGGAA-3 '

(6) 5'-GCTTCAGCCTCTGCTCTCCA-3 '

(7) 5'-GACTGGAGACGAGCTGACAGCA-3 '

As the matrix using ligated genomic DNA, pre-treated with restriction endonuclease HindIII. The amplified PCR fragment has a size of 3500 bp and its structure is determined by the Sanger method on both chains using amplification primers. Promoter region from 1303 bp contains structural elements characteristic of promoters of eukaryotic genes - TATA box (-90 bp from the start codon), transcription region (-65 bp from the start codon) and CreI site (-492 bp from the start codon) . In a similar way, the sequence of the cellobiohydrolase gene is studied in the 3'-untranslated region, which allowed us to identify it as the terminator part of the gene. The general structure of the cbhI gene of P. verruculosum, the promoter, the cbhI gene region encoding the signal peptide and mature cellobiohydrolase I protein, and the terminator is shown in FIG. 1.

To facilitate further steps in cloning homologous and heterologous proteins of the producer strain P.verruculosum, a universal vector is constructed based on two known plasmids pUC19 and pLIC-SGC. The NdeI-HindIII fragment of plasmid pLIC is isolated and cloned into plasmid pUC19, cut at the same restriction sites. The resulting vector was named pUC-LIC. The linearization of the plasmid pUC-LIC is carried out by cutting BseRI, which leads to exposure of two sites for ligation of PCR fragments - 5'-TASTTSAATSSAT-3 'and 5'-AGTAAAGGTGGAT-3'. After processing the vector and the cloned T4 insert with DNA polymerase under conditions of its endonuclease activity (mixed with only one nucleotide), two complementary single-stranded regions are formed on the vector and on the insert, which are respectively ligated. Also, linearization of the pUC-LIC vector removes the SacB gene from the original vector, which provides additional selection of colonies on Petri dishes with sucrose after cloning in pUC-LIC. A schematic representation of the pUC-LIC vector is shown in FIG.

Example 2. Obtaining enzyme preparations with increased cellobiohydrolase activity during fermentation of P.verruculosum strains multicopy of cbhI P.verruculosum gene (cellobiohydrolase I gene, 7th glycoside hydrolase family, molecular weight 66 kDa).

At the first stage, an expression plasmid pCBH1-12 is obtained in which the cloned nucleotide sequence of the complete cbhI of the P. verruculosum gene is connected to homologous regions of the promoter region, signal peptide and terminator region. For this purpose, the following oligonucleotides are synthesized:

(8) 5'-TACTTCCAATCCATGAAGCTTGGAAACGACGCCGACA-3 '

(9) 5'-TATCCACCTTTACTGCGGCCGCATCCATTCAATCTCGTATG-3 '

The resulting PCR fragment is cloned into the pUC-LIC vector at the above ligation sites (see Example 1). Thus, the expression plasmid pCBH1-12 contains all regulatory parts for the expression of the homologous cellobiohydrolase I gene. NotI restriction site is introduced between the ligation site and the vector before linearization of the expression plasmid before transformation into the recipient strain.

The recipient strain P.verruculosum 537 (niaD ^- ) is obtained by selection of clones that are deficient in nitrate reductase and unable to grow on minimal media, where sodium nitrate NaNO ₃ is used as the sole source of nitrogen.

At the second stage, cotransformation of the recipient strain P.verruculosum 537 (niaD ^- ) 10-20 μg of the expression vector plasmid pCBH1-12 and 1-3 μg of the marker plasmid pSTA (niaD ⁺ ) is carried out and clones are screened for transformants with increased secretion of cellobiohydrolase.

The obtained niaD ⁺ transformants are subcultured on plates with minimal medium and a source of nitrogen NaNO ₃ and incubated at 30 ° C for 140 hours until the formation of conidia. Then the transformants are fermented in rocking flasks using an aqueous fermentation medium of the following composition, in g / l: microcrystalline cellulose (MCC) - 40, wheat bran - 10, yeast extract - 10, (NH ₄ ) ₂ SO ₄ - 5, KH ₂ PO ₄ - 5, MgSO ₄ × 7H ₂ O - 0,3, CaCl ₂ × 2H ₂ O - 0,3, pH 4.5. Flasks were incubated on a shaker at 32 ° C and 200 rpm for 144 h. Next, transformants with increased cellobiohydrolase activity compared to the recipient P.verruculosum strain were selected. Cellobiohydrolase activity in the culture fluid is measured by hydrolysis of the MCC.

An analysis in DDS electrophoresis under denaturing conditions revealed an increase in the size of the band corresponding to the zone occupied by cellobiohydrolase I (66 kDa).

At the third stage, the most productive of the transformed P.verruculosum PV-Cbh1P (BKM F-3973D) strains is fermented in a 10-liter fermenter using a nutrient medium of the same composition as in the rocking flasks and feeding with glucose at pH 4.5 and 32 ° C for 144 hours. After fermentation by ultrafiltration of the culture fluid on hollow fibers (with a cutoff limit of 10 kDa) and subsequent freeze drying, dry enzyme preparations are obtained and characterize them by the level of cellobiohydrolase activity.

The activity of cellobiohydrolase in the culture fluid of the most active transformant P.verruculosum PV-Cbh1P (BKM F-3973D) is 35 units / ml, the activity of cellobiohydrolase in a dry preparation is 1200 units / g.

Cellobiohydrolase I has a high specific hydrolytic (saccharifying) activity with respect to insoluble crystalline cellulose substrates (see table).

The specific activity of cellobiohydrolases in relation to microcrystalline cellulose (50 ° C, pH 5), units / mg protein Microorganism Enzymes Cellobiohydrolase I Cellobiohydrolase II Penicillium verruculosum 0.4 0.5 Trichoderm longibrachiatum 0.2 0.3

Example 3. Obtaining enzyme preparations with endoglucanase activity during the fermentation of multicopy P.verruculosum strains using the eglIV gene of Tr.longibrachiatum (endo-β1,4-glucanase IV gene, 61st glycoside hydrolase family, molecular weight 36 kDa).

At the first stage, a DNA fragment with the nucleotide sequence of eglIV gene encoding Tr.longibrachiatum endoglucanase IV is obtained and the expression plasmid pPrCBHI-EGIV_Trich is obtained. For amplification of the eglIV gene, a pair of primers is synthesized:

(10) 5'-CAAACAGAAGCAACCGACACAATGATCCAGAAGCTTTCCAACCTC-3 '

(11) 5'-GAGGAGAAGCCCGGTTAGTTAAGGCACTGGGCGTAGTAG-3 '

Oligonucleotides (10) and (11) are synthesized to construct the plasmid pPrCBHI-EGIV_Trich. Tr.longibrachiatum genomic DNA is used as a template for primary PCR reactions. Since Tr.longibrachiatum is a variant of Tr.reesei, the gene sequence from the cDNA of endoglucanase IV from Tr.reesei, available in the EMBL database (AN: Y 11113), was taken as the basis for creating primers. The nucleotide sequence of the endoglucanase IV gene from Tr.longibrachiatum consists of 1086 base pairs and contains 1 intron (Fig. 3)

Plasmid pPrCBHI-EGIV_Trich contains all the structural elements necessary for expression in P.verruculosum: the cbhI promoter and cbhI terminator. In this construct, a full-length Tr.longibrachiatum endoglucanase IV gene is cloned, including a signal peptide coding sequence.

A schematic map of the expression plasmid pPrCBHI-EGIV_Trich shown in Fig.4.

At the second stage, cotransformation of the recipient strain P.verruculosum 537 (niaD ^- ) 10-20 μg of the expression vector plasmid pPrCBHI-EGIV_Trich and 1-3 μg of the marker plasmid pSTA (niaD ⁺ ) is performed and clones are screened for transformants with increased endoglucanase secretion.

The obtained niaD ⁺ P.verruculosum transformants are subcultured on plates with minimal medium and a source of nitrogen NaNO ₃ , incubated at 30 ° C for 140 hours until conidia is formed. Then, P.verruculosum transformants are fermented in rocking flasks as described in Example 2, and then transformants with increased endoglucanase activity compared to the recipient P.verruculosum strain are selected. Endoglucanase activity in the culture fluid is measured by hydrolysis of the Na salt of carboxymethyl cellulose (CMC).

An analysis in DDS electrophoresis under denaturing conditions revealed that endoglucanase IV occupies a zone corresponding to 36 kDa.

At the third stage, the most productive of the transformed P.verruculosum PV-Egl4T strain (BKM F-3976D) is fermented in a 10-liter fermenter (as described in Example 2).

The activity of endoglucanase in the culture fluid of the strain P.verruculosum PV-Egl4T (BKM F-3976D) at the end of the fermentation is 850 u / ml.

A dry enzyme preparation is obtained by ultrafiltration of a culture fluid on hollow fibers (with a cutoff limit of 10 kDa) and subsequent freeze drying. The activity of endoglucanase in the dry enzyme preparation of the P. verruculosum PV-Egl4T transformant (BKM F-3976D) is 6500 u / g.

Example 4. Obtaining enzyme preparations with xylanase activity in the fermentation of P.verruculosum multicopy strains of the xynIII Tr.longibrachiatum gene (endo-β1,4-xylanase III gene, 10th glycoside hydrolase family, molecular weight 30 kDa).

At the first stage, a DNA fragment is obtained with the nucleotide sequence of the xynIII gene encoding Tr.longibrachiatum xylanase III and the expression plasmid pPrCBHI-XylIII_Trich. To amplify the xynIII gene, a pair of primers is synthesized:

(36) 5'-CAAACAGAAGCAACCGACACAATGAAAGCAAACGTCATCTTGT-3 '

(37) 5'-GAGGAGAAGCCCGGTTATTGTAAGATGCCAACAATGCT-3 '

Tr.longibrachiatum genomic DNA is used as a template for primary PCR reactions. Since Tr.longibrachiatum is a variant of Tr.reesei, the gene sequence from xylanase III cDNA from Tr.reesei, available in the GeneBank database (AN: AB036796), is used as the basis for writing primers. The nucleotide sequence of the xylanase gene from Tr.longibranchiatum consists of 1349 base pairs, contains 2 introns and is completely identical to the sequence of the xylanase gene from Tr.reesei (figure 5).

Plasmid pPrCBHI-XylIII_Trich contains all the structural elements necessary for expression in P.verruclosum: the cbhI promoter and cbhI terminator. In this construct, the full-length Tr.longibrachiatum xylanase III gene is cloned, including the signal peptide coding sequence. A schematic map of the expression plasmid pPrCBHI-XylIII_Trich shown in Fig.6.

At the second stage, cotransformation of the recipient strain P.verruculosum 537 (niaD ^- ) 10-20 μg of the expression vector plasmid pPrCBHI-XylIII_Trich and 1-3 μg of the marker plasmid pSTA (niaD ⁺ ) is carried out and clones are screened for transformants with increased xylanase secretion.

The obtained niaD ⁺ P.verruculosum transformants are subcultured on plates with minimal medium and a source of nitrogen NaNO ₃ , incubated at 30 ° C for 140 hours until conidia is formed.

Then, P.verruculosum transformants are fermented in rocking flasks as described in Example 2, and then transformants with xylanase activity increased compared to the recipient P.verruculosum strain are selected. Xylanase activity in the culture fluid is measured by hydrolysis of birch xylan.

An analysis in DDS electrophoresis under denaturing conditions revealed that xylanase III occupies a zone corresponding to 30 kDa.

At the third stage, the most productive of the transformed P.verruculosum PV-Xyl3T strain (VKM F-3979D) is fermented in a 10-liter fermenter (as described in Example 2).

The xylanase activity in the culture fluid of P.verruculosum PV-Xyl3T strain (BKM F-3979D) at the end of fermentation is 1950 u / ml.

A dry enzyme preparation is obtained by ultrafiltration of a culture fluid on hollow fibers (with a cutoff limit of 10 kDa) and subsequent freeze drying. The xylanase activity in the dry enzyme preparation of the P. verruculosum PV-Xyl3T transformant (In KM F-3979D) is 43600 units / g.

Methods for determining activity. Cellobiohydrolase (avicelase) activity is measured by hydrolysis of the MCC (5 mg / ml) at pH 5.0 (0.1 M acetate buffer) and 50 ° C for 60 minutes. After that, the concentration of reducing sugars (BC) in the reaction mixture is determined by the Nelson-Somogy method. A unit of cellobiohydrolase activity is taken to be such an amount of an enzyme that for 1 minute at a temperature of 50 ° C and a pH of 5.0 when hydrolyzing a 5 mg / ml MCC suspension liberates the amount of BC equivalent to 1 micromole of glucose as determined by the Somogy-Nelson method.

Endoglucanase (CMCase) activity is measured by hydrolysis of CMC (5 mg / ml) at pH 5.0 (0.1 M acetate buffer) and 50 ° C for 10 minutes. The unit of endoglucanase activity is taken to be such an amount of an enzyme that for 1 minute at a temperature of 50 ° C and a pH of 5.0 upon hydrolysis of a CMC solution with a concentration of 5 mg / ml leads to the formation of an amount of BC equivalent to 1 micromole of glucose, determined by the Somogy-Nelson method ( A.P. Sinitsyn, A.V. Gusakov, I.M. Chernoglazov. Bioconversion of lignocellulosic materials, Textbook. - M.: Publishing House of Moscow State University, 1995, p.144-156).

Xylanase activity is measured by hydrolysis of birch xylan (5 mg / ml) at pH 5.0 (0.1 M acetate buffer) and 50 ° C for 10 minutes. A unit of xylanase activity is taken to be such an amount of an enzyme that for 1 minute at a temperature of 50 ° C and a pH of 5.0 upon hydrolysis of a solution of xylan at a concentration of 5 mg / ml leads to the formation of the amount of BC equivalent to 1 micromole of xylose, determined by the Somogy-Nelson method ( A.P. Sinitsyn, A.V. Gusakov, I.M. Chernoglazov. Bioconversion of lignocellulosic materials, Textbook. - M.: Moscow State University, 1995, p.144-156).

Thus, the present invention allows the production of enzyme preparations with high cellobiohydrolase, endoglucanase and xylanase activities by culturing multicopy strains of the mycelial fungus Penicillium verruculosum transformed with DNA fragments with cloned homologous and heterologous genes.

Claims (6)

1. A genetic construct for the expression of target homologous and heterologous enzyme genes in the cells of the mycelial fungus Penicillium verruculosum used as the host, containing the target coding sequence of the enzyme functionally linked to the regulatory elements of the Penicillium verruculosum cellobiohydrolase gene I, which are the promoter and terminator of the cellobiohydrolase gene I, and the signal peptide in this case depends on what the target enzyme is. 2. A method of obtaining a strain of the fungus Penicillium verruculosum, a producer of the enzyme encoded by the nucleotide sequence, comprising transforming the cell of the fungus Penicillium verruculosum with a genetic construct containing the target coding sequence of the enzyme functionally linked to the regulatory elements of the Penicillium verruculosum cellobiohydrolase gene I, which are the promoter and gene promoter and promoter I, and the signal peptide in this case depends on what the target enzyme is. 3. A method for producing an enzyme, preferably cellulase or xylanase, which breaks down cellulose or xylans, respectively, by cultivating microorganisms, characterized in that the method according to claim 2 produces a strain of the fungus Penicillium verruculosum, the resulting strain is cultured and the target product is isolated from the fungal culture fluid. 4. The method of producing the enzyme according to claim 3, characterized in that a strain of Penicillium verruculosum PV-CbhIP (BKM F-3973D) containing multiple copies of the cbhI gene, a highly active cellobiohydrolase I Penicillium verruculosum, is obtained. 5. The method for producing the enzyme according to claim 3, characterized in that a strain of Penicillium verruculosum PV-Egl4T (BKM F-3976D) containing multiple copies of the eglIV gene, endoglucanase IV Trichoderma longibrachiatum, is obtained. 6. The method for producing the enzyme according to claim 3, characterized in that a strain of Penicillium verruculosum PV-Xyl3T (BKM F-3979D) containing multiple copies of the xylIII gene - xylanase III Trichoderma longibrachiatum is obtained.

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