CN112048444A - Laccase-producing Candida utilis expression vector, construction method, recombinant engineering bacteria and application thereof - Google Patents

Abstract

The invention provides a candida utilis yeast expression vector for producing laccase, a construction method thereof, recombinant engineering bacteria and application thereof. The candida utilis yeast expression vector for producing laccase comprises 18S rDNA gene fragments, yeast glyceraldehyde triphosphate dehydrogenase gene promoter sequences (GAP-p), laccase genes, yeast glyceraldehyde triphosphate dehydrogenase gene terminator sequences (GAP-t) and Cycloheximide (CYH) resistance genes. All genetic elements of the whole expression system come from the probiotics Candida utilis and nontoxic and harmless fungi, do not carry antibiotic resistance genes, do not have drifting, diffusion and integration of the resistance genes, do not carry other toxic protein genes, do not bring biological safety hazards to the environment or people and animals, reach food grade, and can be directly added into food, medicines and feeds for application.

Description

Laccase-producing Candida utilis expression vector, construction method thereof, and recombinant engineering bacteria and its application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a laccase-producing Candida utilis expression vector, a construction method thereof, a recombinant engineering bacterium and applications thereof.

Background technique

Inner Mongolia is rich in straw resources, with an annual output of 3.5 billion kilograms of various crop straws, which is equivalent to twice the amount of grass trimmed on natural grasslands in the region. At present, the vast majority of straw is still fed to livestock by simple crushing, pressing into pellets and making silage. The apparent digestibility of dry matter (DM) in simple processed corn stover was 63.38%, the apparent digestibility of neutral detergent fiber (NDF) was 62.06%, and the apparent digestibility of acid detergent fiber (ADF) was 62.06%. 50.36%. The apparent digestibility of neutral detergent fiber in simple crushed wheat straw by beef cattle was 56.87%, and the apparent digestibility of acid detergent fiber was 49.04%. That is to say, in the straw feed, at least nearly 37% of dry matter, 38% to 46% of neutral detergent fiber, and 49% to 51% of acid detergent fiber are lost through feces. my country's corn planting area has continued to increase rapidly. With the increase in corn yield, a large amount of corn stalk is produced, but less than 20% of the corn stalk is effectively processed. Therefore, the key technology to overcome the digestion and utilization rate of straw feed is an effective way to solve the shortage of roughage resources and low utilization rate in animal husbandry production in our region, reduce the cost of breeding, and increase the number of cattle and sheep raised and the economic benefits of breeding.

Due to the low utilization rate of straw nutrients, poor palatability and low utilization value, a large amount of straw is directly returned to the field or incinerated every year, and only a small part is used as feed, which not only causes resource waste, but also pollutes the environment. One of the reasons for the low utilization rate of crop straw as forage grass is that as the crops mature, the degree of lignification is also higher, and the lignin content in the cell wall of the straw also increases.

The basic structural unit of lignin is phenylpropane, which is connected by ether bonds and carbon-carbon bonds to form complex amorphous polymers. Typical lignin is composed of three different alcohols, coniferyl alcohol, sinapyl alcohol and p-coumarol, as precursor substances. Lignin can be tightly cross-linked with hemicellulose molecules to form a hydrophobic network structure, and the entire cell wall becomes a tight network, which increases the mechanical strength of the cell wall and the resistance to microorganisms. At the same time, it also hinders the contact of ruminant microbial hydrolase with cellulose and hemicellulose in the cell wall, thereby reducing the degradation efficiency of cellopolysaccharide. Therefore, lignin is considered to be the main limiting factor inhibiting straw utilization. The lignin content in corn stover can reach 19% to 23%, which directly affects the utilization efficiency of corn stover. The second reason is: acid treatment, alkali treatment, steam explosion, hot water treatment, ammonia fiber explosion, extrusion pretreatment, microwave pretreatment, etc. have been developed and applied. Although these pretreatment methods improve the utilization rate of straw, their The high equipment cost restricts the practical application of straw.

So far, only a few microorganisms can degrade lignin, most of them are fungi, and a few are bacteria, but the latter have only weak lignin degradation ability. Because white rot fungi can efficiently and selectively degrade lignin, they are considered to be the most promising fungi for lignin removal. White rot fungi are known to secrete several major classes of peroxidases involved in lignin degradation: laccase (Laccase; EC 1.10.3.2), manganese peroxidase (MnP; EC 1.11.1.13) (these two enzymes were first described in found in P. chrysogenum), multifunctional peroxidase (first found in Pleurotus eryngii), lignin peroxidase (LiP; EC 1.11.1.14) and various auxiliary enzymes. Some white rot fungi produce the above 3 enzymes that decompose lignin, while most white rot fungi produce only 2 or even 1 enzyme that decomposes lignin, thus indicating that the above 4 enzymes are not involved in the process of decomposing lignin. All are required. Studies have shown that lignin peroxidase, laccase, and manganese peroxidase can all work together with low-molecular-weight mediators, that is, the enzymes produce reaction intermediates that have the ability to attack the structure of lignin, and indirectly cause the oxidation of lignin. reaction, resulting in the decomposition of lignin, which has been proposed as a degradation mechanism of lignin.

White rot fungi have a long growth cycle and low enzyme activity, which are not conducive to industrial production. In the protein expression system, the high efficiency of heterologous host expression can better meet the needs of industrial production. The researchers hope to use genetic engineering technology to increase the production of peroxidase, so they cloned and expressed multiple peroxidase genes of white rot fungi in heterologous hosts. A laccase gene with a full length of 1680 bp was synthesized by homologous cloning technology using P. chrysosporium as a template. The gene was expressed in Escherichia coli, and the laccase activity was nearly 39% higher than that of the original fungus. Sun Yafan expressed the lignin peroxidase gene in Pichia pastoris (Methanol yeast), and the lignin peroxidase activity increased by 122.1%. Wang Jiao expressed the manganese peroxidase gene in Pichia pastoris, and the lignin degradation rate was increased by 31.38%. Since the expression system of Pichia pastoris is relatively mature, there are many studies on the expression of lignin peroxidase gene in this yeast. However, Pichia pastoris is not a food-grade microorganism, and its applications are limited.

SUMMARY OF THE INVENTION

To this end, the present invention provides a laccase-producing Candida utilis genetically engineered bacterium and application thereof, which solves the problems in the prior art that microbial strains heterologously expressing lignin-degrading enzymes do not meet food-grade requirements and are limited in application. technical problem.

In order to solve the above technical problems, the present invention provides a laccase-producing Candida utilis expression vector, comprising 18S rDNA gene fragment-yeast glyceraldehyde triphosphate dehydrogenase gene promoter sequence (GAP-p)-laccase Gene - yeast glyceraldehyde triphosphate dehydrogenase gene terminator sequence (GAP-t) - cycloheximide (CYH) resistance gene.

Preferably, the nucleotide sequence of the laccase gene is SEQ ID No.1.

The present invention also provides a method for constructing a laccase-producing Candida utilis expression vector, comprising the following steps:

(1) Cloning of the laccase (Lac) gene: Extract the total RNA from Trametes yunzhi, and carry out RT-PCR amplification with the extracted total RNA as a template to obtain the target gene and connect it with the pMD19-T vector, transform and extract, to obtain a recombinant plasmid;

(2) Cloning of yeast glyceraldehyde triphosphate dehydrogenase gene (GAP) promoter (GAP-p) and terminator (GAP-t): According to the GenBank database of Candida utilis (C. utilis) GAP-p GAP -t gene sequence combined with pBR322 plasmid sequence features to design primers, using Candida utilis genome as a template, the yeast GAP promoter (GAP-p) fragment and GAP terminator (GAP-t) fragment were obtained by PCR, and the target fragment GAP -p and GAP-t were respectively connected to the vector pMD19-T simple vector to obtain plasmid vectors pT-GP and pT-GT, which were transformed into Escherichia coli, and the positive clones were identified by bacterial liquid PCR and stored for future use;

(3) Cloning of cycloheximide (CYH) resistance gene: Design primers according to the sequence of Candida utilis (C. utilis) CYH sensitive gene (L41) in the GenBank database combined with the sequence characteristics of pBR322 plasmid, and a pair of reverse complements Primers; take the Candida utilis genome as the template, PCR amplify the upstream fragment and downstream fragment of L41, and one end of these two fragments has a base overlapping region; take the two upstream fragments and downstream fragments as templates, and carry out nesting. The CYH resistance gene was obtained by stacking PCR; the target fragment was connected to the vector pMD19-T simple vector to obtain the plasmid vector pT-CYH, transformed into Escherichia coli, and the positive clone was identified by bacterial liquid PCR and stored for subsequent use;

(4) Cloning of the 18S rDNA gene fragment: According to the Candida utilis 18S rDNA gene sequence in the GenBank database combined with the characteristics of the pBR322 plasmid sequence, primers were designed, and the Candida utilis genome was used as the template to obtain 18S rDNA by PCR. Fragment, connect the target fragment to the carrier pMD19-T simple vector to obtain the plasmid vector pT-rD, transform Escherichia coli, the positive clone is identified by bacterial liquid PCR and stored for future use;

(5) Construction of homologous integration expression vector: extract pT-GP, pT-GT and pBR322 plasmids, carry out enzyme digestion respectively, recover the target fragment, and use T4 DNA ligase to connect these 3 fragments at the same time to obtain plasmid pBR-GAP; The pT-CYH and pBR-GAP plasmids were extracted and digested respectively, and the target fragments were recovered and ligated with T4 DNA ligase to obtain the plasmid pBR-G-L; the pBR-G-L plasmid and the laccase (Lac) gene recombinant plasmid were extracted, and enzyme Cut, recover the target fragment, ligate with T4 DNA ligase to obtain the plasmid pBR-G-Q-L; extract the pT-rD and pGQL plasmids, carry out digestion respectively, recover the target fragment, ligate with T4 DNA ligase, and obtain the laccase-producing product. Candida prion expression vector pGQLR.

Preferably, in the cloning of the yeast glyceraldehyde triphosphate dehydrogenase gene (GAP) promoter (GAP-p) and terminator (GAP-t), the primers are designed as follows:

Promoter (GAP-p):

GAP-p1:GGATATCTTTACAGCGAGCACTCAA;

GAP-p2:GCTCTAGAATGTTGTTTGT;

Terminator (GAP-t):

GAP-t1: CTAGCTAGCTATGACTTTTAT;

GAP-t2: GGGATCCTTCATTCATCCCTCACTATCG.

Preferably, in the cloning of the cycloheximide (CYH) resistance gene, the primers are designed as follows:

P4:CGTCGACAGTAAGTATGAAAAGAGC;

RM4:GGGATCCGGTTTGGTCTATGTTGCT;

A Sal I restriction site was introduced into primer P4, and a BamHI restriction site was introduced into primer RM4;

The reverse complementary primers were designed as follows:

P5: AACCAAGCAAGTTTTCCAC;

R5: GTGGAAAACTTGCTTGGGTT.

Preferably, in the cloning of the 18S rDNA gene fragment, the primers are designed as follows:

P3:CGATATCTGCCAGTAGTCATATGC;

R3:CGATATCTGACTTGCGCTTTACTAG;

An EcoR V restriction site was introduced into primer P3, and an EcoR V restriction site was introduced into primer R3.

The present invention also provides a laccase-producing Candida utilis genetically engineered bacterium. The Candida utilis expression vector pGQLR containing the laccase gene is re-spliced to obtain a Candida utilis recombination with the sequence ZHQX1 The recombinant yeast strains were obtained by electrotransformation.

Preferably, primer design is carried out using the pGQLR vector as a template, the fragment sequence is divided into two fragments, and the sequence obtained after re-splicing is ZHQX1, and the designed primers are as follows:

Preferably, the prokaryotic DNA sequence is deleted from the Candida utilis expression vector pGQLR, and the DNA sequence includes the DNA fragment of the bacterial plasmid sequence including the drug resistance marker Amp.

The invention also provides the application of the laccase-producing Candida utilis genetically engineered bacteria in food, pharmacy and livestock and poultry feed.

Beneficial effects:

The laccase-producing Candida utilis genetically engineered bacteria provided by the present invention has all the genetic elements of the entire expression system from the probiotic Candida utilis and non-toxic and harmless fungi, does not carry antibiotic resistance genes, does not There is drift, diffusion and integration of resistance genes, and it does not carry other toxic protein genes, which will not cause biological safety hazards to the environment or humans and animals. It has reached food grade and can be directly added to food, medicine and feed. application. The laccase activity expressed by the Candida utilis engineered bacteria was 10 times higher than that of Trametes prototheca.

Description of drawings

Fig. 1 is the schematic diagram of the identification result of pGQL digestion product according to the embodiment of the present invention;

Fig. 2 is the expression vector pGQLR map constructed by the present invention;

Fig. 3 is the schematic diagram of the identification result of pGQL R enzyme cleavage product according to the embodiment of the present invention;

4 is a schematic diagram of the results of PCR electrophoresis after deletion of prokaryotic sequences in the Candida utilis integrated expression vector according to the embodiment of the present invention;

Figure 5 is a schematic diagram of the identification result of the PCR product of the Candida utilis recombinant ZHQX1 genome according to the embodiment of the present invention;

6 is a schematic diagram of PCR electrophoresis results for detecting the genetic stability of exogenous genes according to the embodiment of the present invention;

FIG. 7 is a schematic diagram of the detection result of the recombinant yeast (ZHQX1) laccase protein by SDS-PAGE of the present invention.

Detailed ways

In order to describe in detail the technical content, achieved objects and effects of the present invention, the following descriptions are given in conjunction with the embodiments.

The reagents and raw materials used in this manual are all commercially available products unless otherwise specified.

The laccase gene of the present invention is derived from Trametes versicolor: BNCC190652.

Example 1

The present embodiment provides a laccase-producing Candida utilis genetically engineered bacterium, comprising:

1. Construct an expression vector containing laccase gene (LAC);

2. Deletion of DNA fragments from prokaryotic microorganisms in the integrated expression vector of Candida utilis;

3. After deletion of prokaryotic DNA sequence, transform into yeast strain.

The specific process is as follows:

1 Construction of expression vector containing laccase gene (LAC)

1.1 Cloning of the laccase gene

Extract total RNA from Trametes versicolor (refer to TIANGEN RNA extraction kit method); take the extracted total RNA from Trametes versicolor as a template, and use BCabest RNA PCR amplification kit for RT-PCR amplification increase. The primer sequences are as follows:

LAC-R1: CAACATGTCGAGGTTTCACTCTC;

LAC-R2: CCATTTACTGGTCGCTGGGGT.

Use E.L.N.ATm Gel Extraction Kit (D2500-01) gel recovery kit to perform gel recovery of RT-PCR products; ligate the target gene with pMD19-T Vector (Treasure Bio) vector; transform the ligated product into Trans-5α (full formula) Gold) competent cells, the recombinant plasmid was obtained for PCR identification, and the nucleotide sequence of the laccase gene was shown in SEQ ID NO.1.

1.2 Cloning of yeast glyceraldehyde triphosphate dehydrogenase gene (GAP) promoter (GAP-p) and terminator (GAP-t)

According to the yeast GAP-p and GAP-t gene sequences published in GenBank combined with the sequence characteristics of pBR322 plasmid, the corresponding primers were designed and appropriate restriction sites were introduced. The primers are used for PCR (completed with a kit) to obtain yeast GAP promoter (GAP-p) fragment and GAP terminator (GAP-t) fragment. The recovered target fragment was connected to the vector pMD19-T simple (Bao Bio) vector (named as pT-GP, pT-GT respectively), the ligation product was transformed into DH5α (full gold) competent cells, and the positive clone was identified by bacterial liquid PCR .

The primer sequences are as follows:

The nucleotide sequence of the cloned promoter sequence GAP-p is shown in SEQ ID NO.2.

The nucleotide sequence of the cloned terminator sequence GAP-t is shown in SEQ ID NO.3.

PCR amplification of GAP-p, GAP-t genes:

The connection system of the target gene and pMD19-T simple (Bao Bio) vector:

After ligation at 16°C overnight, the ligation product was transformed into DH5α (full gold) competent cells.

PCR identification of recombinants

1.3 Cloning of cycloheximide (CYH) resistance gene (using site-directed mutagenesis)

The corresponding primers were designed according to the sequence of CYH sensitive gene (L41) in yeast published in GenBank combined with the sequence characteristics of pBR322 plasmid, and appropriate restriction sites were introduced, and a pair of reverse complementary primers were designed at the same time. First, using the C. utilis genome as a template, the upstream fragment and downstream fragment of L41 were amplified by PCR, and one end of these two fragments had a base overlap region. The CYH resistance gene can be obtained by using the same amount of the two upstream fragments and the downstream fragments as templates, and performing nested PCR. The recovered target fragment was ligated to the vector pMD19-T simple vector (named pT-CYH) and transformed into E. coli, and the positive clones were identified by bacterial liquid PCR.

Primers are designed as follows:

Using yeast genome as template and P4/R5 and P5/RM4 as primers, PCR experiments were carried out.

Reaction conditions:

PCR experiments were performed using the two groups of recovered products as templates:

PCR reaction conditions were the same as above.

The connection system of the target gene and the pMD19-T Vector vector:

Mix gently, centrifuge briefly, and ligate overnight at 16°C. The ligation product was transformed into Trans-5α (full gold) competent cells.

10 monoclonal cells were picked with the inoculating loop and identified by PCR of the bacterial liquid.

The PCR reaction program is the same as above.

The nucleotide sequence of the cloned cycloheximide resistance gene CYH is shown in SEQ ID NO.4.

1.4 Cloning of 18S rDNA gene fragments

The corresponding primers were designed according to the yeast 18S rDNA gene sequence published in GenBank combined with the sequence characteristics of the pBR322 plasmid, and appropriate restriction sites were introduced. Using the C. utilis genome as a template, PCR was performed with the corresponding primers to obtain 18S rDNA fragments. The recovered target fragment was connected to the vector pMD19-T simple vector (named pT-rD) to transform E. coli, and the positive clones were identified by bacterial liquid PCR.

The 18SrDNA gene primers were designed as follows:

PCR amplification reaction system (25μL):

Reaction conditions:

The nucleotide sequence of the cloned 18S rDNA gene fragment is shown in SEQ ID NO.5.

1.5 Construction of pBR322-GAP (pBR-GAP) vector:

GAP-p, GAP-t and pBR322 plasmids were digested with EcoRV/XBa I, XBa I/BamH I and EcoRV/BamH I, respectively, and the target fragments were recovered. Using the 3-fragment ligation method, the 3 fragments were simultaneously ligated with T4 DNA ligase to obtain the plasmid pBR-GAP.

The GAP-p digestion system is as follows:

The GAP-t digestion system is as follows:

The digestion system of PBR322 is as follows:

The gel-recovered and purified target fragment and the vector are ligated by T4 DNA ligase. The 10 μL ligation reaction system is as follows:

After centrifugation and mixing, the ligation product was transformed into DH5α competent cells in a water bath at 16°C overnight.

Pick 10 monoclonal cells and conduct PCR amplification. The reaction system and conditions are as follows:

The bacterial liquid was picked to identify positive clones, placed in a conical flask containing 5 ml of LB medium (+AMP), and cultured at 200 rpm at 37°C overnight. Then the plasmid was extracted and identified by EcoRV and BamHI digestion. The digestion system was as follows:

Digested at 37°C for 2 h, and extracted the correctly digested plasmid for sequencing.

1.6 Construction of pBR322-GAP-L41 (pBR-G-L) vector

The pT-CYH and pBR-GAP plasmids were extracted and digested with the corresponding enzymes introduced into the restriction sites, respectively, and the target fragments were recovered and ligated with T4 DNA ligase to obtain the plasmid pBR-G-L.

The CYH digestion system is as follows:

The pBR-GAP digestion system is as follows:

The gel-recovered and purified target fragment and the vector are ligated by T4 DNA ligase. The 10 μL ligation reaction system is as follows:

After centrifugation and mixing, ligation was performed in a water bath at 16°C overnight, and the ligation product was transformed into DH5α competent cells.

Pick 10 monoclonal cells and conduct PCR amplification. The reaction system and conditions are as follows.

Pre-denaturation at 94°C for 5min, denaturation at 94°C for 30sec

Annealing at 56 °C for 30 sec, extension at 72 °C for 2 min 30 sec for a total of 30 cycles. Extend for 10 min at 72°C.

The bacterial liquid was picked to identify positive clones, placed in a conical flask containing 5 ml of LB medium (+Amp), and cultured at 200 rpm at 37°C overnight. Then the plasmid was extracted for Sal I and BamH I digestion and identification, and the digestion system was as follows:

After digestion at 37°C for 2 h, the correct plasmid was extracted and sequenced and named PBR-G-L.

1.7 Construction of pBR322-GAP-L41-Q (pGQL) vector containing laccase gene (LAC) gene

1) extract the pBR-G-L plasmid, carry out enzyme digestion with XBa I/Nhe I, and recover the target fragment;

The pBR-G-L digestion system is as follows:

2) Cloning and digestion of Lac gene;

The clone system is as follows:

The enzyme digestion system is as follows:

3) The Lac gene fragment and the pBR-G-L digested fragment are ligated by T4 DNA ligase. The 10 μL ligation reaction system is as follows:

After centrifugation and mixing, ligation was performed in a water bath at 16°C overnight, and the ligation product was transformed into DH5α competent cells.

4) PCR identification of recombinant sub-bacteria

Pick 10 monoclonal cells and conduct PCR amplification. The reaction system and conditions are as follows.

5) Enzyme digestion identification

The bacterial liquid was picked to identify positive clones, placed in a conical flask containing 5 ml of LB medium (+AMP), and cultured at 200 rpm at 37°C overnight. Then extract the plasmid for XBa I and Nhe I digestion identification, and the digestion system is as follows:

After digestion at 37°C for 20 min, the plasmid with the correct digestion was extracted and sequenced, and named pBR-G-Q-L (pGQL). The digestion results are shown in Figure 1, where M is Trans 2K II Marker; 1 is the pGQL digestion product.

1.8 Construction of pBR322-GAP-L41-Q-R18 (pGQLR) vector

The pT-rD and pGQL were extracted and digested with the corresponding enzymes introduced into the restriction sites respectively. The target fragments were recovered and ligated with T4 DNA ligase to obtain the homologous integration expression vector (pGQLR). Escherichia coli DH5a was transformed, and the positive clones were identified by enzyme digestion and PCR after the plasmid was extracted.

The 18S rDNA digestion system is as follows:

The pGQL digestion system is as follows:

The gel-recovered and purified target fragment and the vector are ligated by T4 DNA ligase. The 10 μL ligation reaction system is as follows:

After centrifugation and mixing, ligation was performed in a water bath at 16°C overnight, and the ligation product was transformed into competent cells.

Pick 10 monoclonal cells and conduct PCR amplification. The reaction system and conditions are as follows.

The plasmid was identified by EcoRV digestion, and the digestion system was as follows:

Digested at 37°C for 15 min, extracted the correct plasmid for sequencing, named pGQLR, and the expression vector map is shown in Figure 2; the identification result of the digestion product is shown in Figure 3, M is 1kb Marker; 1 is the pGQLR digestion product .

2. Deletion of DNA fragments from prokaryotic microorganisms in the integrated expression vector of Candida utilis

The primer design was carried out with the pGQLR vector as the template, the fragment sequence was divided into two fragments, and the DNA sequence from the prokaryotic (including the DNA fragment of the bacterial plasmid sequence including the drug resistance marker Amp) was deleted by the overlap extension PCR experimental technology. After re-splicing The sequence was named ZHQX1.

The designed primers are as follows:

P1-1S, P1-1AS, P2-1S, P2-1AS were used as primers to carry out two groups of PCR experiments A and B respectively.

The system is:

PCR reaction conditions:

PCR experiments were carried out with two groups of recovered products A and B as templates.

Detected by 1% agarose gel electrophoresis and observed by UV gel imaging system, as shown in Figure 4, M is Trans2K II Marker; QX is the result of PCR electrophoresis after deletion of prokaryotic sequences.

3. After deletion of prokaryotic DNA sequences, transformation into yeast strains

The integrated expression vector (ZHQX1) in which the prokaryotic DNA sequence has been deleted was electroporated to construct a recombinant yeast strain.

Competent preparation: 5ul of Candida utilis was added to a 100ml shake flask containing 3ml of YPD medium (containing glucose), and the bacteria were shaken at 30°C and 100rpm overnight (OD600 was about 6-10). Add 100 μl of bacterial solution from the previous step to a 250 ml shake flask containing 10 ml of YPD medium (containing PTT), and cultivate at 100 rpm and 30° C. until the OD value is 1.0-1.3.

Preparation of treatment solution (1ml):

Aliquot 1 ml of the overnight culture into 1.5 ml EP tubes, centrifuge at 4°C, 10000 g, 1 min, and discard the supernatant. The pellet was washed with sterile water pre-cooled at 4°C, centrifuged under the same conditions, and the supernatant was discarded.

Bacterial treatment: add 1 ml of treatment solution and place at room temperature for 20 min. Centrifuge (same conditions) and discard the supernatant.

1ml of 1M Sorbitol was added, centrifuged (same conditions), and the supernatant was discarded.

Wash twice with 1M Sorbitol to a final volume of approximately 80ul.

Exogenous gene transformation:

Add 1ug exogenous gene, mix well and transfer to a pre-cooled (in ice bath) electric shock cup. 1.5Kv, 25uF, 200Ω for electric transfer. Immediately after shock, add 1 ml of pre-chilled 1M Sorbitol and suspend gently. Aspirate and transfer into a 1.5ml EP tube. Incubate at 28°C for 2h. After centrifugation and concentration, all were spread on YPD solid plate (YPD+PTT+CYH). Incubate at 28°C in the dark. 2-3d start counting and picking out transformants.

The identification results of the Candida utilis recombinant ZHQX1 genome PCR products are shown in Figure 5, M is the DL2000 marker; 1-6 are the Candida utilis recombinant ZHQX1 genome PCR products; 7 is the pGQLR plasmid PCR product (positive control); 8 is the genome PCR product of Candida utilis without the transformed plasmid (negative control 1); 9 is the water PCR product (negative control 2).

Detection of Candida utilis genetically engineered bacteria prepared in this example

1 Enzyme activity assay

Material:

Trametes versicolor: BNCC190652, purchased from Beina Chuanglian Biotechnology Co., Ltd.

pGQLR: This laboratory builds preservation.

Culture medium and reagents:

(1) Enzyme activity medium (g/L):

Glucose: 10g

Ammonium tartrate: 0.1g

KH ₂ PO ₄ : 0.2g

MgSO ₄ .7H ₂ O: 0.5g

MnSO ₄ : 0.035g

CuSO4.5H ₂ O: 0.007g

Add water to 1L and sterilize at 121°C for 20min.

(2) PDA culture medium:

9.76g PDA

200ml water

Autoclave, set aside.

(3) Solution:

20mmol/L H ₂ O ₂ , 10mmol/L veratrol, 50mmol/L tartaric acid buffer (pH3), 50mmol/L succinic acid buffer (pH4.5), 0.4mmol/L guaiacol.

1.2 Methods

Determination of the enzyme activity of laccase: using the guaiacol method.

The buffer solution is usually sodium succinate solution with a final concentration of 50 mmol/L. The pH of the buffer solution was 4.5. The final concentration of the substrate guaiacol was 0.4 mmol/L. The reaction temperature was 30°C, and the reaction time was 30 min. The change in OD value at 465 nm was measured.

1.3 The results are shown in Table 1 below.

Table 1 OD value change at 465nm

(U/L)

It can be seen that the laccase gene cloned by the present invention has the activity of degrading lignin after being expressed by Candida utilis, which is significantly higher than that of the laccase gene derived from Trametes yunzhi.

2 Genetic stability assay of integrated genes

2.1 PCR detection of yeast genome

After the yeast recombinants were inoculated and subcultured with YPD (without CYH) liquid medium, the yeasts on the 1st and 15th days were selected to extract the yeast genome PCR to detect the target gene, and to determine the genetic stability of the exogenous gene.

2.2 Determination of genetic stability of foreign genes

The recombinant yeast genomic DNA was extracted, amplified by PCR with the primers of the target gene, and detected by agarose gel electrophoresis. The results showed that the target gene was stable. The results are shown in Figure 6: M is DL2000 marker; 1 is YPD (CYH-free) medium Yeast (Day15) grown in 2: positive control: PGMLR plasmid PCR product 3: bacterial negative control (1) Candida utilis genome PCR product of untransformed plasmid; 4: negative control (2) water PCR product, by The results showed that the target band was still contained when the yeast was subcultured for 100 generations, indicating that the exogenous gene was not lost, and high genetic stability was maintained during the subculturing process.

3 Detection of recombinant yeast (ZHQX1) laccase protein by SDS-PAGE

The total protein of ZHQX1 was extracted with Yeast Protein Extraction Reagent. The specific operation process is as follows:

① Take 1-5×10 ⁶ liquid yeast cells into a 1.5ml centrifuge tube, centrifuge at 8000g for 2 minutes at 4°C.

② Carefully remove the supernatant, slowly add 1 ml of ice-cold sterilized water to the pellet, and gently pipet repeatedly with a pipette to resuspend the pellet.

③ Centrifuge at 8000g for 2 minutes at 4°C. Carefully remove the supernatant to remove as much liquid as possible.

④Add 25 μL of Yeast Protein Extraction Reagent to the pellet, and gently pipet repeatedly with a pipette to resuspend the pellet.

⑤Put it in a 30°C water bath for more than 30 minutes, and gently shake the centrifuge tube 1 to 2 times during this period.

⑥ Take the centrifuge tube out of the 30°C water bath, centrifuge at 12,000g for 5 minutes at 4°C. Carefully remove the supernatant, add 25 μL of PBS solution and 25 μL of 2×Protein SDS PAGE Loading Buffer to the pellet, and vortex for 2 minutes to resuspend the pellet.

⑦ Take 12.5μL of the above suspension into a new 1.5ml centrifuge tube, incubate at 100°C for 10 minutes, and take 10μL for electrophoresis detection.

SDS-PAGE results are shown in Figure 7, lane M is the protein molecular weight standard; lanes 1, 2, 3, and 4 are four groups of proteins extracted from recombinant yeast (ZHQX1), wherein the protein pointed to by P is a laccase protein.

Candida utilis has been certified by the US FDA as a yeast that can be used as a food additive, which can be used in food, pharmaceutical industry and livestock and poultry feed. The present invention transforms the lignin degrading enzyme gene, that is, the laccase gene, into Candida utilis, and applies it to fermentation straw, which can destroy the encapsulation effect of lignin and expose cellulose and hemicellulose, which is beneficial to rumen microorganisms Degradation and utilization of straw feed; it can be used as a safe single-cell protein to improve the nutrient content of straw feed; its fermented products do not need to be separated and purified, which is time-saving and economical. The heterologous expression of lignin-degrading enzymes through Candida utilis is also to increase its yield and lay the foundation for industrial production.

Obviously, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation manner. For those of ordinary skill in the art, changes or modifications in other different forms can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. And the obvious changes or changes derived from this are still within the protection scope of the present invention.

sequence listing

<110> Inner Mongolia Autonomous Region Academy of Agriculture and Animal Husbandry

<120> Laccase-producing Candida utilis genetically engineered bacteria and its application

<160>5

<170> Patent-In 3.5

<210>1

<211>1571

<212> DNA

<213> Trametes versicolor

<400>1

caacatgtcg aggtttcact ctcttctcgc tttcgtcgtt gcttccctta cggctgtggc 60

ccacgctggt atcggtcctg tcgccgacct caccatcacc aacgcagcgg tcagccccga 120

cgggttttct cgccaggccg tcgtcgtgaa cggcggcacc cctggccctc tcatcaccgg 180

taacatgggg gaccgcttcc agctcaatgt catcgacaac ctcaccaacc acacgatgct 240

gaagagcacc agtattcact ggcacggttt cttccagaag ggcacgaact gggccgacgg 300

tcccgccttc atcaaccagt gcccgatctc atctggtcac tcgttcctgt acgacttcca 360

ggttcctgac caggctggca ccttctggta ccacagtcac ttgtccacgc agtactgtga 420

tggtctgagg ggtccgttcg ttgtttacga cccgaacgac ccagccgccg acttgtacga 480

cgtcgacaac gacgacactg tcattaccct cgtggattgg taccacgtcg ccgcgaagct 540

gggccccgcc ttccctctcg gcgccgatgc taccctcatc aacggtaagg gacgctcccc 600

cagcacgact accgcggacc tctctgttat cagcgtcact ccgggtaaac gctaccgttt 660

ccgcctggtg tccctgtcgt gcgaccccaa ctacacgttc agcatcgatg gtcacaacat 720

gacgatcatc gaaaccgact cgatcaacac ggcgcccctc gtcgtcgact ccattcagat 780

cttcgccgcc cagcgttact ccttcgtgct cgaggccaac caggccgtcg acaactactg 840

gattcgcgct aacccgaact tcggtaatgt cgggttcacc ggcggcatca actcggctat 900

cctccgctac gatggtgccg ctgccgtcga gcccaccacc acgcaaacca cttcgaccga 960

gccgctcaac gaggtcaacc tgcacccgct ggttgccacc gctgttcctg gctctcccgt 1020

tgcgggtggt gttgacctgg ccatcaacat ggcgttcaac ttcaacggca ccaacttttt 1080

catcaacggc gcgtctttca cgcccccgac cgtgcctgtc ctcctccaga tcatcagcgg 1140

cgcgcagaac gcgcaggacc tcctgccctc cggcagcgtc tactcgctcc cctcgaacgc 1200

cgacatcgag atctccttcc ccgccaccgc cgccgcccccc ggcgcgcccc accccttcca 1260

cttgcacggg cacgcgttcg cggtcgtccg cagcgccggc agcacggtct acaactacga 1320

caaccccatc ttccgcgacg tcgtcagcac ggggacgcct gcggccggtg acaacgtcac 1380

catccgcttc cgcaccgaca accccggccc gtggttcctc cactgccaca tcgacttcca 1440

cctcgaggcc ggcttcgccg tcgtgttcgc ggaggacatc cccgacgtcg cgtcggcgaa 1500

ccccgtcccc caggcgtggt ccgacctctg cccgacctac gacgcgctcg accccagcga 1560

ccagtaaatg g 1571

<210>2

<211>971

<212> DNA

<213> Artificial sequences

<400>2

ttacagcgag cactcaaatc tgccctccga gccctccggc cctctcttca acaaactcgc 60

gctgcacttc gtcgtcagtg gtgccaatca cccaacgtgg aggtatcaag aggtgctcca 120

gcccacaaag cgacatcaaa gacaacaacc ctgccggcat acgtcctaca caccctggtg 180

atcgcagaca ttgtacaagg tgccacgcaa taacctacag gcaccgcaca tgacgatggc 240

cttggttgtg caaccagtga cttccacggt ccacgcagca acatgaacca caccacccag 300

aatcgatgcg cgcaacaaca gttgttccgg ttcactcagc cccacagcga gtcgctggca 360

gaacacgagc ctgagggcgg aaagagggta gaggaaagcg caaggacagg ggacaacctg 420

gcccaattga tgtcatataa accctctcga tcaattgagc acactcatcc gccaattgac 480

ccctgttcgc agctccacgc cccatgttcc tcgtccctgg tgtagcttct cccctaaatt 540

ccagcgcttg gttccgccct ccctgtctcc cgggtttaac gaacgtgtgt accatctgat 600

ggtaatccgc tcccgtccgc gcaacacaac tcacaagcag atcacacctc tacacgccgc 660

tgctgatgcg cccaatttaa ttttttttttc tctcaatgta ggggagaagc cttgggagct 720

cccgactccc agttgggcac agctgccacc tcatgacttt tcctgtgtgt gcctgtctga 780

cgttacgtgt gatgtagtgg cccccgtccg gtgtgttttc gcctgttgcg ctgtgccccc 840

cttaaaagta taaaaggaag tgcaattgct gtttgtgttg attgttgatc cttgtttcct 900

ctgttttctc ctcatcacac aagaaaggtt tcttctttcc aacagataca aaacacactt 960

acaaacaaca t 971

<210>3

<211>447

<212> DNA

<213> Artificial sequences

<400>3

tatgactttt atttatggga ttacgttata aattatgatc ctcatggatt atcttattaa 60

gtctccatct tgtagcttgt aatatgatga acactcgtga gttttccagg taattcaccg 120

tgcctcgtcc atgcactttt atcagcctcg acgtcataca ttgcattgtg agtaactggt 180

aaacggcttt ttacgttctg ttgtatatgg ctaaacgctt ctatggcacg gcgctattaa 240

cctgtctgac atttcaacct ggtgttgatg gcttaaacga taatacggtg agatatatag 300

ctaacagaat gggggtgacg cactgattcc actgtatata taggcgatat gtgttgttgg 360

atggacgttt ctttgtctcc tgatccacaa tagtagctca gctccgtgcc aactggttcg 420

ctggtacgat agtgagggat gaatgaa 447

<210>4

<211>1940

<212> DNA

<213> Artificial sequences

<400>4

cgtcgacagt aagtatgaaa agagccaatg ttgagagtct caggaaccac atcgacttct 60

tcgtgccatc ctcccacatt ctgaagccca agaacccaca aatcatcaaa caccaacacg 120

atgcggacgc caacccgagt tgtaacgcca caaagtacgg gtacgaccct gttccaggag 180

ggctcacgcc gcaatcaaca accaaagtcg ccacgatcaa cgccagtatc aagtaaaaga 240

agaatagcat ctccagtctt ccgatagctg tgtacttcga tctgacgttg tagatgatga 300

tgatcatgat cacaagggca ccaatgttga caaaggcgtt accaatctgg aatatcacgg 360

tattggcaac gtctatcgga cgggcgtagc actcagggat gatcccttcg ttcaggtgcg 420

tgaactgctc gttcgtcgtt gccttcacaa cctggcacaa cgggagcggc gtgttgtggc 480

atagcgagtt gaaatcaccg aatgccattg tgttttatcg ttagggagac ctgtttgaag 540

ctgacagcgg gatgaagatg aggaaggaga gcacaacagc tgagcggaag tctctgtgat 600

gcttggtgga ccgggtgtag gtggaatctc cctggtgagc gtacttgcaa cggtgctcag 660

cgacttcttc tcgagaggaa acgtaaacaa agaggtttca atgttgatgt tgatgtgtat 720

ttttgttaca aaagcagaaa ttgtaaacaa aaaggtataa ttagggctct ggtgtaatga 780

tgggcacgtg acgttaccgt gctggtcgat tttagggcta ttggttcgcg tcccgctggt 840

gtccgggtta gcgtgtcaat gtggcgcctc ccgattatta cataagaaaa cacccaccca 900

cgcaacacct ggtgtctgga tgttgacgct ttgtatgcgt gtgtgtgttt tttcttccgt 960

cttgttgggc cactctgcgc gagcgttggc gactcaccgg tgaaatttat cgaaaacttt 1020

caggctcagg cccttttcaa cactaccctt tgagatcaca tcaagcagta atcaaacaca 1080

atgggtatgt gggaaacgac gacgtgtgcg gtgtgtgaat gccattagtg ggatatgtgg 1140

tagtctcgag cgtggatatt atcgataggg atggtgcttg ttctatacgt cttgctggga 1200

aggaagaaag cgatgaagta tgtgggaaga aggggtggtt taagagagga agtagacatg 1260

taacaagtgt gttcagagaa caaggacgga aatatcacct atatgacgta cacatcacga 1320

actgctcctg gaggaagcga caagatgaat atcaacaggc atcatcatat ctctacaatg 1380

gctcgttccc aaagcacacg cacaaacaaa tccgagactt ttgtactaac agctgtatct 1440

ctgacaaata gttaacgttc caaagaccag aagaacctac tgtaagggta aggagtgcag 1500

aaagcacact caacacaagg ttacccagta caaggctggt aaggcttccc tctttgccca 1560

gggtaagcgt cgttatgacc gtaagcaatc cggttacggt ggtcaaacca agcaagtttt 1620

ccacaaaaag gctaaaacca ccaagaaggt tgttttgcgt ttggagtgtg ttgtctgcaa 1680

gaccaaggcc caattggctt tgaagcgttg taagcacttc gagttgggtg gtgacaagaa 1740

gcaaaagggt caagctttgc aattctaagc ttaagacaat tgttgaaagt tttattatta 1800

tcactacact gtgtttttga tgtcatctaa tgtaaaagcg tttatattac cacttggttc 1860

ggtatcctgt agaagaatac ggcctgtagc gtagcattcc cacaggagga tcacagcaac 1920

atagaccaaa cccggatccc 1940

<210>5

<211>1587

<212> DNA

<213> Artificial sequences

<400>5

cgatatctgc cagtagtcat atgcttgtct caaagattaa gccatgcatg tctaagtata 60

agcaatttat acagtgaaac tgcgaatggc tcattaaatc agttatagtt tatttgatag 120

taccttacta cttggataac cgtggtaatt ctagagctaa tacatgctaa aaaccccgac 180

tgcttgggag gggtgtattt attagataaa aaatcaatgc cctcgggctc tttgatgatt 240

cataataact tgtcgaatcg catggcttta cgccggcgat ggttcattca aatttctgcc 300

ctatcaactg tcgatggtag gatagtggcc taccatggtg gcaacgggta acggggaata 360

agggttcgat tccggagagg gagcctgaga aacggctacc acatccaagg aaggcagcag 420

gcgcgcaaat tacccaatcc taattcaggg aggtagtgac aataaataac gatacagggc 480

ccttctgggt cttgtaattg gaatgagtac aatgtaaata ccttaacgag gaacaattgg 540

agggcaagtc tggtgccagc agccgcggta attccagctc caatagcgta tattaaagtt 600

gttgcagtta aaaagctcgt agttgaactt tgggcctggc aggccggtcc gctttttggc 660

gagtactgac cctgccgggc ctttccttct ggctaccctc ccctctggag aggcgaacca 720

ggacttttac tttgaaaaaa ttagagtgtt caaagcaggc ctttgctcga atatattagc 780

atggaataat agaataggac gtttggttct attttgttgg tttctaggac catcgtaatg 840

attaataggg acggtcgggg gcatcagtat tcagttgtca gaggtgaaat tcttggattt 900

actgaagact aactactgcg aaagcatttg ccaaggacgt tttcattaat caagaacgaa 960

agttagggga tcgaagatga tcagataccg tcgtagtctt aaccataaac tatgccgact 1020

agggatcggg tgttgttttt ataatgactc actcggcacc ttacgagaaa tcaaagtctt 1080

tgggttctgg ggggagtatg gtcgcaaggc tgaaacttaa aggaattgac ggaagggcac 1140

caccaggagt ggagcctgcg gcttaatttg actcaacacg gggaaactca ccaggtccag 1200

acacaataag gattgacaga ttgagagctc tttcttgatt ttgtgggtgg tggtgcatgg 1260

ccgttcttag ttggtggagt gatttgtctg cttaattgcg ataacgaacg agaccttaac 1320

ctactaaata gcgcgactag cttttgctgg tgctgacgct tcttagaggg actatcgatt 1380

tcaagtcgat ggaagtttga ggcaataaca ggtctgtgat gcccttagac gttctgggcc 1440

gcacgcgcgc tacactgacg gagccagcga gtctagcctt ggccgagagg tcatgggtaa 1500

tcttgtgaaa ctccgtcgtg ctggggatag agcattgcaa ttattgctct tcaacgagga 1560

attcctagta agcgcaagtc agatatc 1587

Claims (10)

1. The Candida utilis expression vector for producing laccase is characterized by comprising an 18S rDNA gene fragment, a yeast glyceraldehyde triphosphate dehydrogenase gene promoter sequence (GAP-p), a laccase gene, a yeast glyceraldehyde triphosphate dehydrogenase gene terminator sequence (GAP-t) -Cycloheximide (CYH) resistance gene.

2. The Candida utilis expression vector for producing laccase of claim 1, wherein the nucleotide sequence of the laccase gene is SEQ ID No. 1.

3. A method for constructing a candida utilis expression vector for producing laccase, which is characterized in that,

(1) cloning of laccase (Lac) Gene: extracting total RNA in trametes versicolor, performing RT-PCR amplification by using the extracted total RNA as a template to obtain a target gene, connecting the target gene with a pMD19-T vector, and performing transformation extraction to obtain a recombinant plasmid;

(2) cloning of the Yeast glyceraldehyde triphosphate dehydrogenase Gene (GAP) promoter (GAP-p) and terminator (GAP-t): designing a primer according to the combination of a Candida utilis (C.utilis) GAP-p GAP-T gene sequence and a pBR322 plasmid sequence characteristic in a GenBank database, obtaining a GAP promoter (GAP-p) fragment and a GAP terminator (GAP-T) fragment of the yeast by PCR by taking a Candida utilis genome as a template, respectively connecting target fragments GAP-p and GAP-T to a vector pMD19-T simple vector to obtain plasmid vectors pT-GP and pT-GT, transforming escherichia coli, and carrying out bacteria liquid PCR identification and storing for later use on a positive clone;

(3) cloning of Cycloheximide (CYH) resistance Gene: designing a primer and a pair of reverse complementary primers according to the combination of a Candida utilis (C.utilis) CYH sensitive gene (L41) sequence in a GenBank database and the sequence characteristics of a pBR322 plasmid; taking a candida utilis genome as a template, amplifying an upstream fragment and a downstream fragment of L41 by PCR, wherein one end of the 2 fragments has a base overlapping region; mixing the two upstream fragments and the two downstream fragments in equal amount to form a template, and performing intussusception PCR to obtain a CYH resistance gene; connecting the target fragment to a vector pMD19-T simple vector to obtain a plasmid vector pT-CYH, transforming escherichia coli, and carrying out bacteria liquid PCR identification and preservation on positive clones for later use;

(4) cloning of 18S rDNA gene fragment: designing a primer according to the combination of a candida utilis (C.utilis)18S rDNA gene sequence in a GenBank database and a pBR322 plasmid sequence characteristic, taking a candida utilis genome as a template, obtaining an 18S rDNA fragment through PCR, connecting a target fragment to a vector pMD19-T simple vector to obtain a plasmid vector pT-rD, transforming escherichia coli, and carrying out bacteria liquid PCR identification and preservation on positive clones for later use;

(5) construction of homologous integration expression vector: extracting pT-GP, pT-GT and pBR322 plasmids, respectively carrying out enzyme digestion, recovering target fragments, and simultaneously connecting the 3 fragments by using T4DNA ligase to obtain a plasmid pBR-GAP; extracting pT-CYH and pBR-GAP plasmids, performing enzyme digestion respectively, recovering target fragments, and connecting with T4DNA ligase to obtain a plasmid pBR-G-L; extracting pBR-G-L plasmid and laccase (Lac) gene recombinant plasmid, performing enzyme digestion respectively, recovering target fragments, and connecting with T4DNA ligase to obtain plasmid pBR-G-Q-L; and extracting pT-rD and pGQL plasmids, performing enzyme digestion respectively, recovering target fragments, and connecting the target fragments by using T4DNA ligase to obtain the laccase-producing Candida utilis expression vector pGQLR.

4. The method for constructing the Candida utilis expression vector for producing laccase of claim 3, wherein primers used in cloning the promoter (GAP-p) and terminator (GAP-t) of glyceraldehyde triphosphate dehydrogenase Gene (GAP) of yeast are designed as follows:

promoter (GAP-p):

GAP-p1:GGATATCTTACAGCGAGCACTCAA；

GAP-p2:GCTCTAGAATGTTGTTTGT；

terminator (GAP-t):

GAP-t1：CTAGCTAGCTATGACTTTTAT；

GAP-t2：GGGATCCTTCATTCATCCCTCACTATCG。

5. the method for constructing the candida utilis expression vector for producing laccase according to claim 3, wherein in the cloning of the Cycloheximide (CYH) resistance gene, primers are designed as follows:

P 4:CGTCGACAGTAAGTATGAAAAGAGC；

RM4:GGGATCCGG GTTTGGTCTATGTTGCT；

introducing a SalI enzyme cutting site into a primer P4, and introducing a BamHI enzyme cutting site into a primer RM 4;

the reverse complementary primer was designed as follows:

P5:AACCAAGCAAGTTTTCCAC；

R5:GTGGAAAACTTGCTTGGTT。

6. the method for constructing the candida utilis expression vector for producing laccase according to claim 3, wherein in the cloning of the 18S rDNA gene fragment, primers are designed as follows:

P3:CGATATCTGCCAGTAGTCATATGC；

R3:CGATATCTGACTTGCGCTTACTAG；

an EcoRV cleavage site was introduced into primer P3 and into primer R3.

7. A Candida utilis gene engineering strain for producing laccase is characterized in that the Candida utilis expression vector pGQLR containing laccase gene of any one of claims 1 to 6 is spliced again to obtain a Candida utilis recon with a sequence of ZHQX1, and the recon is subjected to electric transformation to construct a recombined yeast strain.

8. The candida utilis gene engineering strain producing laccase of claim 7, wherein a pGQLR vector is used as a template for primer design, a fragment sequence is divided into two fragments, and the sequence obtained after splicing again is zhhqx 1, and the design primers are as follows:

9. the genetically engineered candida utilis strain producing laccase of claim 7, wherein a prokaryotic DNA sequence including a DNA fragment of a bacterial plasmid sequence including a drug resistance marker Amp is deleted from the candida utilis expression vector pGQLR.

10. Use of the genetically engineered candida utilis strain producing laccase according to any one of claims 1 to 9 in food, pharmaceutical and livestock feed.

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