CN110343624B

CN110343624B - Recombinant strain and application thereof in improving yield of cellulase

Info

Publication number: CN110343624B
Application number: CN201810287531.1A
Authority: CN
Inventors: 董志扬; 林洁; 陈秀珍; 宋冰冉; 张吓妹
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2018-04-03
Filing date: 2018-04-03
Publication date: 2021-06-15
Anticipated expiration: 2038-04-03
Also published as: CN110343624A

Abstract

The invention discloses a recombinant bacterium and application thereof in improving the yield of cellulase. The recombinant strain protected by the invention is obtained by introducing specific DNA molecules into trichoderma reesei. The specific DNA molecule has a coding gene of a trichoderma reesei hemoglobin structural domain. The invention identifies the hemoglobin structural domain of trichoderma reesei for the first time, successfully expresses the hemoglobin structural domain in trichoderma reesei strains for the first time, improves the strain fermentation efficiency, and obviously improves the total extracellular protein yield and the activity of main cellulase. The invention has great value for the production of the cellulase and has great significance for reducing the cost and improving the yield.

Description

Recombinant strain and application thereof in improving yield of cellulase

Technical Field

The invention relates to a recombinant bacterium and application thereof in improving yield of cellulase.

Background

Lignocellulose biomass is the most abundant renewable resource on the earth, and main polysaccharide components of the lignocellulose biomass, namely cellulose and hemicellulose, can be efficiently degraded into oligosaccharide and monosaccharide under the action of cellulase and hemicellulase, so that the lignocellulose biomass can be used for the production of multiple industries such as food, feed, biological energy sources and the like. The cellulase plays a key role in the process, and the yield and the activity of the cellulase are directly related to the cost and the application of related products such as food, feed, biological energy sources and the like.

The filamentous fungus Trichoderma reesei is a main industrial strain for producing cellulase, has strong secretion capacity of the cellulase and hemicellulase, and more than 90 percent of cellulase products are produced by the Trichoderma reesei globally. At present, the industry mainly applies the filamentous fungi submerged fermentation technology to produce the cellulase. Research has shown that oxygen is a key factor for producing cellulase from trichoderma reesei. In the process of submerged fermentation, fermentation liquor is very viscous, local oxygen supply deficiency is easily caused, cell growth is slowed down and even cells die, and meanwhile, the yield of cellulase is obviously reduced. The traditional solution method has the disadvantages of high stirring speed or increased ventilation capacity, high energy consumption and high cost, and can not effectively solve the problem of insufficient oxygen supply.

Disclosure of Invention

The invention aims to provide a recombinant bacterium and application thereof in improving the yield of cellulase.

The invention provides a method for promoting trichoderma reesei to produce cellulase, which comprises the following steps: introducing specific DNA molecules into trichoderma reesei, thereby promoting the trichoderma reesei to produce cellulase; the specific DNA molecule has a coding gene of a trichoderma reesei hemoglobin structural domain.

The invention also provides a method for increasing the yield of cellulase produced by trichoderma reesei, which comprises the following steps: introducing specific DNA molecules into trichoderma reesei, thereby promoting the trichoderma reesei to produce cellulase; the specific DNA molecule has a coding gene of a trichoderma reesei hemoglobin structural domain.

The trichoderma reesei hemoglobin domain is (a1) or (a2) as follows:

(a1) a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;

(a2) and (b) a protein obtained by substituting and/or deleting and/or adding (a1) one or more amino acid residues and having the same function.

The encoding gene of the trichoderma reesei hemoglobin domain is a DNA molecule as described in any one of (b1) to (b4) below:

(b1) the coding region is DNA molecule shown as 2311 th to 2775 th nucleotides from 5' end of sequence 1 in the sequence table;

(b2) DNA molecule shown in sequence 1 in the sequence table;

(b3) a DNA molecule that hybridizes under stringent conditions to the DNA sequence defined in (b1) or (b2) and encodes a trichoderma reesei hemoglobin domain;

(b4) and (b) a DNA molecule which has more than 90% homology with the DNA sequence defined in (b1) or (b2) or (b3) and encodes a hemoglobin domain of Trichoderma reesei.

The stringent conditions can be hybridization and washing with 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS solution at 65 ℃ in DNA or RNA hybridization experiments.

Any one of the above specific DNA molecules has a gpdA promoter and a gene encoding a hemoglobin domain of trichoderma reesei, and expression of the gene encoding the hemoglobin domain of trichoderma reesei is promoted by the gpdA promoter.

Any one of the gpdA promoters can be a DNA molecule shown by 1 st to 2310 th nucleotides from the 5' end of a sequence 1 in a sequence table.

Any of the specific DNA molecules described above further comprises a TrpC terminator.

The TrpC terminator can be a DNA molecule shown by 2776-3545 th nucleotides from the 5' tail end of a sequence 1 in a sequence table.

Any one of the specific DNA molecules can be specifically shown as a sequence 1 in a sequence table.

The invention also protects any one of the specific DNA molecules.

The invention also protects the application of any one of the specific DNA molecules or the recombinant expression vector containing the specific DNA molecule, which is (c1) or (c 2):

(c1) promoting Trichoderma reesei to produce cellulase;

(c2) the yield of cellulase produced by trichoderma reesei is improved.

The recombinant expression vector can be specifically obtained by recombining the encoding gene of the trichoderma reesei hemoglobin domain and an expression vector with a gpdA promoter by a recombinant cloning method.

The recombinant expression vector can be specifically a recombinant expression vector obtained by recombining the encoding gene of the trichoderma reesei hemoglobin domain and a pNOM102 plasmid by a recombinant cloning method.

The invention also protects a recombinant bacterium, which is obtained by introducing any one of the specific DNA molecules into trichoderma reesei.

The preparation method of the recombinant bacterium comprises the following steps:

(d1) adding the specific DNA molecules into a trichoderma reesei protoplast solution, adding a PEG 4000 solution, and standing for 20min at room temperature;

(d2) adding a PEG 4000 solution to the mixed solution obtained in the step (d 1);

(d3) mixing the mixed solution obtained in the step (d2) with a sorbitol aqueous solution, and inoculating the mixture to a MM solid medium containing sorbitol for culture to obtain a transformant;

(d4) inoculating the transformant obtained in the step (d3) into a PDA solid culture medium for culture, and eluting with sterile water to obtain a spore suspension;

(d5) inoculating the spore suspension obtained in the step (d4) into a MM solid medium containing Triton X-100 for culture to obtain a recombinant bacterium.

The (d1) can be specifically that the pyr4 marker gene fragment and the specific DNA molecule are added into the trichoderma reesei protoplast solution, then the PEG 4000 solution is added, and the mixture is kept standing for 20min at room temperature.

The molar ratio of the pyr4 marker gene fragment to the specific DNA molecule may specifically be 1: 4.

The pyr4 marker gene fragment can be specifically shown as a sequence 3 in a sequence table.

The proportion of the pyr4 marker gene fragment, the specific DNA molecule and the trichoderma reesei protoplast solution can be 1 mol: 4 mol: 0.2 mL. The trichoderma reesei protoplast solutionThe concentration of protoplast can be 4 × 10⁷-8×10⁷one/mL.

In the step (d1), the PEG 4000 solution may be a 50% (volume percentage) PEG 4000 solution. The proportion relationship between the PEG 4000 solution and the protoplast solution can be specifically 0.2 mL: 50 μ L.

In the step (d2), the PEG 4000 solution may be a 50% (volume percentage) PEG 4000 solution. The proportion relationship between the PEG 4000 solution and the protoplast solution can be specifically 0.2 mL: 1 mL.

In the step (d3), the sorbitol concentration in the aqueous sorbitol solution may be 1.0M. The final concentration of sorbitol in the sorbitol-containing MM solid medium may specifically be 1.0M. The culture temperature may specifically be 30 ℃. The culture time may be specifically 5 days.

In the above (d4), the culture temperature may specifically be 30 ℃. The culture time is 4-7 days. The concentration of the spore suspension may be specifically 10⁷one/mL.

The (d5) can be specifically the spore suspension obtained in the (d4) is diluted 10³-10⁶After doubling, inoculating the strain to a MM solid culture medium containing Triton X-100 for culture to obtain a recombinant strain. The volume percentage of Triton X-100 in the MM solid medium containing Triton X-100 can be specifically 0.1%. The degree of culture may specifically be 30 ℃. The culture time may be specifically 5 days.

The preparation method of the trichoderma reesei protoplast solution comprises the following steps:

(e1) inoculating trichoderma reesei into a spore production culture medium for culturing to obtain spore suspension;

(e2) inoculating the spore suspension obtained in the step (e2) into an MM liquid culture medium for culture, and centrifugally collecting thallus precipitates;

(e3) the cell pellet obtained in step (e2) was washed 3 times with sterile water and 1 time with an aqueous solution of magnesium sulfate.

(e4) Resuspending the thallus precipitate treated in the step (e3) by using a lysis solution for a lysis reaction, and collecting a protoplast precipitate after the reaction is finished;

(e5) resuspending the protoplast pellet obtained in step (e4) with an aqueous sorbitol solution to obtain a protoplast solution.

In the (e1), the spore-forming medium consists of a solute and a solvent; the solute and the concentration thereof in the spore production culture medium are 200g/L of potato, 10g/L of glucose and 20g/L of agar powder; the solvent is water.

In the step (e1), the concentration of the spore suspension is 10⁷-10⁸one/mL.

In the step (e2), the volume ratio of the spore suspension to the MM liquid medium is 1: 20. The culture conditions may specifically be 200rpm, 28 ℃. The incubation time may be specifically 14 hours.

In the step (e3), the magnesium sulfate concentration in the aqueous magnesium sulfate solution is 1.2M.

In the step (e4), the lysate is composed of a lywallzyme and a magnesium sulfate aqueous solution of cellulase. The concentration of magnesium sulfate in the magnesium sulfate aqueous solution was 1.2M. The proportioning relation of the muramidase, the cellulase and the magnesium sulfate aqueous solution can be specifically 150 mg: 15 mg: 15 mL.

In the (e4), the reaction temperature of the cleavage reaction may be specifically 30 ℃. The reaction time of the cleavage reaction may be specifically 1.5 hours. And (3) stopping the reaction by adopting a sorbitol aqueous solution when the cracking reaction is finished. The sorbitol concentration in the sorbitol aqueous solution was 0.6M.

In the step (e4), the method for collecting the protoplast precipitate comprises the steps of taking the reaction system, filtering the reaction system by using a 200-mesh sieve to remove residual hyphae, centrifuging the filtrate at the room temperature of 3000rpm for 10min, and collecting the protoplast precipitate.

In the step (e5), the sorbitol concentration in the aqueous sorbitol solution is 1.0M.

The invention also protects the application of any recombinant bacterium in the preparation of cellulase.

The invention also provides a method for producing cellulase, which comprises the following steps: culturing any one of the recombinant bacteria to obtain the cellulase.

Any of the above trichoderma reesei may specifically be trichoderma reesei Tu 6.

The invention identifies the hemoglobin structural domain of trichoderma reesei for the first time, successfully expresses the hemoglobin structural domain in trichoderma reesei strains for the first time, improves the strain fermentation efficiency, and obviously improves the total extracellular protein yield and the activity of main cellulase. The invention has great value for the production of the cellulase and has great significance for reducing the cost and improving the yield.

Drawings

FIG. 1 shows the result of PCR identification of recombinant bacterium TrHb 1. FIG. 1A is a schematic diagram of PCR identification, FIG. 1B is a PCR product electrophoresis result, lane 1 is an amplification result using Trichoderma reesei TU-6 genome DNA as a template, lane 2 is an amplification result using recombinant bacteria TrHb1 genome DNA as a template, and lane 3 is an amplification result using recombinant expression vector pNOM102-TrHb1 as a template.

FIG. 2 shows the expression of the hemoglobin domain TrHb1 in the transformed strain detected by western blot experiment. Lane 1 shows the results of the intracellular protein western of Trichoderma reesei TU-6, and lane 2 shows the results of the intracellular protein western of recombinant bacterium TrHb 1.

FIG. 3 shows the result of the difference spectrum analysis of the recombinant bacterium TrHb1 and Trichoderma reesei TU-6 CO.

FIG. 4 shows SDS-PAGE analysis of recombinant strain TrHb1 and Trichoderma reesei TU-6 fermentation at different time points. C represents Trichoderma reesei TU-6, and T represents recombinant bacterium TrHb 1.

FIG. 5 shows the measurement of the concentration of extracellular protein in cellulose induction medium fermentation of Trichoderma reesei TU-6 and recombinant strain TrHb 1.

FIG. 6 shows the determination of the enzyme activity of the fermentation filter paper of the cellulose induction culture medium of Trichoderma reesei TU-6 and recombinant bacterium TrHb 1.

FIG. 7 shows the enzyme activity determination conditions of CMC-Na fermentation of cellulose induction culture medium by Trichoderma reesei TU-6 and recombinant bacterium TrHb 1.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

pNOM102 plasmid: reference documents: punt P J, Dingense M A, Kuyvenhoven A, et al, functional elements in the promoter region of the Aspergillus nidulans gpdA Gene encoding for glycine-3-phosphate dehydrogenase [ J ]. Gene,1990,93(1): 101-9.; the public is available from the institute for microorganisms of the Chinese academy of sciences.

plasmid pSK-pyr 4: the invention patent is as follows: a preparation method and application of Trichoderma reesei strain with high yield of Penicillium yangqi alpha-galactosidase are disclosed (application No. 201510684461. X); references Qin L N, Cai F R, Dong X R, et al, advanced production of heterologous lipase in Trichoderma reesei by RNAi mediated gene amplification of an endogenous high level expressed gene [ J ]. Bioresource technology,2012,109: 116-; the public is available from the institute for microorganisms of the Chinese academy of sciences.

Trichoderma reesei TU-6: ATCC, No.: MYA-256.

And (3) muramidase: SIGMA company, cat #: L1412-5G.

CELLULASE (cellulose "ONOZUKA" R-10): yakult, Japan, Cat No.: 130918-01.

Spore production culture medium: 200g of potato, 10g of glucose, 20g of agar powder and distilled water, wherein the volume is constant to 1L, and the potato is sterilized by high-pressure steam at 115 ℃ for 20 min.

MM liquid medium: (NH)₄)₂SO₄ 0.5g、KH₂PO₄ 1.5g、MgSO₄ 0.06g、CaCl₂ 0.06g、FeSO₄·7H₂O 0.5mg、MnSO₄·H₂O 0.16mg、ZnSO₄·7H₂O 0.14mg、CoCl₂0.2mg, adding distilled water to 100mL, adjusting pH to 5.0, and sterilizing with 115 deg.C high pressure steam for 20 min.

MM solid medium: (NH)₄)₂SO₄ 0.5g、KH₂PO₄ 1.5g、MgSO₄ 0.06g、CaCl₂ 0.06g、FeSO₄·7H₂O 0.5mg、MnSO₄·H₂O 0.16mg、ZnSO₄·7H₂O 0.14mg、CoCl₂0.2mg, adding distilled water to a constant volume of 100mL, and adjusting the pH value to5.0 g of agar powder, and sterilizing for 20min by high-pressure steam at 115 ℃.

Fermentation medium: 1% (mass percent) of microcrystalline cellulose is added into the MM liquid culture medium.

Recombinant cloning kit:

MultiS recombinant kit, Nanjing Novozam Biotech, Inc.

The primer information used in the following examples is shown in Table 1.

TABLE 1 primer information

Example 1 obtaining of recombinant bacteria

Construction of recombinant expression vector pNOM102-TrHb1

1. And (3) performing PCR amplification by using the genome DNA of the Trichoderma reesei TU-6 as a template and adopting a primer pair consisting of a primer Trhb1-F and a primer Trhb1-R to obtain a PCR amplification product (Trhb1 gene fragment).

2. The pNOM102 plasmid is used as a template, and PCR amplification is carried out by adopting a primer pair consisting of a primer trpC-F and a primer gpd-R, so as to obtain an expression vector of about 5728bp containing a gpdA promoter and a TrpC terminator.

3. And (3) recombining the PCR amplification product (Trhb1 gene fragment) obtained in the step (1) and the expression vector obtained in the step (2) by using a recombinant cloning kit to obtain a recombinant expression vector pNOM102-TrHb1 (which is verified by sequencing).

Preparation of trichoderma reesei protoplast

1. Inoculating Trichoderma reesei TU-6 into a spore production culture medium for culturing, and eluting spores with deionized water to obtain spore suspension (the concentration is 10)⁷-10⁸one/mL).

2. 5mL of the spore suspension obtained in step 1 was inoculated into 100mL of MM liquid medium, cultured at 28 ℃ for 14 hours at 200rpm, and centrifuged to collect cell pellets.

3. The cell pellet obtained in step 2 was washed 3 times with sterile water and 1 time with 1.2M magnesium sulfate aqueous solution.

4. The cells obtained in step 3 were collected, 15mL of a lysis buffer (150 mg of muramidase and 15mg of cellulase were added to 15mL of a 1.2M magnesium sulfate aqueous solution) was added thereto, and the reaction was cleaved at 30 ℃ for 1.5 hours, followed by addition of 15mL of a 0.6M sorbitol aqueous solution to terminate the reaction.

5. And (4) after the step 4 is finished, taking the reaction system, filtering the reaction system by using a 200-mesh sieve to remove residual hyphae, centrifuging the filtrate at the room temperature of 3000rpm for 10min, and collecting a protoplast precipitate.

6. The protoplast pellet obtained in step 5 was resuspended in 10mL of 1.0M aqueous sorbitol solution, centrifuged at 3000rpm at room temperature for 10 minutes, and the protoplast pellet was collected.

7. And 6, repeating the step.

8. Resuspending the protoplast pellet obtained in step 7 with 200. mu.L of 1.0M sorbitol aqueous solution to obtain a protoplast solution (4X 10)⁷-8×10⁷one/mL).

Preparation of recombinant bacteria

1. And (3) taking the recombinant expression vector pNOM102-TrHb1 prepared in the step one as a template, and adopting a primer M13F and a primer M13R to carry out amplification to obtain a PCR product (TrHb1 expression fragment). The TrHb1 expression fragment is shown as a sequence 1 in a sequence table. The TrHb1 expression fragment is shown as a sequence 1 in a sequence table.

In the sequence 1, the 1 st-2310 th nucleotides from the 5' end are gpdA promoter segments, the 2311 st-2775 th nucleotides are Trhb1 gene segments, and the 2776 st-3545 th nucleotides are TrpC terminator segments. The protein shown by the nucleotide coding sequence 2 from 2311-2772 th site of the 5' end of the sequence 1 in the sequence table.

2. The plasmid pSK-pyr4 is used as a template, a primer M13F and a primer M13R are used for amplification to obtain a PCR product, and the PCR product (pyr4 marker gene fragment) and the pyr4 marker gene fragment are shown as a sequence 3 in a sequence table.

3. Mixing 4 mol of TrHb1 expression fragment obtained in

step

1 and 1 mol of pyr4 marker gene fragment obtained in step 2, adding the mixture into 0.2mL of the protoplast solution obtained in step two, adding 50. mu.L of 50% (volume percent) PEG 4000 solution, and carrying out ice bath for 30 min.

4. To the mixed solution obtained in step 3, 1mL of 50% (volume percent) PEG 4000 solution was added and left at room temperature for 20 min.

5. To the mixed solution obtained in step 4, 1mL of a 1.0M aqueous sorbitol solution was added.

6. Taking 0.5mL of the mixed solution obtained in the step 5, mixing the mixed solution with 4mL of 1.0M sorbitol aqueous solution, then paving the mixed solution on an MM solid culture medium containing 1.0M sorbitol, and culturing the mixed solution in a constant temperature incubator at 30 ℃ for 5 days to obtain a transformant.

7. Inoculating the transformant obtained in the step 6 to a PDA solid culture medium, culturing for 4-7 days in a constant-temperature incubator at 30 ℃, eluting spores on the culture medium with sterile water after hyphae grow to obtain a spore suspension (the concentration is 10)⁷one/mL), the gradient was diluted 10³-10⁶And (3) coating the multiplied spore suspension on an MM solid culture medium containing 0.1 percent (volume percentage) of Triton X-100, culturing for 5 days in a constant-temperature incubator at 30 ℃, and picking a single colony to obtain the recombinant bacterium TrHb 1.

8. And (3) taking Trichoderma reesei TU-6 and the recombinant bacterium TrHb1 obtained in the step 7, extracting genome DNA, carrying out PCR identification by using the genome DNA as a template and using a primer TF and a primer TR, and setting a positive control by using a recombinant expression vector pNOM102-TrHb1 as the template. A schematic representation of the PCR identification is shown in FIG. 1A. The results are shown in FIG. 1B. The result shows that the recombinant bacterium TrHb1 amplifies a specific band of about 2.2kb, which is the same as the amplification result of the recombinant expression vector pNOM102-TrHb1, but the specific band is not amplified by the Trichoderma reesei TU-6. The band of about 2.2kb in

lanes

2 and 3 was recovered and sequenced as shown at positions 996-3222 from the 5' end of sequence 1 of the sequence listing. The result shows that the recombinant bacterium TrHb1 is successfully constructed.

9. The pNOM102 plasmid is used as a template, and a primer M13F and a primer M13R are adopted for amplification to obtain a PCR product.

10. And (3) replacing the TrHb1 expression fragment with the PCR product obtained in the step (9), and operating according to the steps (3-7) to obtain a control recombinant bacterium.

Fourth, TrHb1 protein expression identification of recombinant bacteria

The strains to be tested are as follows: trichoderma reesei TU-6, recombinant bacterium TrHb1 and a control recombinant bacterium.

1. Inoculating the strain to be tested into a spore production culture medium for culture, eluting spores by deionized water to obtain spore suspension (the concentration is about 10)⁷one/mL).

2. 1mL of the spore suspension was inoculated into 50mL of MM liquid medium containing 2% (mass percent) glucose, cultured at 28 ℃ for 48 hours at 200rpm, and filtered with sterile gauze to collect mycelia.

3. And (3) adding liquid nitrogen into the mycelium precipitate obtained in the step (2) for grinding, then using 30mL of 50mM potassium phosphate buffer solution with pH of 7.0 to re-suspend the thalli, centrifuging at 8000rpm for 40min, and taking the supernatant (namely the intracellular protein extracting solution).

4. And (4) carrying out Western blot detection on the intracellular protein extracting solution obtained in the step (3).

The results are shown in FIG. 2. The result shows that the recombinant bacterium TrHb1 has an obvious specific band at about 17kDa and Trichoderma reesei TU-6 has no specific band, which indicates that the TrHb1 protein is successfully expressed in the recombinant bacterium. The control recombinant bacteria also did not produce specific bands.

5. And (3) taking 2mL of the intracellular protein extracting solution obtained in the step (3), adding 20mg of sodium dithionite, introducing CO gas for 5min, and measuring the activity of TrHb1 by spectral scanning of a spectrophotometer.

The results are shown in FIG. 3. The result shows that the protein extracting solution in the recombinant bacterium TrHb1 has a characteristic absorption peak at the wavelength of 420nm but does not exist in Trichoderma reesei TU-6, which indicates that the TrHb1 gene is successfully expressed in Trichoderma reesei and can enable normal functions in cells. The control recombinant strain has no characteristic absorption peak at the wavelength of 420 nm.

Example 2 fermentation application of recombinant bacteria

The strains to be detected are as follows: trichoderma reesei TU-6, recombinant bacterium TrHb1 and a control recombinant bacterium.

1. Suspending spore of the strain with deionized water to obtain spore suspension (concentration of 10)⁷one/mL).

2. 4mL of the spore suspension obtained in step 1 was inoculated into 400mL of MM liquid medium containing 2% (mass percent) glucose, cultured at 28 ℃ for 48 hours at 200rpm, and the fermentation broth was centrifuged to collect mycelia.

3. The mycelia obtained in step 2 were washed with sterile water and inoculated into 100mL of a fermentation medium containing 1% (mass percent) of crystalline cellulose Avicel, cultured at 28 ℃ and 200rpm, and measured at 48h, 72h, 96h, 120h and 144h as follows:

(1) the fermentation system was centrifuged to take the supernatant, and the protein concentration in the supernatant (extracellular protein concentration) was determined and subjected to SDS-PAGE electrophoretic analysis. The results of SDS-PAGE analysis are shown in FIG. 4. The results of extracellular protein concentration measurement are shown in FIG. 5. The results show that extracellular protein secretion is obviously different, the extracellular protein concentration of the recombinant bacterium TrHb1 is obviously improved compared with that of Trichoderma reesei TU-6, the protein concentration measurement result also shows that the extracellular protein of the recombinant bacterium TrHb1 is obviously improved compared with that of Trichoderma reesei TU-6, and the extracellular protein concentration of the recombinant bacterium TrHb1 is improved by 81% in 120 th hour compared with that of Trichoderma reesei TU-6. The statistical results of the control recombinant bacteria and the Trichoderma reesei TU-6 have no significant difference.

(2) Centrifuging the fermentation system to obtain supernatant, and measuring the filter paper enzyme activity and carboxymethylcellulose sodium (CMC-Na) enzyme activity OF the supernatant according to IUPAC (International Unit OF PURE AND APPLIED CHEMISTRY) standard method, reference: ghose T K.measurement of cellular activities [ J ]. Pure & Applied Chemistry,1987,59(2): 257-.

The statistical result of the filter paper enzyme activity is shown in figure 6, and the statistical result of the carboxymethyl cellulose sodium enzyme activity is shown in figure 7.

Results show that the cellulase activity in the recombinant strain TrHb1 fermentation broth is also obviously improved, the filter paper enzyme activity of the recombinant strain TrHb1 fermentation broth is improved by about 36% compared with Trichoderma reesei TU-6 at 120 hours, and the CMC-Na enzyme activity is not obviously improved. The statistical results of the control recombinant bacteria and the Trichoderma reesei TU-6 have no significant difference.

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> recombinant bacterium and application thereof in improving yield of cellulase

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3545

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gaattccctt gtatctctac acacaggctc aaatcaataa gaagaacggt tcgtcttttt 60

cgtttatatc ttgcatcgtc ccaaagctat tggcgggata ttctgtttgc agttggctga 120

cttgaagtaa tctctgcaga tctttcgaca ctgaaatacg tcgagcctgc tccgcttgga 180

agcggcgagg agcctcgtcc tgtcacaact accaacatgg agtacgataa gggccagttc 240

cgccagctca ttaagagcca gttcatgggc gttggcatga tggccgtcat gcatctgtac 300

ttcaagtaca ccaaccctct tctgatccag tcgatcatcc cgctgaaggg cgctttcgaa 360

tcgaatctgg ttaagatcca cgtcttcggg aagccagcga ctggtgacct ccagcgtccc 420

tttaaggctg ccaacagctt tctcagccag ggccagccca agaccgacaa ggcctccctc 480

cagaacgccg agaagaactg gaggggtggt gtcaaggagg agtaagctcc ttattgaagt 540

cggaggacgg agcggtgtca agaggatatt cttcgctctg tattatagat aagatgatga 600

ggaattggag gtagcatagc ttcatttgga tttgctttcc aggctgagac tctagcttgg 660

agcatagagg gtccctttgg ctttcaatat tctcaagtat ctcgagtttg aacttattcc 720

cgtgaacctt ttattcacca atgagcattg gaatgaacat gaatctgagg actgcaatcg 780

ccatgaggtt ttcgaaatac atccggatgt cgaaggcttg gggcacctgc gttggttgaa 840

tttagaacgt ggcactattg atcatccgat agctctgcaa agggcgttgc acaatgcaag 900

tcaaacgttg ctagcagttc caggtggaat gttatgatga gcattgtatt aaatcaggag 960

atatagcatg atctctagtt agctcaccac aaaagtcaga cggcgtaacc aaaagtcaca 1020

caacacaagc tgtaaggatt tcggcacggc tacggaagac ggagaagccc accttcagtg 1080

gactcgagta ccatttaatt ctatttgtgt ttgatcgaga cctaatacag cccctacaac 1140

gaccatcaaa gtcgtatagc taccagtgag gaagtggact caaatcgact tcagcaacat 1200

ctcctggata aactttaagc ctaaactata cagaataaga tggtggagag cttataccga 1260

gctcccaaat ctgtccagat catggttgac cggtgcctgg atcttcctat agaatcatcc 1320

ttattcgttg acctagctga ttctggagtg acccagaggg tcatgacttg agcctaaaat 1380

ccgccgcctc caccatttgt agaaaaatgt gacgaactcg tgagctctgt acagtgaccg 1440

gtgactcttt ctggcatgcg gagagacgga cggacgcaga gagaagggct gagtaataag 1500

cgccactgcg ccagacagct ctggcggctc tgaggtgcag tggatgatta ttaatccggg 1560

accggccgcc cctccgcccc gaagtggaaa ggctggtgtg cccctcgttg accaagaatc 1620

tattgcatca tcggagaata tggagcttca tcgaatcacc ggcagtaagc gaaggagaat 1680

gtgaagccag gggtgtatag ccgtcggcga aatagcatgc cattaaccta ggtacagaag 1740

tccaattgct tccgatctgg taaaagattc acgagatagt accttctccg aagtaggtag 1800

agcgagtacc cggcgcgtaa gctccctaat tggcccatcc ggcatctgta gggcgtccaa 1860

atatcgtgcc tctcctgctt tgcccggtgt atgaaaccgg aaaggccgct caggagctgg 1920

ccagcggcgc agaccgggaa cacaagctgg cagtcgaccc atccggtgct ctgcactcga 1980

cctgctgagg tccctcagtc cctggtaggc agctttgccc cgtctgtccg cccggtgtgt 2040

cggcggggtt gacaaggtcg ttgcgtcagt ccaacatttg ttgccatatt ttcctgctct 2100

ccccaccagc tgctcttttc ttttctcttt cttttcccat cttcagtata ttcatcttcc 2160

catccaagaa cctttatttc ccctaagtaa gtactttgct acatccatac tccatccttc 2220

ccatccctta ttcctttgaa cctttcagtt cgagctttcc cacttcatcg cagcttgact 2280

aacagctacc ccgcttgagc agacatcaca atggccaccc tcaccgcaga ccaaatcgcc 2340

atcatcaagt ccaccgtgcc catcatccgc gagcacggca ccaccgtcac aaccaccttc 2400

tacgcaaaca tgctcgccgc ccaccccgag ctcaagaact acttctccct ccgcaaccag 2460

caaacgggag cccagcaggc cgccctcgcc aactcggtcc tcgccgcggc aacctacatc 2520

gacaacctgg ccgtcatcgc cggcgccgtc gagcgcatcg cccagaagca cgcctcgctc 2580

ttcatcaagc ccgagcacta ccccatcgtc ggcaagtacc tcatcggcgc ctttgagcag 2640

atcctcggcg acgccttcac cccggagatc aaggacgcct gggtcaccgc ctacggcatc 2700

ctggccgaca tcttcatcaa gcgcgagcag cagctctacg ccgaggccgg ctggcatcat 2760

catcatcatc attaaggatc cacttaacgt tactgaaatc atcaaacagc ttgacgaatc 2820

tggatataag atcgttggtg tcgatgtcag ctccggagtt gagacaaatg gtgttcagga 2880

tctcgataag atacgttcat ttgtccaagc agcaaagagt gccttctagt gatttaatag 2940

ctccatgtca acaagaataa aacgcgtttt cgggtttacc tcttccagat acagctcatc 3000

tgcaatgcat taatgcattg actgcaacct agtaacgcct tncaggctcc ggcgaagaga 3060

agaatagctt agcagagcta ttttcatttt cgggagacga gatcaagcag atcaacggtc 3120

gtcaagagac ctacgagact gaggaatccg ctcttggctc cacgcgacta tatatttgtc 3180

tctaattgta ctttgacatg ctcctcttct ttactctgat agcttgacta tgaaaattcc 3240

gtcaccagcn cctgggttcg caaagataat tgcatgtttc ttccttgaac tctcaagcct 3300

acaggacaca cattcatcgt aggtataaac ctcgaaatca nttcctacta agatggtata 3360

caatagtaac catgcatggt tgcctagtga atgctccgta acacccaata cgccggccga 3420

aactttttta caactctcct atgagtcgtt tacccagaat gcacaggtac acttgtttag 3480

aggtaatcct tctttctaga agtcctcgtg tactgtgtaa gcgcccactc cacatctcca 3540

ctcga 3545

<210> 2

<211> 154

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ala Thr Leu Thr Ala Asp Gln Ile Ala Ile Ile Lys Ser Thr Val

1 5 10 15

Pro Ile Ile Arg Glu His Gly Thr Thr Val Thr Thr Thr Phe Tyr Ala

20 25 30

Asn Met Leu Ala Ala His Pro Glu Leu Lys Asn Tyr Phe Ser Leu Arg

35 40 45

Asn Gln Gln Thr Gly Ala Gln Gln Ala Ala Leu Ala Asn Ser Val Leu

50 55 60

Ala Ala Ala Thr Tyr Ile Asp Asn Leu Ala Val Ile Ala Gly Ala Val

65 70 75 80

Glu Arg Ile Ala Gln Lys His Ala Ser Leu Phe Ile Lys Pro Glu His

85 90 95

Tyr Pro Ile Val Gly Lys Tyr Leu Ile Gly Ala Phe Glu Gln Ile Leu

100 105 110

Gly Asp Ala Phe Thr Pro Glu Ile Lys Asp Ala Trp Val Thr Ala Tyr

115 120 125

Gly Ile Leu Ala Asp Ile Phe Ile Lys Arg Glu Gln Gln Leu Tyr Ala

130 135 140

Glu Ala Gly Trp His His His His His His

145 150

<210> 3

<211> 2908

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gactagactg acccccccgg ttgggcccct cgtcccgtct ccaacagagc accaccagac 60

aaagacccct gcccgcgcga atccagaccc ccccagcaat tccgggcctc gttgatcctc 120

ctctactgta gttgtacata catacctacc gactgcattg cattggtaca gctgcaggca 180

cttccaggca cggccaccaa attgcagcgg cccttgcttg cttgcttggt tcgagaacta 240

ggatctgtgt cttttgcctt gccttgtctt gtctgggttc ctgctcgtct gcggcaatcg 300

gaacgccgcc agtgcggtgc caagcaaacc agccaaggta ggtacctacc actaggcttc 360

ttttctcgtt gtctcactct ctcttttcct ctttgtcctc tcttatcccc atcttttctc 420

tctctctctg ctcctttcct aaccacttcc ctacctttct ctttttcctt ttcttgtcat 480

ctccatcttg gctgacgaaa aaggtctgac tgggtaggta ttatctggca gacttgtgtg 540

tatcattcac cctatttctg cttcatagta catgtactgt acctgaacgg ctcaaccgct 600

atttacgact cttatttttt tgtggcgttg gtcacgtttg ccagctgttg tccgtctttc 660

tagggctcct caaacttgac ctgaccgagc tccctttctg gacccggtgg gcttcacttc 720

cagctgctga gcgacctgag ccgaacatcc tcagtccttg tccagcgcaa ttcattttct 780

ttccttttct ttttttttat tcctttcttt acttttattc tctctttttc tcctcttcct 840

cttcttcttc tttctcctcc tcctccatat cctcactctc gtctccctca ttactaccct 900

ctcggctcct caggtccacc aaccctcccg cacccaaacc tctgccgctg aaacccattc 960

ggtggtcgcc gttttttttt tttttttttt ctcaccccca aagtcgcaat atcgggtatc 1020

gccgccggca ttgaatcgcc ttctccgcta gcatcgacta ctgctgctct gctctcgttg 1080

ccagcgctgc tccctagaat tttgaccagg ggacgagccc gacattaaag caactccctc 1140

gcctcgagac gactcggatc gcacgaaatt ctcccaatcg ccgacagttc ctactcctct 1200

tcctcccgca cggctgtcgc gcttccaacg tcattcgcac agcagaattg tgccatctct 1260

ctcttttttt tccccccctc taaaccgcca caacggcacc ctaagggtta aactatccaa 1320

ccagccgcag cctcagcctc tctcagcctc atcagccatg gcaccacacc cgacgctcaa 1380

ggccaccttc gcggccagga gcgagacggc gacgcacccg ctgacggctt acctgttcaa 1440

gctcatggac ctcaaggcgt ccaacctgtg cctgagcgcc gacgtgccga cagcgcgcga 1500

gctgctgtac ctggccgaca agattggccc gtcgattgtc gtgctcaaga cgcactacga 1560

catggtctcg ggctgggact tccacccgga gacgggcacg ggagcccagc tggcgtcgct 1620

ggcgcgcaag cacggcttcc tcatcttcga ggaccgcaag tttggcgaca ttggccacac 1680

cgtcgagctg cagtacacgg gcgggtcggc gcgcatcatc gactgggcgc acattgtcaa 1740

cgtcaacatg gtgcccggca aggcgtcggt ggcctcgctg gcccagggcg ccaagcgctg 1800

gctcgagcgc tacccctgcg aggtcaagac gtccgtcacc gtcggcacgc ccaccatgga 1860

ctcgtttgac gacgacgccg actccaggga cgccgagccc gccggcgccg tcaacggcat 1920

gggctccatt ggcgtcctgg acaagcccat ctactcgaac cggtccggcg acggccgcaa 1980

gggcagcatc gtctccatca ccaccgtcac ccagcagtac gagtccgtct cctcgccccg 2040

gttaacaaag gccatcgccg agggcgacga gtcgctcttc ccgggcatcg aggaggcgcc 2100

gctgagccgc ggcctcctga tcctcgccca aatgtccagc cagggcaact tcatgaacaa 2160

ggagtacacg caggcctgcg tcgaggccgc ccgggagcac aaggactttg tcatgggctt 2220

catctcgcag gagacgctca acaccgagcc cgacgatgcc tttatccaca tgacgcccgg 2280

ctgccagctg ccccccgaag acgaggacca gcagaccaac ggatcggtcg gtggagacgg 2340

ccagggccag cagtacaaca cgccgcacaa gctgattggc atcgccggca gcgacattgc 2400

cattgtgggc cggggcatcc tcaaggcctc agaccccgta gaggaggcag agcggtaccg 2460

atcagcagcg tggaaagcct acaccgagag gctgctgcga taggggaggg aagggaagaa 2520

agaagtaaag aaaggcattt agcaagaagg gggaaaaggg agggaggaca aacggagctg 2580

agaaagagct cttgtccaaa gcccggcatc atagaatgca gctgtattta ggcgacctct 2640

ttttccatct tgtcgatttt tgttatgacg taccagttgg gatgatggat gattgtaccc 2700

cagctgcgat tgatgtgtat ctttgcatgc aacaacacgc gatggcggag gcgaactgca 2760

cattggaagg ttcatatatg gtcctgacat atctggtgga tctggaagca tggaattgta 2820

tttttgattt ggcatttgct tttgcgcgtg gagggaacat atcaccctcg ggcatttttc 2880

atttggtagg atggtttgga tgcagttg 2908

Claims

1. A method for improving the yield of cellulase produced by Trichoderma reesei comprises the following steps: introducing specific DNA molecules into trichoderma reesei, thereby promoting the trichoderma reesei to produce cellulase; the specific DNA molecule has a coding gene of a trichoderma reesei hemoglobin structural domain;

the specific DNA molecule has a gpdA promoter and a gene encoding a Trichoderma reesei hemoglobin domain, and the expression of the gene encoding the Trichoderma reesei hemoglobin domain is promoted by the gpdA promoter;

the trichoderma reesei hemoglobin structural domain is a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table.

2. The method of claim 1, wherein: the encoding gene of the trichoderma reesei hemoglobin structural domain is a DNA molecule shown by nucleotides 2311 to 2775 from the 5' end of a sequence 1 in a sequence table.

3. A specific DNA molecule shown in a sequence 1 in a sequence table.

4. Use of the specific DNA molecule of claim 3 or a recombinant expression vector comprising the specific DNA molecule of claim 3 for increasing the yield of cellulase produced by Trichoderma reesei.

5. A recombinant bacterium obtained by introducing the specific DNA molecule according to claim 3 into Trichoderma reesei.

6. Use of the recombinant bacterium of claim 5 for the production of cellulase.

7. A method of producing cellulase comprising the steps of: culturing the recombinant strain of claim 5 to obtain cellulase.