Trichoderma reesei engineering bacterium for secreting and expressing neutral cellulase and construction method of trichoderma reesei engineering bacterium

Abstract

The invention discloses a trichoderma reesei engineering bacterium for secreting and expressing neutral cellulase and a construction method of the trichoderma reesei engineering bacterium, and belongs to the technical field of genetic engineering. In a trichoderma reesei engineering bacterium, two endogenous acidic cellulase genes cbh1 and cbh2 in the trichoderma reesei are sequentially replaced by genes for coding neutral cellulase, and the trichoderma reesei is converted into the engineering bacterium for secreting and expressing the neutral cellulase; the amino acid sequence of neutral cellulose is as shown in SEQ ID NO.1, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.2. The trichoderma reesei engineering strain for secreting and expressing the neutral cellulase is successfully constructed and has a wide pH application range, and a new strain and method are provided for production of the neutral cellulase in the fields of textile and the like.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to an engineering bacterium of Trichoderma reesei capable of secreting and expressing neutral cellulase and a construction method thereof. Background Art

[0002] Neutral cellulase generally refers to cellulase that adapts to neutral and slightly alkaline environments. Its optimum pH value is usually around 7.0, and it can also have good activity in the pH range of 4.0-8.0. Acid cellulase adapts to acidic environments with lower pH values, and its optimum pH range is generally 4.5-5.5. Its activity is greatly reduced above pH 6.0. Neutral cellulase and acid cellulase differ in many aspects, as follows:

[0003] (1) Different sources. Neutral cellulase: The most common and widely used, generally produced by bacterial culture. The yield of bacterial cellulase is relatively high, and most of the produced enzymes are secreted into the culture medium, so extraction and purification are relatively easy. Acid cellulase: Generally extracted from fungi, and the yield of fungal cellulase is also relatively high.

[0004] (2) Different application fields. Neutral cellulase: widely used in the textile printing and dyeing industry. It can be used for the stripping and patterning of denim and the softening process of pure cotton fabrics. It can make the fabrics have lasting anti-pilling properties. The treated fabrics have clear patterns, strong three-dimensional sense, low back staining, and little damage to the fabric strength. It is also used in the food industry, such as in some food processing processes to improve the texture and taste of food. Acid cellulase: commonly used in the fields of feed and biorefining, such as completely hydrolyzing cellulose in biomass into glucose. It is also used in the brewing industry, such as improving the utilization rate of raw materials.

[0005] (3) Different action characteristics. Neutral cellulase: The reaction is relatively mild, with little damage to fabrics, low sensitivity to pH fluctuations, and good process reproducibility. Acidic cellulase: The treatment effect on cotton fabrics is relatively strong, but improper treatment may lead to problems such as decreased fabric strength and significant weight loss, and there may be "re-staining" during the treatment process, that is, the indigo dye will be deposited on the white part of the clothing.

[0006] Trichoderma reesei is a mesophilic saprophytic filamentous fungus found on rotten cotton clothes and canvas tents. The isolated strain was identified as Trichoderma reesei QM6a. Starting from the original strain QM6a, a series of mutagenesis breeding was carried out to screen out some high-yield cellulase strains, such as RUT-C30. Trichoderma reesei is widely used because it has the ability to naturally secrete a large amount of endogenous proteins into the extracellular space, and the endogenous cellulase can produce up to 100g / L after fermentation. Trichoderma reesei has become a common host for expressing homologous and heterologous enzyme proteins due to its ability to efficiently express enzyme proteins. The cellulases produced by Trichoderma reesei are all acid cellulases. The two endogenous acid cellulase genes with the highest expression levels are cbh1 and cbh2. The cellulase produced by Trichoderma reesei is an acidic cellulase, which has a strong degradation effect on cotton cloth and can achieve a good stone washing effect in a short time, but it is also accompanied by a strong back-dyeing phenomenon, that is, the indigo dye hydrolyzed by the cellulase is very easy to re-adsorb on the surface of the cotton fiber for re-dyeing, which causes the contrast between the white yarn and the blue yarn formed after the indigo falls off to decrease, reducing the three-dimensional sense and beauty of the fabric. Neutral and alkaline cellulases are widely available, but the yield is low. Compared with acidic cellulases, they have a weaker binding effect on cotton cloth, and little or no back-dyeing occurs.

[0007] Neutral cellulase is usually obtained by expression of Pichia pastoris, but Pichia pastoris is not a food safety strain and does not meet food safety standards; and methanol is required to induce gene expression, which is toxic and flammable and explosive. Trichoderma reesei is a food safety strain, but it expresses too many endogenous acid cellulases, especially cbh1 and cbh2. There are no reports on using heterologous neutral cellulase genes to replace the endogenous acid cellulases cbh1 and cbh2 of Trichoderma reesei to transform Trichoderma reesei into a strain that produces neutral cellulase. Summary of the invention

[0008] The purpose of the present invention is to provide an engineered bacterium of Trichoderma reesei that secretes and expresses neutral cellulase and a method for constructing the same, so as to solve the problems existing in the above-mentioned prior art. By replacing the endogenous acid cellulase genes cbh1 and cbh2 of Trichoderma reesei with a neutral cellulase encoding gene, an engineered bacterium of Trichoderma reesei that secretes and expresses neutral cellulase is successfully constructed. The engineered bacterium can be used for the production of neutral cellulase.

[0009] To achieve the above object, the present invention provides the following solutions:

[0010] The invention provides an engineered Trichoderma reesei that secretes and expresses neutral cellulase, which is obtained by heterologously expressing neutral cellulase in Trichoderma reesei. The heterologous expression of neutral cellulase comprises the steps of sequentially replacing endogenous acid cellulase genes cbh1 and cbh2 in the Trichoderma reesei with coding genes of the neutral cellulase.

[0011] Preferably, the amino acid sequence of the neutral cellulase is shown as SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO.2.

[0012] Preferably, the Trichoderma reesei includes Trichoderma reesei RUT-C30.

[0013] The present invention also provides a method for constructing the engineered Trichoderma reesei, comprising the steps of using the coding gene of neutral cellulase to sequentially replace the endogenous acid cellulase genes cbh1 and cbh2 in Trichoderma reesei.

[0014] Preferably, the sequential replacement method comprises the following steps:

[0015] The coding gene of neutral cellulase was respectively combined with expression plasmid I and expression plasmid II to construct recombinant plasmid I and recombinant plasmid II;

[0016] The recombinant plasmid I is transferred into Trichoderma reesei, and the gene encoding neutral cellulase replaces the cbh1 gene to obtain the first-generation Trichoderma reesei; the first-generation Trichoderma reesei with the highest neutral cellulase activity is screened as the host by utilizing the lack of hygromycin resistance, and the recombinant plasmid II is transferred into the host, and the gene encoding neutral cellulase replaces the cbh2 gene to obtain the second-generation Trichoderma reesei; the second-generation Trichoderma reesei with the highest neutral cellulase activity is screened by utilizing the lack of hygromycin resistance, which is the engineered Trichoderma reesei.

[0017] Preferably, the expression plasmid I comprises the promoter and terminator of the endogenous acid cellulase gene cbh1, and the expression plasmid II comprises the promoter and terminator of the endogenous acid cellulase gene cbh2.

[0018] Preferably, the recombinant plasmid I comprises the promoter and terminator of the endogenous acid cellulase gene cbh1, and the coding gene of the neutral cellulase, and the recombinant plasmid II comprises the promoter and terminator of the endogenous acid cellulase gene cbh2, and the coding gene of the neutral cellulase.

[0019] The present invention also provides application of the engineered Trichoderma reesei in producing neutral cellulase.

[0020] The present invention also provides a method for producing neutral cellulase, comprising the steps of fermenting and culturing the engineered Trichoderma reesei and obtaining the neutral cellulase from the supernatant of the fermentation liquid.

[0021] The present invention also provides the application of the engineered Trichoderma reesei or the neutral cellulase obtained by the method in the field of textiles.

[0022] Preferably, it is applied to the stone washing process.

[0023] The present invention discloses the following technical effects:

[0024] The present invention uses the Trichoderma reesei expression plasmid to construct a neutral cellulase expression module, transforms the coding gene of the neutral cellulase into the Trichoderma reesei genome, and sequentially replaces the two endogenous acid cellulase genes (cbh1 and cbh2) with the highest secretion expression in Trichoderma reesei. The present invention successfully constructs a Trichoderma reesei engineering strain that secretes and expresses neutral cellulase. The engineering strain can be used for the production of neutral cellulase, and the produced neutral cellulase meets food safety standards, has no methanol toxicity and flammable and explosive hazards, and will be more suitable for the needs of actual food, feed and textile industries.

[0025] The present invention also applies the neutral cellulase expressed by the constructed Trichoderma reesei engineering strain to the stone washing process, and finds that the produced neutral cellulase can significantly increase the stone washing effect of denim, providing a scientific basis for applying the Trichoderma reesei engineering strain or the neutral cellulase produced by it to the field of placement. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 A diagram showing the construction of the neutral cellulase expression module of the present invention;

[0028] Figure 2 Flow chart (A) for the construction of the engineered strain of Trichoderma reesei of the present invention and its corresponding enzyme activity (B);

[0029] Figure 3 The denim is treated with an enzyme solution produced by the engineered strain Tr-Cel-2 of Trichoderma reesei of the present invention. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The main culture media and reagents involved in the following examples are:

[0036] (1) LB medium formula: 5 g yeast powder, 10 g peptone, 10 g sodium chloride, dilute to 1 L with tap water, natural pH.

[0037] (2) Formula of glucose or xylose PDA medium: 200 g potato, 20 g glucose (or xylose), 20 g agar, dilute to 1 L with tap water, natural pH.

[0038] (3) Formula of Trichoderma reesei fermentation medium (1 L): lactose 40 g, ammonium sulfate 6 g, cellulose powder 10 g, bran 5 g, corn steep liquor 15 g, peptone 3 g, yeast powder 2 g, KH 2 PO 4 2.0 g, CaCl2 0.34 g, MgSO 4 7H 2 O 0.3g, Mandels trace element solution 1mL and Tween 80 1mL.

[0039] (4) Formula of Mandels trace element solution (1000×): FeSO 4 7H 2 O 5g, CoCl·6H 2 O 2g, ZnSO 4 7H 2 O 1.4 g, MnSO 4 ·H 2 O 1.6g, dilute to 1L with purified water.

[0040] (5) The plasmid extraction kit was purchased from AXYGEN, the gel recovery kit was purchased from MAGEN, the seamless cloning kit was purchased from Quanshijin, and the DNA restriction endonuclease and ligase were purchased from NEB. Similar products from other companies may also be used instead.

[0041] The Trichoderma reesei starting strain used in the following examples is Trichoderma reesei RUT-C30 (ATCC 56765, which can be purchased through conventional commercial channels). This protocol is also applicable to other Trichoderma reesei strains, such as QM9414 (ATCC 26921), QM6a (ATCC 13631), RL-P37 (NRRL 15709) and NG14 (ATCC 56767).

[0042] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).

[0043] Some of the experimental methods involved in the following examples:

[0044] 1. Agrobacterium conjugative transfer of Trichoderma reesei: The expression module was electroporated into Agrobacterium tumefaciens, and then the successfully electroporated Agrobacterium was co-cultured with the Trichoderma reesei host strain in IM plate medium (Covert et al. Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol. Res. 105(3): 259-264) for Agrobacterium tumefaciens-mediated conjugative transfer. After 2 days of co-cultivation, the cells were screened in glucose PDA plates supplemented with cefotaxime (300 μg / mL) and hygromycin B (75 μg / mL) until hyphae and spores grew, and then PCR verification was performed to prove that the grown bacteria were correct transformants.

[0045] 2. Trichoderma reesei resistance marker deletion scheme: After the hygromycin resistance marker is eliminated in each round of engineered strains according to the literature scheme (Zhang et al. Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN 1.0. Scientific Reports. 2016, 6: 20761), the next round of genetic modification can be carried out. The principle is as follows: by culturing and passage on xylose PDA plates, hygromycin resistance will be lost, and the deletion efficiency is close to 70-100%; the deleted strain cannot grow on PDA plates with hygromycin, and this feature can be used to verify the deleted strain.

[0046] 3. The principle and method of determining the activity of neutral cellulase are as follows:

[0047] Under certain conditions, neutral cellulase can hydrolyze sodium carboxymethyl cellulose (CMCNa) to produce reducing sugars. The amount of reducing sugars is measured by the DNS method to calculate its enzyme activity.

[0048] 3.1 Definition of the activity unit of neutral (acid) cellulase: 1 mL of enzyme solution, at 50°C and pH 7.0 (pH 5.0 for acid cellulase), hydrolyzes CMCNa to produce 1 μmol of reducing sugar per minute, which is 1 international unit of neutral (acid) cellulase, expressed as U / mL.

[0049] 3.2 Reagents and solutions:

[0050] (1) CMCNa solution (pH 7.0): Dissolve CMCNa in pH 7.0 phosphate buffer (50 mM) to a concentration of 1% and dissolve until clear.

[0051] (2) DNS solution (1 L): 6.3 g of 3,5-dinitrosalicylic acid and 262 mL of NaOH solution (2 M) were added to 500 mL of a solution containing 182 g of sodium potassium tartrate, followed by 5 g of sodium sulfite and 5 g of crystalline phenol. Stir to dissolve the mixture and then cool and add ddH 2 O is diluted to 1L, stored in a brown bottle for 7 days, and can be used after precipitation and filtration.

[0052] 3.3 Drawing of glucose standard curve:

[0053] 1) Prepare 1 mg / mL glucose standard solution;

[0054] 2) Take 0 μL, 20 μL, 40 μL, 60 μL, 80 μL and 100 μL of glucose standard solution into EP tubes respectively, add ddH 2 O until the total volume in the tube is 200 μL;

[0055] 3) Add 300 μL of DNS solution to each tube (keep away from light), mix well and place in a thermostat, set the temperature to 99°C, and the reaction time to 10 min;

[0056] 4) Take 40 μL of the above reaction solution and put it into a 96-well plate, then add 160 μL of ddH 2 O, mix by pipetting and read the absorbance at 540 nm using a microplate reader;

[0057] 5) Subtract the absorbance value of the blank sample from the absorbance value of the glucose sample. Set up three parallels for each concentration and take the average of the measured absorbance values. This average is used as the horizontal axis and the corresponding molar amount of glucose is used as the vertical axis. Then draw a standard curve and obtain a linear equation.

[0058] 3.4 Enzyme activity measurement:

[0059] 1) Take 180 μL of CMCNa solution (1%) and dispense it into EP tubes, then add 20 μL of enzyme solution (it can be diluted with pH 7.0 phosphate buffer (50 mM) according to experience, and the enzyme solution is kept on ice throughout the process), set a blank control (no enzyme solution), mix thoroughly and place in a constant temperature instrument, set the temperature to 50°C, and the reaction time is 15 min;

[0060] 2) After 15 minutes of reaction, add 300 μL of DNS solution to each EP tube, mix well and place in a constant temperature instrument, set the temperature to 99°C for color development, and the reaction time is 10 minutes;

[0061] 3) Take 40 μL of the above reaction solution and put it into a 96-well plate, then add 160 μL of ddHO 2 O, mix by pipetting and read the absorbance at 540 nm using a microplate reader;

[0062] 4) The absorbance value of the control group was subtracted from the absorbance value of the experimental group, and then the activity of the neutral cellulase was calculated according to the previous glucose standard curve.

[0063] 5) When measuring the activity of acid cellulase, the phosphate buffer was changed to a pH 5.0 formula, and the CMCNa solution was prepared with a pH 5.0 phosphate buffer (50 mM) to dilute the enzyme solution.

[0064] The expression plasmid I and expression plasmid II (CBH1T1.0 and CBH2T1.0) involved in the following examples respectively contain the promoter sequence of the cellulase gene to be replaced, the deletable hygromycin resistance marker sequence and the terminator sequence of the cellulase gene to be replaced.

[0065] Example 1 Construction of recombinant plasmids I and II for neutral cellulase genes

[0066] (1) The method for obtaining the neutral cellulase encoding gene is: provide the amino acid sequence (SEQ ID NO.1), and entrust a conventional gene company to synthesize the corresponding nucleotide coding sequence. The synthesis rule can be based on the codon preference of the host. Because of the difference in hosts, the codon preference is also different. According to the preference of Trichoderma reesei codon, the final synthesized DNA sequence is shown in SEQ ID NO.2.

[0067] (2) Provide the gene company with an empty expression plasmid without a gene connected, and inform the company of the restriction site where the expression gene needs to be connected, as well as the DNA sequences upstream and downstream of the restriction site, to avoid frameshift mutations. The gene can then be directly synthesized on the provided plasmid to construct the final expression plasmid for expressing neutral cellulase, eliminating the steps of PCR, restriction digestion, and ligation.

[0068] (3) The two Trichoderma reesei plasmids constructed in Example 1 of the present invention are the expression plasmids I and II constructed by the inventors in the early stage, namely, CBH1T1.0 and CBH2T1.0 (disclosed in the patent application document with application number 202211596367.5). A gene synthesis company was commissioned to recombinant SEQ ID NO.2 into the XbaI restriction site (TCTAGA) after the promoter of the CBH1T1.0 plasmid to construct recombinant plasmid I, namely, CBH1T1.0-Cel; at the same time, SEQ ID NO.2 was recombinant into the SwaI restriction site (ATTTAAAT) after the promoter of the CBH2T1.0 plasmid to construct recombinant plasmid II, namely, CBH21T1.0-Cel. It should be noted that after the neutral cellulase gene was recombined and inserted into the CBH1T1.0 plasmid, only 2 bases (TC) remained in the 6-base sequence (TCTAGA) of the XbaI restriction site, followed by the start codon (ATG) of the neutral cellulase gene. After the neutral cellulase gene was inserted into the CBH2T1.0 plasmid, all sequences of the SwaI restriction site (8 bases - ATTTAAAT) were completely removed and no residues remained on the plasmid. After the neutral cellulase gene was successfully inserted into the expression plasmid, it became the neutral cellulase recombinant plasmids I and II - CBH1T1.0-Cel and CBH1T1.0-Cel. Figure 1 shown.

[0069] Example 2 Construction of an engineered strain of Trichoderma reesei expressing neutral cellulase

[0070] The neutral cellulase recombinant plasmids I and II in Example 1 are sequentially transformed into Trichoderma reesei, and the transformants finally obtained are the engineered strains of Trichoderma reesei. The present invention utilizes the Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum (Covert et al. Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol. Res. 105 (3): 259-264) to transform the recombinant plasmid I (CBH1T1.0-Cel) into the Trichoderma reesei strain RUT-C30 (ATCC 56765), and the neutral cellulase will replace the cellulase cbh1 to obtain the first generation Trichoderma reesei process strain Tr-Cel-1. After culturing in xylose PDA medium and deleting the hygromycin resistance marker, the strain with the highest neutral cellulase activity is selected as the host. Then, the recombinant plasmid II (CBH2T1.0-Cel) was transformed into the first generation Trichoderma reesei process strain Tr-Cel-1 to obtain the second generation Trichoderma reesei process strain Tr-Cel-2, and the neutral cellulase replaced the cellulase cbh2. After the resistance marker was deleted, the strain with the highest neutral cellulase activity was selected as the final Trichoderma reesei engineering strain Tr-Cel-2 expressing neutral cellulase.

[0071] In the engineered strain, the two acid cellulases cbh1 and cbh2 were replaced by neutral cellulase genes. The entire construction process is as follows Figure 2 As shown in A. The engineered strain Tr-Cel-2 of Trichoderma reesei was fermented in Trichoderma reesei fermentation medium to induce the secretory expression of neutral cellulase. The fermenter culture method refers to the literature "Chen et al. Engineering of Trichoderma reesei for enhanced degradation of lignocellulosic biomass by truncation of the cellulase activator ACE3. Biotechnol Biofuels. 2020, 13: 62". After the fermentation, the fermentation broth was taken, centrifuged at 12500 rpm for 10 minutes, and the supernatant was taken, which was the crude enzyme solution of neutral cellulase. The activity of neutral cellulase was measured under pH 7.0, as shown Figure 2 As shown in B. As can be seen from the figure, as the transformation process progresses, the activity (pH7.0) of the neutral cellulase produced by the strain in the shake flask fermentation gradually increases. The engineered strain also has good activity at pH5.0, indicating that the engineered strain has a wide pH range; while the activity of the starting strain of Trichoderma reesei at pH7.0 drops to only about 20% of that at pH5.0, and does not have a wide pH range.

[0072] Example 3 Application of neutral cellulase

[0073] Denim washed with cellulase can achieve a good effect similar to that of washing with pumice. In order to characterize the application effect of the cellulase preparation produced in the present invention under alkaline environment, acid cellulase produced by Trichoderma reesei RUT-C30 was used as a control experiment.

[0074] Experimental group: supernatant of fermentation broth of neutral cellulase engineered strain Tr-Cel-2 in a 7L tank.

[0075] Control group: supernatant of fermentation broth of the starting strain of Trichoderma reesei in a 7L tank.

[0076] Blank group: tap water.

[0077] The biological stone washing conditions were as follows: 0.5 kg blue denim (108 × 56 threads / inch), 50 L water, 40 °C, pH 7.0 (experimental group and water) or pH 5.0 (control group), and drum washing machine reaction for 90 min. The final cellulase activity in the washing machine was 0.33 IU / mL (the enzyme activity of the experimental group was measured at pH 7.0; or the enzyme activity of the control group was measured at pH 5.0). After rolling reaction for 90 min in a 100 L industrial washing machine produced by Hangxing Washing Machinery Co., Ltd. (Taizhou, China), the denim was taken out, gently rinsed with clean water to remove the enzyme solution, and dried in an oven at 60 °C to observe the stone washing effect.

[0078] Depend on Figure 3 As is known: tap water treatment cannot produce patterns; adding cellulase can produce patterns on denim. Although cellulase with the same activity (0.33IU / mL, pH 5.0 or 7.0) was added, the application effect of the enzyme solution produced by the neutral cellulase engineering strain Tr-Cel-2 was better than that produced by the starting strain of Trichoderma reesei, whether in terms of the length of the white stripes or the contrast between the blue and white yarns. Denim washed with the enzyme solution of Tr-Cel-2 can produce longer, whiter, and clearer stripes, with a more obvious sense of aging. This fully proves that the neutral cellulase preparation produced by the neutral cellulase engineering strain constructed by the method provided by the present invention has a good application effect in the biological stone washing process. This is due to the lower backstaining of the neutral cellulase and the wide pH application range of the enzyme preparation disclosed in the present invention.

[0079] From the results of the above examples, it can be seen that the present invention provides an engineered strain that secretes and expresses neutral cellulase, and a method for producing neutral cellulase.

[0080] In summary, the present invention discloses a neutral cellulase gene sequence, and discloses a process of genetically modifying Trichoderma reesei to successfully construct an engineered strain that secretes and expresses neutral cellulase.

[0081] SEQ ID NO.1:

[0082] MRSSPLLPSAVVAALPVLALAADGRSTRYWDCCKPSCGWAKKAPVNQPVFSCNANFQRITDFDAKSGCEPGGVAYSCADQTPWAVNDDFALGFAATSIAGSNEAGWCCACYELTFTS GPVAGKKMVVQSTSTGGDLGSNHFDLNIPGGGVGIFDGCTPQFGGLPGQRYGGISSRNECDRFPDALKPGCYWRFDWFKNADNPSFSFRQVQCPAELVARTGCRRNDDGNFPAVQIP.

[0083] SEQ ID NO.2:

[0084] .

[0085] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An engineered bacterium of Trichoderma reesei that secretes and expresses neutral cellulase, characterized in that: The neutral cellulase is obtained by heterologously expressing neutral cellulase in Trichoderma reesei. The heterologous expression of neutral cellulase comprises the steps of replacing the endogenous acid cellulase genes cbh1 and cbh2 in the Trichoderma reesei with the coding gene of the neutral cellulase in sequence.

2. The engineered Trichoderma reesei according to claim 1, characterized in that The amino acid sequence of the neutral cellulase is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

3. The engineered Trichoderma reesei according to claim 1, characterized in that The Trichoderma reesei includes Trichoderma reesei RUT-C30.

4. A method for constructing an engineered strain of Trichoderma reesei according to any one of claims 1 to 3, characterized in that: The invention comprises the steps of using the coding gene of neutral cellulase to sequentially replace the endogenous acid cellulase genes cbh1 and cbh2 in Trichoderma reesei.

5. The construction method according to claim 4, characterized in that: The method of sequential replacement comprises the following steps: The coding gene of neutral cellulase was respectively combined with expression plasmid I and expression plasmid II to construct recombinant plasmid I and recombinant plasmid II; The recombinant plasmid I is transferred into Trichoderma reesei, and the gene encoding neutral cellulase replaces the cbh1 gene to obtain the first-generation Trichoderma reesei; the first-generation Trichoderma reesei with the highest neutral cellulase activity is screened as the host by utilizing the lack of hygromycin resistance, and the recombinant plasmid II is transferred into the host, and the gene encoding neutral cellulase replaces the cbh2 gene to obtain the second-generation Trichoderma reesei; the second-generation Trichoderma reesei with the highest neutral cellulase activity is screened by utilizing the lack of hygromycin resistance, which is the engineered Trichoderma reesei.

6. The method according to claim 5, characterized in that The recombinant plasmid I comprises the promoter and terminator of the endogenous acid cellulase gene cbh1 and the coding gene of the neutral cellulase, and the recombinant plasmid II comprises the promoter and terminator of the endogenous acid cellulase gene cbh2 and the coding gene of the neutral cellulase.

7. Use of the engineered Trichoderma reesei according to any one of claims 1 to 3 in producing neutral cellulase.

8. A method for producing neutral cellulase, characterized in that: The method comprises the steps of fermenting and culturing the engineered Trichoderma reesei according to any one of claims 1 to 3, and obtaining neutral cellulase from the supernatant of the fermentation liquid.

9. Use of the engineered Trichoderma reesei according to any one of claims 1 to 3 or the neutral cellulase obtained by the method according to claim 8 in the textile field.

10. The use according to claim 9, characterized in that Applied to stone washing process.

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