A Trichoderma reesei engineered bacterium for producing lipase and its construction method and application

Abstract

The present invention discloses an engineered Trichoderma reesei for producing lipase, and a construction method and application thereof, and belongs to the fields of molecular biology and biotechnology. The engineered Trichoderma reesei heterologously expresses a lipase having an amino acid sequence as shown in SEQ ID NO.1; in the engineered Trichoderma reesei, the genes of the original cellulases cbh1 and cbh2 and xylanases xyn1 and xyn2 in Trichoderma reesei are replaced in sequence with genes encoding the lipase; the nucleotide sequence of the gene encoding the lipase is shown in SEQ ID NO.2. The present invention successfully constructs an engineered Trichoderma reesei strain that secretes and expresses lipase. The engineered strain can be used for the production of food-grade lipase, and the produced lipase meets food safety standards, and can significantly improve the specific volume and whiteness of baked goods (such as bread), which will be more suitable for actual food industry needs.

Description

Technical Field

[0001] The invention relates to the fields of molecular biology and biotechnology, and in particular to an engineering bacterium of Trichoderma reesei for producing lipase, a construction method and application thereof. Background Art

[0002] Lipase, also known as triglyceride hydrolase, can catalyze the hydrolysis of triglycerides to form diglycerides, monoglycerides or glycerol. Lipase plays an important role in improving flour quality, including: (1) Increasing the volume of bread and strengthening gluten: soft and strong bread tastes better, and the expanded volume makes it more marketable. The mechanism of action of lipase is that triglyceride lipase decomposes the non-polar lipids (triglyceride lipids) contained in flour into polar lipids (mono / diglycerides). This decomposition can make the flour form a molecular structure with surfactant properties, which can better combine with water and gluten to form a stronger gluten network, increase the strength and stirring resistance of the dough, and the ability of bread to expand rapidly in the oven, increase the volume of baked products, make the texture delicate and uniform, the core soft, and the taste better. (2) Whitening steamed buns: The whiteness of steamed buns is an important indicator of flour quality. If the surface of steamed buns is relatively white and smooth, it means that the flour used is of good quality, and this type of steamed buns is also more attractive to consumers. The mechanism of action of lipase in whitening steamed bread is that lipase decomposes fat, releasing the pigment dissolved in fat, and the pigment is oxidized and faded by oxygen in the air, achieving a secondary whitening effect. This application characteristic of lipase is widely used in whitening steamed bread, especially after the ban on the use of benzoyl peroxide in flour, this application has been more recognized. In addition, the mono / diglyceride produced by lipase hydrolyzing fat can combine with starch to play a lubricating and separating role, thereby effectively blocking the recrystallization between starch particles and delaying starch aging.

[0003] Trichoderma reesei is a thermophilic saprophytic filamentous fungus. During World War II, it was found on rotten cotton clothes and canvas tents by American soldiers stationed in the Solomon Islands. 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 also become a common host for expressing homologous and heterologous proteins due to its diverse post-translational modifications. The cellulases produced by Trichoderma reesei are widely used in various industries. For example, the two cellulase promoters with the highest expression levels, cbh1 and cbh2, have been successfully used to express heterologous proteins in Trichoderma reesei.

[0004] Lipase is usually obtained by expressing Pichia pastoris, but Pichia pastoris is not a food-safe strain and requires methanol to induce gene expression, which does not meet food safety standards. Trichoderma reesei is a food-safe strain, but it expresses too many endogenous enzymes, especially cellulase and xylanase, which are not conducive to dough forming and cannot be used in baked goods. Summary of the invention

[0005] The purpose of the present invention is to provide an engineered Trichoderma reesei for producing lipase and a construction method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention successfully constructs an engineered Trichoderma reesei strain that secretes and expresses lipase. The engineered strain can be used for the production of food-grade lipase, and the produced lipase can significantly improve the specific volume and whiteness of baked foods (such as bread).

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an engineered Trichoderma reesei for producing lipase, wherein the engineered Trichoderma reesei heterologously expresses a lipase having an amino acid sequence as shown in SEQ ID NO.1; in the engineered Trichoderma reesei, the genes of the original cellulases cbh1 and cbh2 and the xylanases xyn1 and xyn2 in Trichoderma reesei are replaced in sequence with genes encoding the lipase;

[0008] The nucleotide sequence of the gene encoding the lipase is shown in SEQ ID NO.2.

[0009] The present invention also provides a set of plasmids for constructing the engineered bacteria of Trichoderma reesei, which sequentially include plasmids for replacing genes of cellulases cbh1 and cbh2 and xylanases xyn1 and xyn2, and the plasmids sequentially include the promoter sequence of the cellulase or xylanase gene to be replaced, the gene sequence encoding the lipase and the terminator sequence of the cellulase or xylanase gene to be replaced.

[0010] The present invention also provides a method for constructing the engineered Trichoderma reesei, comprising the steps of using the plasmid to sequentially replace the gene sequences of the original cellulases cbh1 and cbh2 and xylanases xyn1 and xyn2 in Trichoderma reesei with the gene sequences encoding the lipase.

[0011] Furthermore, when replacing the original gene sequences of cellulases cbh1 and cbh2 and xylanases xyn1 and xyn2 in Trichoderma reesei in sequence, after each replacement of a gene sequence, the strain with the highest lipase activity among the obtained process strains is used as the host for the next replacement until all the gene sequences of cellulases cbh1 and cbh2 and xylanases xyn1 and xyn2 are replaced.

[0012] The present invention also provides application of the Trichoderma reesei engineering bacteria in producing lipase.

[0013] The present invention also provides a method for producing lipase, comprising fermenting and culturing the engineered Trichoderma reesei to obtain the lipase.

[0014] The present invention also provides application of the lipase obtained by the method in preparing baked food.

[0015] Furthermore, the baked food comprises bread.

[0016] The invention also provides application of the lipase obtained by the method in improving the specific volume and whiteness of baked food.

[0017] Furthermore, the baked food comprises bread.

[0018] The present invention discloses the following technical effects:

[0019] The present invention utilizes the Trichoderma reesei expression plasmid to construct a lipase expression module, transforms the coding gene of the lipase into the Trichoderma reesei genome, and sequentially replaces the two cellulase genes (cbh1 and cbh2) with the highest secretion expression in Trichoderma reesei, as well as two xylanase genes (xyn1 and xyn2). The present invention successfully constructs a Trichoderma reesei engineering strain that secretes and expresses lipase. The engineering strain can be used for the production of food-grade lipase, and the produced lipase meets food safety standards, and can significantly improve the specific volume and whiteness of baked goods (such as bread), which will be more suitable for actual food industry needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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.

[0021] Figure 1 This is a diagram for constructing the Trichoderma reesei expression plasmid (A) and the lipase expression module (B) in the present invention;

[0022] Figure 2 Flow chart (A) of the strain construction of the present invention and its corresponding enzyme activity (B);

[0023] Figure 3 The figure shows the effect of different lipase addition amounts on the specific volume of bread in Example 4 of the present invention. DETAILED DESCRIPTION

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] In an embodiment of the method of the present invention, the formula of the culture medium and reagents is as follows:

[0030] (1) Luria Bertani (LB) medium formula: 5 g yeast powder, 10 g peptone, 10 g sodium chloride, dilute to 1 L with tap water, natural pH.

[0031] (2) Glucose or xylose PDA medium formula: 200 g potato, 20 g glucose (or xylose), 20 g agar, dilute to 1 L with tap water, natural pH.

[0032] (3) Formula of Trichoderma reesei fermentation medium (1 L): 30 g glucose, 10 g lactose, 14 g ammonium gluconate, 10 g cellulose powder, 5 g bran, 5 g corn steep liquor, 3 g peptone, 2 g yeast powder, 2.0 g KH2PO4, 0.34 g CaCl2, 0.3 g MgSO4·7H2O, 1 mL Mandels trace element solution and 1 mL Tween 80.

[0033] (4) Formula of Mandels trace element solution (1000×): FeSO4·7H2O 5 g, CoCl·6H2O 2 g, ZnSO4·7H2O 1.4 g, MnSO4·H2O 1.6 g, and pure water to make up to 1 L.

[0034] (5) 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 on IM plate culture 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 on glucose PDA plates supplemented with cefotaxime (300 μg / mL) and hygromycin B (75 μg / mL) until hyphae and spores grew. PCR verification was then performed to prove that the grown bacteria were the correct transformants.

[0035] (6) 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 in xylose PDA plates, hygromycin resistance will be lost, and the deletion efficiency is close to 70-100%; the deleted strain cannot grow in PDA plates with hygromycin, and this feature can be used to verify the deleted strain.

[0036] The Trichoderma reesei starting strain used in the embodiment of the method of the present invention is Trichoderma reesei RUT-C30 (ATCC56765). This scheme is also applicable to other Trichoderma reesei strains, such as QM9414 (ATCC 26921), QM6a (ATCC 13631), RL-P37 (NRRL 15709) and NG14 (ATCC 56767).

[0037] 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 can also be used instead.

[0038] The experimental methods in the following examples where specific conditions are not specified were generally carried out under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).

[0039] The amino acid sequence of the lipase protein in the following examples is shown in SEQ ID NO.1.

[0040] SEQ ID NO.1:

[0041] MVSFISISQGVSLCLLVSSMMLGSSAVPVAGHKGSVKATNGTDFQLPPLISSRCTPSHPETTGDPDAEAYYINKSVQCYQAHGGNYTALIKRDTETVGGMTLDLPENPPPIPATSTAPSSDSGEVVTATAAQIKELTNYAGVAATAYCRSVVPGTKWDCKQCLKYVPDGKLIKTFTSLHTDTNGFILRSDAQKT IYVTFRGTNSFRSAITDMVFTFSDYSPVKGAKVHAGFLFSYNQVVKDYFPVVQDQLTAYPDYKVIVTGHSLGGAQALLAGMDLYQREKRLSPKNLSIYTVGCPRVGNNAFAYYVDSTGIPFHRTVHKRDIVPHVPPQAFGYLHPGVESWIKEDPADVQICTSNIETKQCSNSIVPFTSIADHHLTYFGINEGSCL.

[0042] The nucleotide sequence encoding the lipase protein is shown in SEQ ID NO.2, which is a nucleotide sequence optimized according to the codon preference of Trichoderma reesei.

[0043] SEQ ID NO.2:

[0044]

[0045] The nucleotide sequence of the promoter Pxyn1 of the xyn1 gene is shown in SEQ ID NO.3.

[0046] SEQ ID NO.3:

[0047]

[0048] The terminator Tcbh1 nucleotide sequence of the cbh1 gene is shown in SEQ ID NO.4.

[0049] SEQ ID NO.4:

[0050] GTTCTGTTGATGTTGACTTGGAGTGGATGAGGGGTTTGAGCTGGTATGTAGTATTGGGGTGGTTAGTGAGTTAACTTGACAGACTGCACTTTGGCAACAGAGCCGACGATTAAGAGATTGCTGTCATGTAACTAAAGTAGCCTGCCTTTGACGCTGTATGCTCATGATACATGCGTGACATCGAAATATATCAGCCAAAGTATCCGTCCGGCGACATGCCCATCAACTATATTGAAGTCAGAAACACACTGTCCCTCTTCCCTCCTATGCTTTTACAAGCTGCTCCTCTATCCGCCCCCACAGTCCCTTGTTCATATACCCCGAAAGCCAAAAGTTTCCATCCTTGTCCTTGCCCATGATCGGGAAGCCGTTTGGTAGCACGATACCCCACTGATTATTCTGTATATAGATCGGTGAACCCGATTTCCCACCCTCCCTACTGGGCTGAAGCACAGCTGCAGAAAAGTCCAAGTCGAACAGCTTTGCCTTGCCCCAATTTGACAACGTAATCATGTGCATGTTGCCGTTGCCGAAGAAAGGCGGAATCCTCCCGCTAGATCCTCGCCACATAGCGAAAAAGGCTTCTACCTGAGACCGAGTTCCCAGTTCTTGAATCGCGGTTCGAGTAGCAGCAGCAATATAACTCAGCGGCTTC.

[0051] The promoter Pxyn2 nucleotide sequence of the xyn2 gene is shown in SEQ ID NO.5.

[0052] SEQ ID NO.5:

[0053]

[0054] The nucleotide sequence of the terminator Txyn2 of the xyn2 gene is shown in SEQ ID NO.6.

[0055] SEQ ID NO.6:

[0056] AGGGGGCTCTTCTTTTGTGATGTGTGAAAAAAAAAAAAAGGATGGTGGATAAAAGGGGGTTGAAGGGCTTGTGTTCTTTGGCTCTGTAGAGGCTCTAGGGGGTTGGATTGGGCTGACTATGGTCTCCCCTTTGTATGCTACACACTGATCAGACGATCGTAGTCAGTTGAGAGATGAGCTTCATTGGTCGATATGAGTGCTTTGAGACTGGACACTTGCGTGACTGCATGTTCCGTTTCTTGTGATAATTGCCGTGAAGATGTGTCTGCTGGTATGACGACAAAGTAGCATCAGCAGCAAAGTATGGCGAGGACTACTAGGCCCAAGATATTGTCGGCTTCGTCAAAGTGAGAAATTTTAGGGTCTCCATTGATTACATTCATCCGTTTCAGAAGCACGATATAGAGGAGTAGCACCATCAGGTCGGAAAGGTCGTCAACAGCTTGAACAAGTCATACTAAGGTATGTTAGTGTGGTCGTGGTTGTCGGTATCAGTAGGTATCATTGGAGTATGTGTTATATACAGATTAATGGGACCAATGCATCACGCATCAGGACATGCCCTGGCAATCCTCCCTCTTACAAATCGCGATGAGGCTTGGGTTTGGGGGTAATAGTAGCGGTCGTAAAGGTGGTGGTGCCAGTAGCGCACACGCAGCCCTGGCCGGAAATGCTGGTAATGGGAGGAGATTTGCAGGTGGGCTCGGTGGTAAAGGAGGCGTAGGTGCTGGTGGGCTCAACCGTGGGCTCAACCAGCTCAGCCTTGACCAGGACGGGGATGATCTGGCCTGTGGACAGGCTGAAGGAGCGGTGGTTGAGCACGGTGAGAATGGCGCCAATCTCGCCCGAGTGGCTGGTGATGCTAATCCAGGTGTTGTCGTCGTG。

[0057] The determination principle and method of lipase activity in the following examples are as follows:

[0058] Under certain conditions, lipase can hydrolyze triglycerides into fatty acids, diglycerides, monoglycerides and glycerol. The released fatty acids can be neutralized and titrated with a standard alkaline solution. The end point of the reaction can be indicated by a pH meter or phenolphthalein indicator solution. The enzyme activity can be calculated based on the amount of alkali consumed. The reaction formula is: RCOOH+NaOH→RCOONa+H2O.

[0059] 1. Definition of enzyme activity unit: 1 mL of enzyme solution or 1 g of solid enzyme powder, at 40°C and pH 7.5, hydrolyzes fat to produce 1 micromole (μmol) of fatty acids per minute, which is 1 international unit of lipase, expressed as U / mL or U / g.

[0060] 2. Reagents and solutions:

[0061] (1) Polyvinyl alcohol (PVA): degree of polymerization 1750±50 (Sinopharm Group Shanghai Chemical Reagent Company).

[0062] (2) Olive oil (Sinopharm Group Shanghai Chemical Reagent Company).

[0063] (3) 95% (V / V) ethanol (GB679).

[0064] (4) Substrate solution: Weigh 40g of polyvinyl alcohol (PVA), add 800mL of water, heat and stir in a boiling water bath until completely dissolved, and then cool and dilute to 1000mL. Filter with a clean double-layer gauze and take the filtrate, which is the 4% PVA solution. Measure 150mL of 4% PVA solution, add 50mL of olive oil, and process with a high-speed tissue crusher for 6min (2 times, 3min each time, with a rest of 5min in between) to obtain a milky white PVA emulsion. The solution should be prepared before use. If the solution is stored in a refrigerator, it is valid for one week.

[0065] (5) pH 7.5 phosphate buffer: Weigh 1.96 g of potassium dihydrogen phosphate (KH2PO4) and 39.62 g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), dissolve in water, calibrate to 7.5±0.01 with a pH meter, and then make up to 500 mL.

[0066] (6) 0.05 mol / L sodium hydroxide standard solution: prepared and calibrated in accordance with GB 601.

[0067] (7) 0.5 mol / L sodium hydroxide titration solution: prepared and calibrated in accordance with GB 601.

[0068] If the lipase is liquid, directly measure 1mL of enzyme liquid. If the lipase is solid, weigh 1g of enzyme powder and dissolve it in several times: first dissolve it with 10mL of pH 7.5 phosphate buffer in (5), and pound it with a glass rod, carefully pour the supernatant into a volumetric flask, add 5-10mL of buffer to the sediment, pound it and pour the supernatant into a volumetric flask, repeat this 3-4 times, and finally transfer all of it into a volumetric flask, make the volume up to 100mL with buffer, shake it well, transfer it to a tissue pounder and pound it for 3 minutes before use for determination. When determining the enzyme powder, refer to the following table 1 for the dilution multiple, control the enzyme solution concentration, and the difference in alkali consumption between the sample and the control is within the range of 1-2mL. Note: When aspirating the sample, the enzyme solution should be shaken well before taking it.

[0069] Table 1 Dilution multiples of enzyme activity determination

[0070] Estimated enzyme activity (U / g) Dilution multiple 4000 600 5000 750 6000 900

[0071] 3. Enzyme activity measurement:

[0072] Take two 100mL beakers, add 4.00mL of substrate solution and 5.00mL of pH 7.5 phosphate buffer to the first blank control cup A and the second sample test cup B, then add 15.0mL of 95% ethanol to cup A, and preheat in a 40±0.2℃ water bath for 5min. Then add 1.00mL of the enzyme solution to be tested to cups A and B, mix well and start timing immediately, react in a 40±0.2℃ water bath for 15min, immediately add 15.0mL of 95% ethanol to cup B to terminate the reaction, and take out;

[0073] Place a rotor in a beaker and place it on an electromagnetic stirrer. Titrate with 0.5 mol / L sodium hydroxide titration solution while stirring until pH = 10.3, which is the end point. Record the volume of 0.5 mol / L sodium hydroxide titration solution consumed.

[0074] Calculate the enzyme activity of the sample using the following formula:

[0075] X=(BA)×C / 0.05×50×1 / 15×n

[0076] =200 / 3×(BA)×C×n;

[0077] Where:

[0078] X—enzyme activity of the sample, U / g or U / mL;

[0079] B—The volume of sodium hydroxide standard solution consumed when titrating the sample, mL;

[0080] A—the volume of sodium hydroxide standard solution consumed when titrating the blank, mL;

[0081] C—the actual concentration of sodium hydroxide standard titration solution, 0.5 mol / L;

[0082] 0.05—conversion coefficient of sodium hydroxide standard solution concentration;

[0083] 1.00mL of 50-0.05mol / L sodium hydroxide solution is equivalent to 50 micromoles (μmol) of fatty acid;

[0084] 1 / 15—reaction time 15min, calculated as 1min;

[0085] n—dilution multiple. For 1g of solid enzyme, add 1000mL of buffer, which is a 1000-fold dilution; for 1mL of liquid enzyme, add 99mL of buffer, which is a 100-fold dilution.

[0086] The results are expressed as integers.

[0087] Example 1 Construction of XYN1T1.0 and XYN2T1.0 plasmids

[0088] The backbone of the vector constructed in the present invention is LmL2.0a (disclosed in the document "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"), which contains a Cre-loxP element and can realize the reuse of the screening marker.

[0089] 1. Construction of expression plasmid XYN1T1.0

[0090] (1) Primers Pxyn1-F and Pxyn1-R were used to amplify the promoter sequence Pxyn1 (such as SEQ ID NO. 3) of the xyn1 gene using the Trichoderma reesei genome as a template, excluding the homology arm sequences on the primers, a total of 1164 bp.

[0091] Pxyn1-F (SEQ ID NO.7):

[0092] 5'-GATTACGAATTCTTAATTAATCAGCGAACAAATCAGCGATT-3';

[0093] Pxyn1-R (SEQ ID NO.8):

[0094] 5'-ATTATACGAAGTTATCTAGATGATTATTTGTGCGTGTTTTCC-3'.

[0095] Amplification reaction system: 10×PCRBuffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mM MgSO4 3μL; primers (10μM each) 1.5μL; Trichoderma reesei genomic template (20ng) 1μL; KOD-Plus-Neo (1U / μL) 1μL.

[0096] Reaction program: 94°C for 2 min; 98°C for 10 sec, 58°C for 30 sec, 68°C for 45 sec, for 30 cycles; 68°C for 5 min.

[0097] (2) Primers Txyn1-1 and Txyn1-2 were used to amplify the terminator sequence Tcbh1 (such as SEQ ID NO. 4) of the cbh1 gene using the Trichoderma reesei genome as a template, excluding the homology arm sequences on the primers, a total of 655 bp.

[0098] Txyn1-1 (SEQ ID NO.9):

[0099] 5'-ACTAGTGAGCTCATTTGTTCTGTTGATGTTGACTTGGA-3';

[0100] Txyn1-2 (SEQ ID NO.10):

[0101] 5'-AGTGCCAAGCTTATTTGAAGCCGCTGAGTTATATTGC-3'.

[0102] Amplification reaction system: 10×PCRBuffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mM MgSO4 3μL; primers (10μM each) 1.5μL; Trichoderma reesei genomic template (20ng) 1μL; KOD-Plus-Neo (1U / μL) 1μL.

[0103] Reaction program: 94°C for 2 min; 98°C for 10 sec, 58°C for 30 sec, 68°C for 35 sec, for 30 cycles; 68°C for 5 min.

[0104] (3) An expression vector was constructed using LmL2.0a as the backbone. First, double digestion was performed using restriction endonucleases PacI / XbaI, homologous recombination was performed using Vazyme One Step Clone Kit, and the promoter sequence Pxyn1 of xyn1 was connected. Then, single digestion was performed using restriction endonuclease SwaI, homologous recombination was performed using Vazyme One Step Clone Kit, and the terminator sequence Txyn1 of xyn1 was connected. This is the expression plasmid XYN1T1.0 ( Figure 1 (A).

[0105] 2. Construction of expression plasmid XYN2T1.0

[0106] (1) Primers Pxyn2-F and Pxyn2-R were used to amplify the promoter sequence Pxyn2 (such as SEQ ID NO. 5) of the xyn2 gene using the Trichoderma reesei genome as a template, excluding the homology arm sequences on the primers, a total of 1187 bp.

[0107] Pxyn2-F (SEQ ID NO.11):

[0108] 5'-GATTACGAATTCTTAATTAATTCAACTCAACCGACCTTCCA-3';

[0109] Pxyn2-R (SEQ ID NO.12):

[0110] 5'-TACATTATACGAAGTTATTTAAATGTTGATGTCTTCTTGCTTCAGCTA-3'.

[0111] Amplification reaction system: 10×PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mM MgSO4 3μL; primers (10μM each) 1.5μL; Trichoderma reesei genomic template (20ng) 1μL; KOD-Plus-Neo (1U / μL) 1μL.

[0112] Reaction program: 94°C for 2 min; 98°C for 10 sec, 58°C for 30 sec, 68°C for 45 sec, for 30 cycles; 68°C for 5 min.

[0113] (2) Primers Txyn2-1 and Txyn2-2 were used to amplify the terminator sequence Txyn2 (such as SEQ ID NO. 6) of the xyn2 gene using the Trichoderma reesei genome as a template, excluding the homology arm sequences on the primers, a total of 885 bp.

[0114] Txyn2-1 (SEQ ID NO.13):

[0115] 5'-ACTAGTGAGTCATTTAGGGGGCTCTTCTTTTGTGAT-3';

[0116] Txyn2-2 (SEQ ID NO.14):

[0117] 5'-AGTGCCAAGCTTATTTCACGACGACAACACCTGGAT-3'.

[0118] Amplification reaction system: 10×PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mM MgSO4 3μL; primers (10μM each) 1.5μL; Trichoderma reesei genomic template (20ng) 1μL; KOD-Plus-Neo (1U / μL) 1μL.

[0119] Reaction program: 94°C for 2 min; 98°C for 10 sec, 58°C for 30 sec, 68°C for 30 sec, for 30 cycles; 68°C for 5 min.

[0120] (3) An expression vector was constructed using LmL2.0a as the backbone. First, restriction endonuclease SwaI was used for single digestion, and homologous recombination was performed using the Vazyme One Step Clone Kit to connect the terminator sequence Txyn2 of xyn2. Then, restriction endonucleases PacI / XbaI were used for double digestion, and homologous recombination was performed using the Vazyme One Step Clone Kit to connect the promoter sequence Pxyn2 of xyn2, thus obtaining the expression plasmid XYN2T1.0( Figure 1 (A).

[0121] The expression vector used here mainly provides the function of Agrobacterium conjugation transfer site, so, in addition to LmL2.0a, any of the following Agrobacterium binary vectors can be used, such as pZP100, pPZP201, pPZP201B, pPZP201BK, pBI121, pCAMBIA, pPK2 or LmL2.1, but not limited to these vectors.

[0122] Example 2 Construction of the expression module of the lipase gene

[0123] (1) The method for obtaining the lipase 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.

[0124] (2) Provide the gene company with an empty expression plasmid without a gene connected, and inform the gene 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 lipase, eliminating the steps of PCR, restriction digestion, and ligation.

[0125] (3) The two Trichoderma reesei plasmids XYN1T1.0 and XYN2T1.0 constructed in Example 1 of the present invention, and the two expression plasmids CBH1T1.0 and CBH2T1.0 constructed by the inventors in the early stage (disclosed in the patent application document with application number 202211596367.5), a total of four expression plasmids. A gene synthesis company was commissioned to recombinantly insert SEQ ID NO.2 into the XbaI restriction site (TCTAGA) after the promoter of the XYN1T1.0 and CBH1T1.0 plasmids, and to recombinantly insert SEQ ID NO.2 into the SwaI restriction site (ATTTAAAT) after the promoter of the XYN2T1.0 and CBH2T1.0 plasmids. It should be noted that after the XYN2T1.0 and CBH2T1.0 plasmids were recombined and inserted into the lipase gene, all sequences of the SwaI restriction site (8 bases - ATTTAAAT) were completely eliminated and did not remain on the plasmid. After the lipase gene is successfully inserted into the expression plasmid, it will be four lipase expression modules, such as Figure 1 As shown in B.

[0126] Example 3 Construction of an engineered strain of Trichoderma reesei expressing lipase

[0127] The four lipase expression modules in Example 2 are sequentially transformed into Trichoderma reesei, and the transformants finally obtained are the engineering 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 expression module 1 (CBH1T1.0-lip) into the Trichoderma reesei strain RUT-C30 (ATCC 56765), and the lipase will replace the cellulase cbh1, and the first generation Trichoderma reesei process strain Tr-lip-1 is obtained. After culturing in xylose PDA medium and deleting the hygromycin resistance marker, the strain with the highest lipase activity is selected as the host. Then, the expression module 2 (CBH2T1.0-lip) is transformed into the first generation Trichoderma reesei process strain Tr-lip-1 to obtain the second generation Trichoderma reesei process strain Tr-lip-2, and the lipase will replace the cellulase cbh2. After the resistance marker is deleted, the strain with the highest lipase activity is selected as the host. The expression module 3 (XYN1T1.0-lip) is transformed into the second-generation Trichoderma reesei process strain Tr-lip-2, and the lipase will replace the xylanase xyn1 to obtain the third-generation Trichoderma reesei process strain Tr-lip-3. After the resistance marker is deleted, the strain with the highest lipase activity is selected as the host. The expression module 4 (XYN2T1.0-lip) is transformed into the third-generation Trichoderma reesei process strain Tr-lip-3, and the lipase will replace the xylanase xyn2 to obtain the fourth-generation transformant. After the resistance marker is deleted, the strain with the highest lipase activity is selected, which is the final Trichoderma reesei engineering strain Tr-lip-4. In the engineering strain, the two cellulases cbh1 and cbh2, and the two xylanases xyn1 and xyn2 are replaced by lipase genes. The entire construction process is as follows. Figure 2 As shown in A. As the transformation process progresses, the activity of lipase produced by the strain in shake flask fermentation gradually increases, as shown in Figure 2 As shown in B.

[0128] The engineered strain of Trichoderma reesei Tr-lip-4 was fermented in the fermentation medium of Trichoderma reesei to induce the secretory expression of lipase. The fermentation tank 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 is completed, the fermentation broth is taken, centrifuged at 12500rpm for 10min, and the supernatant is taken, which is the crude lipase enzyme solution. The crude enzyme solution is spray-dried, and a universal protective agent is added to make a solid lipase powder with an enzyme powder activity of 100000U / g.

[0129] Example 4 Application of lipase

[0130] The lipase powder prepared in Example 3 was used to carry out bread baking experiments, with no lipase added as a control.

[0131] The dough formula for making bread in this embodiment is: 1000g wheat flour (flour), 10g yeast powder, 16g salt, 580g water, 60g sugar and 30g vegetable oil. The added amounts of lipase in the control group and the experimental group are 0 and 15ppm, respectively, with 3 replicates in each group.

[0132] The bread preparation steps are as follows: add the dough ingredients of the above formula into a dough mixing bowl, stir for 18 minutes to form a uniform dough, put the kneaded dough on a scale, divide it into 50g / piece, put it into a square mold, ferment it at 38℃ for 60 minutes, put the fermented dough into a 180℃ oven and bake it for 10 minutes to obtain the finished bread.

[0133] The prepared bread was cooled at room temperature for 2 hours, and the mass and volume of each experimental group of bread were measured. The specific volume of the bread was calculated as the volume of the bread / the mass of the bread (mL / g). The results are as follows: Figure 3 The whiteness of the bread was measured by sensory testing, and the results are shown in Table 2.

[0134] Table 2 Effect of different lipase addition amounts on the whiteness of baked goods

[0135]

[0136] Note: + represents dark color with low whiteness, ++ represents light color with high whiteness.

[0137] according to Figure 3 The data in Table 2 show that the lipase provided by the method can significantly improve the specific volume and whiteness of baked goods.

[0138] From the results of the above examples, it can be seen that the lipase produced by the secretory lipase-expressing engineered strain provided by the present invention can be applied to the dough processing process in the food industry to improve the quality of baked foods.

[0139] In summary, the present invention discloses a lipase sequence and discloses the process of genetically modifying Trichoderma reesei to successfully construct an engineered strain that secretes and expresses lipase. The lipase produced by the engineered strain can be used in the food industry to improve the quality of baked foods.

[0140] 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 for producing lipase, characterized in that: The engineered Trichoderma reesei heterologously expresses a lipase having an amino acid sequence as shown in SEQ ID NO.1; in the engineered Trichoderma reesei, the genes of the original cellulases cbh1 and cbh2 and xylanases xyn1 and xyn2 in Trichoderma reesei are replaced in sequence with genes encoding the lipase; The nucleotide sequence of the gene encoding the lipase is shown in SEQ ID NO.2; The Trichoderma reesei is Trichoderma reesei RUT-C30 with strain number ATCC 56765.

2. A set of plasmids for constructing the engineered bacteria of Trichoderma reesei according to claim 1, characterized in that: Includes a plasmid for replacing a cellulase gene and a plasmid for replacing a xylanase gene; The plasmid for replacing the cellulase gene sequentially comprises the promoter sequence of the cellulase gene to be replaced, the gene sequence encoding the lipase and the terminator sequence of the cellulase gene to be replaced; The plasmid for replacing the xylanase gene sequentially comprises the promoter sequence of the xylanase gene to be replaced, the gene sequence encoding the lipase and the terminator sequence of the xylanase gene to be replaced; The cellulase gene to be replaced is cbh1 or cbh2, and the xylanase gene to be replaced is xyn1 or xyn2; The gene sequence encoding the lipase is shown in SEQ ID NO.

2.

3. A method for constructing the engineered strain of Trichoderma reesei according to claim 1, characterized in that: The method comprises the steps of using the plasmid according to claim 2 to sequentially replace the gene sequences of the original cellulases cbh1 and cbh2 and xylanases xyn1 and xyn2 in Trichoderma reesei with the gene sequences encoding the lipase.

4. The construction method according to claim 3, characterized in that: When replacing the original gene sequences of cellulases cbh1 and cbh2 and xylanases xyn1 and xyn2 in Trichoderma reesei in sequence, after each gene sequence is replaced, the strain with the highest lipase activity among the obtained process strains is used as the host for the next replacement until all the gene sequences of cellulases cbh1 and cbh2 and xylanases xyn1 and xyn2 are replaced.

5. Use of the engineered Trichoderma reesei according to claim 1 in producing lipase.

6. A method for producing lipase, characterized in that: The method comprises fermenting and culturing the engineered Trichoderma reesei according to claim 1 to obtain the lipase.

7. Use of the lipase obtained by the method according to claim 6 in preparing baked goods.

8. The use according to claim 7, characterized in that: The baked food is bread.

9. Use of the lipase obtained by the method according to claim 6 in improving the specific volume and whiteness of baked goods.

10. The use according to claim 9, characterized in that: The baked food is bread.

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