A bidirectional promoter BDP1 of Trichoderma reesei and its application

Abstract

The present invention relates to a Trichoderma reesei bidirectional promoter BDP1 and an application thereof. The nucleotide sequence of the bidirectional promoter BDP1 is shown in SEQ ID NO.1. The Trichoderma reesei bidirectional promoter BDP1 is discovered for the first time in the present invention. By verifying the function of the promoter, it is found that the Trichoderma reesei bidirectional promoter BDP1 has a bidirectional start function, which has great advantages over unidirectional promoters and has more application prospects. The present invention also uses the promoter to construct a bidirectional expression cassette of cellulose endonuclease and exonuclease, and then constructs a Trichoderma reesei engineered bacterium with high cellulase production. Compared with the starting strain, the Trichoderma reesei engineered bacterium with high cellulase production provided by the present invention increases the expression amount of extracellular cellulase by more than 17%, and has important application value for the production of cellulase in industry.

Description

Technical Field

[0001] The invention relates to a Trichoderma reesei bidirectional promoter BDP1 and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] Degradable cellulose and hemicellulose are the main components of biomass resources. Their development and utilization have important strategic significance for my country's core social issues such as economic development and environmental protection. There are some microorganisms in nature that can use cellulose and hemicellulose as carbon sources and energy sources, playing an important role in the carbon cycle. Among them, the representative strain Trichoderma reesei can produce a variety of cellulases and hemicellulases. It is a filamentous fungus widely used in industrial production.

[0003] Since Trichoderma reesei itself has a strong protein secretion ability, it is also studied and used as a protein expression chassis. Gene transcription is the first link in the protein synthesis process, and the promoter is the primary essential element of gene transcription. At present, a variety of promoter elements have been discovered in Trichoderma reesei, including constitutive gene gpd1, cdna1, tcu1 promoters and inducible gene cbh1 promoters. However, these known promoters are all unidirectional promoters, that is, a promoter can only start the expression of a gene in a single direction. Trichoderma reesei is a filamentous fungus, and its growth and genetic transformation operation cycle is long. When multiple genes need to be expressed at the same time, it is necessary to use a unidirectional promoter to construct the strain in sequence, which greatly increases the time and material costs. In addition, the repeated use of the same promoter can also cause genetic instability. Studies have found that bidirectional promoters are widely present in mammals, plants and microorganisms. Bidirectional promoters have bidirectional initiation activity, can transcribe and express two genes at the same time, and are particularly suitable for the expression of multiple genes. They can greatly simplify the protein expression process, save time, and have many advantages. However, there is no identification and description of the bidirectional promoter of Trichoderma reesei in the prior art. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a Trichoderma reesei bidirectional promoter BDP1 and applications thereof.

[0005] The technical solution of the present invention is as follows:

[0006] A bidirectional promoter BDP1 of Trichoderma reesei, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The application of the Trichoderma reesei bidirectional promoter BDP1 as a promoter.

[0008] The application of the Trichoderma reesei bidirectional promoter BDP1 in improving the extracellular cellulase production of Trichoderma reesei strains.

[0009] A recombinant vector, wherein the recombinant plasmid vector contains a gene expression box constructed by a bidirectional promoter BDP1 of Trichoderma reesei.

[0010] The invention discloses an engineered Trichoderma reesei with high cellulase production. The engineered Trichoderma reesei with high cellulase production is constructed by using Trichoderma reesei QM9414-OEypr1-Δsor1 as a starting strain and replacing the pyr4 gene position region on the Trichoderma reesei chromatin with a gene expression cassette containing the Trichoderma reesei bidirectional promoter BDP1.

[0011] Preferably according to the present invention, the yield of cellulase expressed by the engineered Trichoderma reesei is increased by 17-37%.

[0012] The technical features of the present invention are as follows:

[0013] The bidirectional promoter BDP1 described in the present invention is from Trichoderma reesei. The sequence characteristics of the bidirectional promoter BDP1 and the example of bidirectional expression of fluorescent protein and cellulase are provided in the embodiments of the present invention. The bidirectional promoter consists of 767bp nucleotides. By constructing a promoter BDP1-driven green fluorescence and red fluorescent protein expression strain and a bidirectional promoter BDP1-driven two cellulase expression strains, it can be shown that the promoter can simultaneously express green fluorescence and red fluorescent protein, and can also simultaneously express the extracellular cellulase exo-nuclease encoded by the Ctcbh1 gene and the extracellular cellulase endo-nuclease encoded by the eg1 gene, and has a bidirectional activation function.

[0014] Beneficial effects:

[0015] The present invention first discovered the bidirectional promoter BDP1 of Trichoderma reesei. By verifying the function of the promoter, it was found that the bidirectional promoter BDP1 of Trichoderma reesei has a bidirectional activation function and can express two cellulases at the same time. Compared with the starting strain, the expression amount of extracellular cellulase of the high-yield cellulase-producing Trichoderma reesei engineered bacteria provided by the present invention is increased by more than 17%, which has great advantages over unidirectional promoters, has more application prospects, and has important application value for the production of cellulases in industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 , Schematic diagram of the bidirectional promoter BDP1 driving the green fluorescent protein and red fluorescent protein expression cassettes.

[0017] Figure 2 , Schematic diagram of the bidirectional promoter BDP1 driving the green fluorescent protein and red fluorescent protein expression vector.

[0018] Figure 3, agarose gel electrophoresis diagram verifying the schematic diagram of the construction of the red and green fluorescent protein expression strain driven by the bidirectional promoter BDP1.

[0019] Figure 4 , Schematic diagram of the simultaneous expression of red and green fluorescent proteins in the constructed strain detected by fluorescence microscopy;

[0020] In the figure, A is the visual field of the constructed strain under the bright field of the microscope, B is the green fluorescence visual field produced under the green fluorescence excitation wavelength, C is the red fluorescence visual field produced under the red fluorescence excitation wavelength, and D is the yellow fluorescence visual field produced by the superposition of red fluorescence and green fluorescence.

[0021] Figure 5 , schematic diagram of the bidirectional promoter BDP1 driving two cellulase expression cassettes.

[0022] Figure 6 , Schematic diagram of two cellulase expression vectors driven by the bidirectional promoter BDP1.

[0023] Figure 7 , agarose gel electrophoresis diagram verifying the schematic diagram of the construction of two cellulase expression strains driven by the bidirectional promoter BDP1.

[0024] Figure 8 , analysis of extracellular fermentation broth enzyme activity of two cellulase expression strains driven by the bidirectional promoter BDP1;

[0025] In the figure, A is the analysis of cellulase exo-nuclease activity in extracellular fermentation broth, and B is the analysis of cellulase endo-nuclease activity in extracellular fermentation broth.

[0026] Fig. 9 , analysis of extracellular fermentation broth enzyme activity of two cellulase expression strains driven by bidirectional promoter BDP1 under cellulose culture conditions;

[0027] In the figure: A is the analysis of cellulase exonuclease activity in extracellular fermentation broth, B is the analysis of cellulase endoenuclease activity in extracellular fermentation broth; 1 represents the control strain Trichoderma reesei OEypr1-Δsor1, and 2 represents the two cellulase expression strains driven by the bidirectional promoter BDP1. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with the examples and drawings, but the scope of protection of the present invention is not limited thereto. The drugs and reagents involved in the examples are all common commercially available products unless otherwise specified; the experimental operations involved in the examples are all routine operations in the art unless otherwise specified. The primers in the examples are all synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0029] The starting strain Trichoderma reesei QM9414 was purchased from the American Type Culture Collection with the accession number of ATCC 26921.

[0030] Example 1: Construction of green fluorescent protein and red fluorescent protein expression vector driven by promoter BDP1

[0031] (1) Construction of the expression cassette of reporter genes gfp and mCherry driven by promoter BDP1

[0032] The fungal DNA extraction kit (EZNA TM Fungal DNA Mini Kit, OMEGA) was used to extract genomic DNA of Trichoderma reesei QM9414, and genomic DNA was used as a template and BDP1-F1 / BDP1-R1 were used as upstream and downstream primers to obtain a fragment of about 0.8 kb that may be a bidirectional promoter, and the fragment was named BDP1; primers Tcbh2-F / Tcbh2-R were used as upstream and downstream primers to obtain a terminator Tcbh2 fragment of about 1 kb by PCR amplification; plasmid Ptcu1-clp1-gfp-TtrpC was used as a template and primer pair gfp-F / gfp-R were used as upstream and downstream primers to obtain a green fluorescent protein encoding gene gfp of about 0.7 kb by PCR amplification; primer pair TtrpC-F / TtrpC-R were used as upstream and downstream primers to obtain a terminator TtrpC fragment of about 0.75 kb by PCR amplification. Using plasmid Ptcu1-mCherry-h2b as a template, primers mCherry-F / mCherry-R were designed, and the red fluorescent protein encoding gene mCherry was obtained by PCR amplification. Using the fusion PCR method, the three fragments of TtrpC, gfp and BDP1 were first connected and fused into one fragment in sequence, and then the two fragments of mCherry and Tcbh2 were connected and fused into one fragment. Finally, the above two fusion fragments were connected and fused into one fragment, and the expression cassette TtrpC-gfp-BDP1-mCherry-Tcbh2 of the reporter gene gfp and mCherry driven by the promoter BDP1 was obtained.

[0033] The construction process of the above plasmids Ptcu1-clp1-gfp-TtrpC and Ptcu1-mCherry-h2b can be found in the literature CLP1, a Novel Plant Homeo Domain Protein, Participates in Regulating Cellulase Gene Expression in the Filamentous Fungus Trichoderma reesei. Lei Wang, Renfei Yang, Yanli Cao, Fanglin Zheng, Xiangfeng Meng, Yaohua Zhong, Guanjun Chen, Weixin Zhang, Weifeng Liu. Front Microbiol. 2019 Aug 6; 10: 1700. doi: 10.3389 / fmicb.2019.01700.

[0034] Wherein, the above primer sequences are as follows:

[0035] BDP1-F1:

[0036] 5'-ACCACCCTCAACGACGGAAGATGGTGAGCAAGGGCGAGGAGCTG-3',

[0037] BDP1-R1:5'-TCCTCGCCCTTGCTCACCATCTTCCGTCGTTGAGGGTGGTCG-3';

[0038] TtrpC-F:5'-GTTGACCTGCAGGTTTAAACTCGAGTGGAGATGTGGAGTGGG-3',

[0039] TtrpC-R:5'-TGGACGAGCTGTACAAGTAACTGAAATCATCAAACAGCTTGACG-3';

[0040] gfp-F:5'-TGGACGAGCTGTACAAGTAACTGAAATCATCAAACAGCTTGACG-3',

[0041] gfp-R:5'-AAGCTTGTTTGATGATTTCAGTTACTTGTACAGTCGTCCATGCC-3';

[0042] mCherry-F:5'-GCCAGCTTGCGCAGACAGTCATGGTGAGCAAGGGCGAGGAGG-3',

[0043] mCherry-R:5'-AAGCCTGTTTGATGATTTCAGCTACTTGTACAGTCGTCCATGCCG-3';

[0044] Tcbh2-F:5'-TGGACGAGCTGTACAAGTAGGGCTTTCGTGACCGGGCTTCAAACA-3',

[0045] Tcbh2-R:5'-GCAGGTCGACGATTAGCTTTTTTACGCCCTCGAAGATCTCGCTCAG-3';

[0046] The PCR amplification system was as follows: 2 μL of 10 μM upstream primer, 2 μL of 10 μM downstream primer, 10 ng of genomic DNA, 5 μL of 2.5 mM dNTP mixture, 10 μL of 5× TransStart Fast Pfu buffer, 1 μL of TransStart Fast Pfu DNA polymerase, and ddH2O was added to 50 μL.

[0047] The PCR amplification procedure was as follows: pre-denaturation at 98°C for 5 min; denaturation at 95°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 2 min, 28 cycles; final extension at 72°C for 10 min; after the completion of PCR, 1% agarose gel electrophoresis was performed and the DNA fragments were recovered.

[0048] The fusion PCR amplification system was as follows: 20 ng of each of the two fragments to be connected, 8 μL of 2.5 mM dNTP mixture, 20 μL of 5× TransStart FastPfu buffer, 1 μL of TransStart FastPfu DNA polymerase, 5 μL of 10 μM upstream primer, 5 μL of 10 μM downstream primer, and ddH2O to make up to 100 μL;

[0049] The fusion PCR amplification procedure was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 sec, annealing at 53°C for 6 min, extension at 72°C for 5 min, 28 cycles; final extension at 72°C for 10 min; after PCR completion, 1% agarose gel electrophoresis was performed and DNA fragments were recovered.

[0050] Schematic diagram of the promoter BDP1 driving the reporter gene gfp and mCherry expression cassette as shown in Figure 1As shown, the promoter BDP1 is flanked by gene sequences of gfp and mCherry, respectively, and the Trichoderma reesei trpC and cbh2 gene terminators are used as gene transcription termination elements, respectively.

[0051] (2) Construction of site-specific expression vector driven by promoter BDP1 for reporter genes gfp and mCherry

[0052] Using the genomic DNA of Trichoderma reesei QM9414 as a template, primers pyr4Up-F / pyr4Up-R were designed, and the upstream homology arm fragment of the pyr4 gene was obtained by PCR amplification, which was about 2.3 kb; primers pyr4Down-F / pyr4Down-R were designed, and the downstream homology arm fragment of the pyr4 gene was obtained by PCR amplification, which was about 2.3 kb. The sulfonylurea resistance gene sur was synthesized by conventional gene synthesis at Suzhou Genewise Biotechnology Co., Ltd., and the synthetic sequence was used as a template, and the sur gene expression cassette fragment was amplified using primers surF / surR. The screening marker sur fragment is connected to the downstream homology arm fragment of the pyr4 gene by fusion PCR, and then the fusion fragment and the upstream homology arm fragment of the pyr4 gene are simultaneously connected to the BamHI / HindⅢ site of the pUC19 plasmid, and finally the expression cassette TtrpC-gfp-BDP1-mCherry-Tcbh2 described in step (1) is connected to the MssI / BamHI site of the plasmid, and finally the site-directed expression vector pUC19-gfp-BDP1-mCherry is obtained. In this vector, the upstream homology arm and downstream homology arm fragments of the pyr4 gene are located on both sides, and the expression cassette TtrpC-gfp-BDP1-mCherry-Tcbh2 and the resistance screening gene sur are in the middle. Among them, the pUC19 plasmid is a commonly used plasmid purchased from TAKARA, and the resistance screening gene sur is a commonly used screening marker.

[0053] The primer sequences are as follows:

[0054] pyr4 Up-F:5'-AAACGACGGCAGTGAATTCGGTCTCGTGCGTGCCGACAGCC-3',

[0055] pyr4Up-R:5'-CGTTGGCACGTCGAGGATCCCCGGTTTAAACTCGATGCTAGCGGAGAAGGCGATTC-3';

[0056] pyr4 Down-F:5'-TTGCATGCTCTCACGTCGACGAAGTAAAGAAAGGCATTTAGC-3',

[0057] pyr4 Down-R:5'-ACCATGATTACGCCAAGCTTAATGTCAGCCCCAAATCCACCC-3';

[0058] sur-F:5'-AGCTCGGTACCCGGGGATCCTCGACGTGCCAACGCCA-3',

[0059] sur-R:5'-TAAATGCCTTCTTTACTTCGTCGACGTGAGAGCATGCAATTCC-3';

[0060] The PCR amplification system and amplification procedure are as described above.

[0061] Example 2: Construction of green fluorescent protein and red fluorescent protein expression strain driven by promoter BDP1

[0062] (1) Preparation of Trichoderma reesei protoplasts

[0063] The concentration was 5 × 10 7 ~5×10 8 1 mL of a spore suspension of Trichoderma reesei OEypr1-Δsor1 of 10 / mL was inoculated into 50 mL of MM medium, and cultured at 28°C and 160 rpm for 16 h to obtain a mycelial culture; the mycelial culture was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and then a cell wall lysing enzyme solution was added, resuspended, and enzymatically hydrolyzed at 30°C and 60 rpm for 2.5 to 3 h. When a large number of free protoplasts appeared, the filtrate was filtered using a G2 funnel, and the filtrate was centrifuged at 4°C and 3500 rpm for 5 min to collect the protoplasts. The collected protoplasts were washed twice with 15 mL of precooled STC solution, and then resuspended in the precooled STC solution to make the protoplast concentration 5×10 7 ~5×10 8 All the above operations were performed on ice.

[0064] Among them, Trichoderma reesei OEypr1-Δsor1 is to overexpress the transcriptional regulatory factor ypr1 in Trichoderma reesei QM9414-Δpyr4 and knock out the sor1 gene. The strain construction method refers to the literature Influences of genetically perturbing synthesis of the typical yellow pigment on conidiation, cell wall integrity, stress tolerance, and cellulase production in Trichoderma reesei. Weixin Zhang, Ning An, Junqi Guo, Zhixing Wang, Xiangfeng Meng, Weifeng Liu. J Microbiol. 2021Apr;59(4):426-434. doi:10.1007 / s12275-021-0433-0. Overexpression of the transcriptional regulatory factor ypr1 can promote the increase of the transcription level of genes near the promoter BDP1 on the genome, and has a promoting effect on the activation activity of BDP1. The growth of this strain is significantly improved compared with QM9414-Δpyr4 and QM9414 strains, and it does not produce the yellow pigment of the secondary product characteristic of Trichoderma reesei. It is a good chassis strain for expressing secreted proteins. QM9414-Δpyr4 replaces the pyr4 gene with the ptrA screening marker in QM9414, thereby knocking out the pyr4 gene so that the strain can use the commonly used screening marker pyr4. The strain construction method refers to the literature A novel transcriptional regulator RXE1modulates the essential transactivator XYR1 and cellulase gene expression in Trichoderma reesei. Wang L, Lv X, Cao Y, Zheng F, Meng X, Shen Y, Chen G, Liu W, Zhang W. Appl Microbiol Biotechnol. 2019 Jun; 103 (11): 4511-4523.

[0065] The components of MM medium are as follows (all in mass percentage): 1% glucose, 0.5% (NH4)2SO4, 1.5% KH2PO4, 0.1% peptone, pH adjusted to 5.0-5.5 with NaOH, 0.6 g / L MgSO4, 0.4 g / L CaCl2, trace elements (final concentration 1×, mother solution 1000×), 10 mM uridine were added before use; 1000× trace element mother solution formula: FeSO4·7H2O 0.5 g, MnSO4·H2O 0.16 g, ZnSO4·7H2O 0.14 g, CoC l2 2H2O 0.2g, dilute to 100mL with ddH2O, filter and sterilize;

[0066] The composition of the cell wall lysing enzyme solution is as follows: 0.24 g of cell wall lysing enzyme was dissolved in 5.7 mL of 0.7 M NaCl solution, filtered and sterilized, and 0.3 mL of pH 5.8 PBS buffer was added;

[0067] The composition of the STC solution was as follows: 0.7 M D-sorbitol, 0.05 M CaCl2, 10 mM Tris-HCl, pH 7.5.

[0068] (2) Transformation of site-directed expression vector pUC19-gfp-BDP1-mCherry and regeneration of transformants

[0069] Take 150 μL of the site-directed expression vector pUC19-gfp-BDP1-mCherry obtained in Example 1, use EcoRI restriction endonuclease to linearize, mix with 150 μL of Trichoderma reesei OEypr1-Δsor1 protoplasts prepared in step (1), and add to the bottom of a 10 mL centrifuge tube. After ice bathing for 20 minutes, add 1.5 mL of pre-cooled PTC solution dropwise, ice bath for 20 minutes, and then place at room temperature for 20 minutes, then add 2 mL of STC solution and mix to obtain a transformation solution; spread the transformation solution on a regeneration medium plate without uridine, and culture at 28° C. for 5-7 days.

[0070] The components of the PTC solution are as follows: 60% PEG 4000 (polyethylene glycol 4000), 10 mM Tris-HCl, 10 mM CaCl2, pH 7.5 in a mass volume ratio (w / v);

[0071] The components of the regeneration medium are as follows (all in mass percentage): 1% glucose, 0.5% (NH4)2SO4, 1.5% KH2PO4, 1M D-sorbitol, 1.5% agar powder, pH adjusted to 5.0-5.5 with NaOH, 0.6 g / LMgSO4, 0.4 g / LCaCl2 were added before use, and trace elements (final concentration 1×, mother solution 1000×) were added. The formula of 1000× trace element mother solution is the same as above.

[0072] The colonies grown on the above regeneration medium plates were transferred to screening medium plates containing sulfonylurea antibiotics at a final concentration of 2‰, and cultured at 28°C for 5-7 days to obtain transformants.

[0073] The components of the screening medium are as follows (all in mass percentage): 1% glucose, 0.5% (NH4)2SO4, 1.5% KH2PO4, 1.5% agar powder, pH adjusted to 5.0-5.5 with NaOH, 0.6 g / L MgSO4, 0.4 g / L CaCl2 were added before use, and trace elements (final concentration 1×, mother solution 1000×) were added. The formula of 1000× trace element mother solution is the same as above.

[0074] Schematic diagram of the process of BDP1 bidirectionally driving the expression of red and green fluorescent proteins in the Trichoderma genome. Figure 2 As shown, the site-directed expression vector pUC19-gfp-BDP1-mCherry contains the upstream homology arm sequence and the downstream homology arm sequence of the pyr4 gene. After transformation into the protoplasts of Trichoderma reesei, the upstream and downstream homology arms undergo homologous recombination with the upstream and downstream regions of the pyr4 gene in the genome, and the TtrpC-gfp-BDP1-mCherry-Tcbh2 expression cassette and the sur selection marker in the expression vector pUC19-gfp-BDP1-mCherry are integrated into the pyr4 position of the Trichoderma reesei genome, replacing the original ptrA selection marker expression cassette on the genome.

[0075] (3) Verification and acquisition of expression strains

[0076] The genomic DNA of the transformants was extracted, and the downstream homology arm sequence anchor verification was performed using PCR technology with the genomic DNA as a template. The primers were pyr4-anchor-F and pyr4-anchor-R, and the amplified length was about 2.3 kb. The primer sequences are as follows:

[0077] pyr4-anchor-F:5'-CCTTCCGACCCTCTTTCTTACA-3',

[0078] pyr4-anchor-R:5'-CAACAGGACGCTCTCAACATCA-3'

[0079] The transformants with correctly anchored upstream and downstream homologous arms were purified by single spore isolation, and internal gene verification was performed using PCR technology and ptrA-F / ptrA-R primers.

[0080] The primer sequences are as follows:

[0081] ptrA-F:5'-TGGTAACGAAATGTAAAAGCTAGGAG-3',

[0082] ptrA-R:5'-CTATCATGGGGTGACGATGAGCCGC-3'.

[0083] The above verification PCR reaction system was 20 μL. For the specific reaction system and reaction conditions, please refer to the instruction manual of 2×Taq MasterMix (purchased from Nanjing Novezan Biotechnology Co., Ltd.).

[0084] Verification results are as follows Figure 3 As shown, in the anchored PCR verification, the control strain Trichoderma OEypr1-Δsor1 had no target band, while the bidirectional fluorescent protein expression strain had a bright target band with a band size of approximately 2.3 kb, which was consistent with the expected band size; in the verification of the internal gene, the control strain Trichoderma OEypr1-Δsor1 had a bright target band with a band size of approximately 1.5 kb, which was consistent with the expected band size, while the bidirectional fluorescent protein expression strain had no such band, indicating that the bidirectional fluorescent protein expression strain was successfully obtained.

[0085] Example 3 Detection of green fluorescence and red fluorescent protein expression

[0086] The correctly transformed spores obtained in Example 2 were prepared into a spore suspension, which was inoculated into MM liquid culture medium and cultured in a shaker at 30° C. for 48 h. Mycelium samples were taken out and the expression of red and green fluorescent proteins was observed using a fluorescence microscope.

[0087] The microscopic observation method is as follows:

[0088] Take out the cultured hyphae from the operating table, use the tip of a gun to absorb the bacterial liquid or pick up hyphae and apply it on the slide, and cover with a coverslip; turn on the microscope power, camera, computer, white light power supply, and fluorescence stimulator in sequence, and wait for the stimulator to be turned on for 5 minutes before conducting fluorescence observation; first use a low-power microscope (10×) to find the field of view under white light, find the hyphae and focus, then switch to a high-power microscope (40×, 60× or 100×, of which 60× and 100× lenses are oil lenses and need to add cedar oil) for observation, fine-tune the fine focus screw to make the image clear, and adjust the amount of light entering to make the field of view to the appropriate brightness; use the corresponding fluorescence observation filter, turn off the white light, turn on the stimulator, and observe the fluorescence of the hyphae; after selecting the field of view, first shoot under white light, keep the field of view unchanged, switch to shooting under fluorescence, adjust the appropriate exposure time, and take pictures for storage.

[0089] Wherein, the components of MM medium are as described in Example 2.

[0090] Observation results such as Figure 4 As shown, under the corresponding excitation wavelengths of red and green fluorescence, bright red fluorescence and green fluorescence were observed, respectively, while there was no such fluorescence in the control strain, which indicated that the promoter BDP1 had a bidirectional promoter function and could simultaneously initiate the transcriptional expression of two fluorescent protein genes.

[0091] Example 4: Construction of a strain expressing two cellulases driven by the bidirectional promoter BDP1

[0092] (1) Construction of expression cassettes of the cellulase exonuclease gene Ct-cbh1 and the endonuclease gene eg1 driven by the bidirectional promoter BDP1

[0093] The cbh1 gene (Ct-cbh1) from Chaetomium thermophilum was simultaneously expressed in Trichoderma reesei using the bidirectional promoter BDP1. The pUC19-gfp-BDP1-mCherry plasmid constructed in Example 2 was used as a template, and BDP1-F2 / BDP1-R2 were used as upstream and downstream primers to obtain the bidirectional promoter BDP1 fragment by PCR amplification. Using QM9414 genomic DNA as a template, the primers eg1-F / eg1-R were used to amplify the full length of the endogenous cellulase gene eg1 and its terminator fragment, which was about 2.6 kb in length; the terminator TtrpC fragment of about 0.7 kb was obtained by PCR amplification using the primer pair Ct-cbh1 TtrpC-F / Ct-cbh1 TtrpC-R. The full-length sequence of Ct-cbh1 cDNA was synthesized by Genewise Biotech Co., Ltd., and the synthetic sequence was used as a template to amplify the cDNA fragment of Ct-cbh1 with the primer pair Ct-cbh1-F / Ct-cbh1-R. The amplified TtrpC, Ct-cbh1 and bidirectional promoter BDP1 fragments were fused and connected by the fusion PCR method, and then the fusion fragment was connected with the eg1 and its terminator fragment to obtain the bidirectional cellulase gene expression cassette TtrpC-Ct-cbh1-BDP1-eg1-Teg1 driven by the bidirectional promoter BDP1.

[0094] The PCR system and PCR procedure are the same as those in Example 1.

[0095] Wherein, the sequence of the above primers is as follows:

[0096] Ct-cbh1TtrpC-F:5'-CGCTAGCATCGAGTTTAAACTCGAGTGGAGATGTGGAGTGGG-3',

[0097] Ct-cbh1TtrpC-R:5'-GCGAACTTCTTGTACATCATCTGAAATCATCAAACAGCTTGACG-3';

[0098] Ct-cbh1-F:5'-AAGCTTGTTTGATGATTTCAGTTACAGGCACTGGCTGTACCAGGGG-3',

[0099] Ct-cbh1-R:5'-ACCACCCTCAACGACGGAAGATGATGTACAAGAAGTTCGCCGCTCTCG-3';

[0100] BDP1-F2:5'-GCGAACTTCTTGTACATCATCTTCCGTCGTTGAGGGTGGTCG-3',

[0101] BDP1-R2:5'-ATGGCGCCCTCAGTTACACTGACTGTCTGCGCAAGCTGGCGAAC-3';

[0102] eg1-F:5'-GCCAGCTTGCGCAGACAGTCATGGCGCCCTCAGTTACACTGCCG-3',

[0103] eg1-R:5'-CGTTGGCACGTCGAGGATCCGATTTTCCCCTGACTCTGGGCTCGG-3'.

[0104] The schematic diagram of the expression cassette of the bidirectional promoter BDP1 driving the exonuclease gene Ct-cbh1 and the endonuclease gene eg1 is shown in Figure 5 As shown, the bidirectional promoter BDP1 is flanked by gene sequences of Ct-cbh1 and eg1, respectively, and the endogenous terminators of Trichoderma reesei trpC and eg1 genes are used as gene transcription termination elements, respectively.

[0105] (2) Construction of site-specific expression vector of two cellulase genes Ct-cbh1 and eg1 driven by bidirectional promoter BDP1

[0106] The pUC19-gfp-BDP1-mCherry plasmid constructed in Example 2 was treated with restriction endonucleases Mss I and BamH I, the bidirectional fluorescent protein gene expression cassette was cut out, and the expression cassette TtrpC-Ct-cbh1-BDP1-eg1-Teg1 constructed in step (1) was connected to obtain the pyr4 site-directed expression vector pUC19-Ctcbh1-BDP1-eg1.

[0107] (3) Construction of expression strains driven by the bidirectional promoter BDP1 for two cellulase genes Ct-cbh1 and eg1

[0108] The vector pUC19-Ctcbh1-BDP1-eg1 constructed in step (2) was linearized with EcoRI and transformed with Trichoderma reesei OEypr1-Δsor1. The protoplast preparation, transformation and transformant screening methods of Trichoderma reesei OEypr1-Δsor1 were the same as those described in Example 2.

[0109] Schematic diagram of the process of bidirectional promoter BDP1 driving the site-specific expression of cellulase genes Ct-cbh1 and eg1 in the Trichoderma genome Figure 6As shown, the site-directed expression vector pUC19-Ctcbh1-BDP1-eg1 contains the upstream homology arm sequence and the downstream homology arm sequence of the pyr4 gene. After transformation into the protoplasts of Trichoderma reesei, the upstream and downstream homology arms undergo homologous recombination with the upstream and downstream regions of the pyr4 gene in the genome, and the TtrpC-Ct-cbh1-BDP1-eg1 expression cassette and the sur selection marker in the expression vector pUC19-Ctcbh1-BDP1-eg1 are integrated into the pyr4 position of the Trichoderma reesei genome, replacing the original ptrA selection marker expression cassette on the genome.

[0110] The genomic DNA of the transformant was extracted, and the downstream homology arm sequence anchor verification was performed using PCR technology with the genomic DNA as a template. The primers were pyr4-anchor-F and pyr4-anchor-R, and the amplified length was about 2.3 kb. The primer sequences and PCR method were as described in Example 2.

[0111] The transformants with correctly anchored upstream and downstream homology arms were purified by single spore separation, and internal gene verification was performed by PCR technology and ptrA-F / ptrA-R primers. The primer sequences and PCR method are as described in Example 2.

[0112] Verification results are as follows Figure 7 As shown, in the anchored PCR verification, the control strain Trichoderma OEypr1-Δsor1 had no target band, while the bidirectional cellulase expression strain had a bright target band with a band size of approximately 2.3 kb, which was consistent with the expected band size; in the verification of the internal gene, the control strain Trichoderma OEypr1-Δsor1 had a bright target band with a band size of approximately 1.5 kb, which was consistent with the expected band size, while the BDP1-driven cellulase expression strain did not have this band, indicating that the bidirectional cellulase expression strain was successfully obtained.

[0113] Example 5: Analysis of the expression of two cellulases driven by the bidirectional promoter BDP1

[0114] Equal amounts of spores of the bidirectional cellulase expression strain constructed in Example 4 and the control strains OEypr1-Δsor1 and OEypr1-Δpyr4 were inoculated into MA liquid culture medium containing 1% glucose, and cultured at 28° C. and 180 rpm for a corresponding period of time. The fermentation broth was then taken for cellulase activity detection.

[0115] The formula of MA liquid culture medium is as follows: add 17.907g Na2HPO4·12H2O, 1.4g (NH4)2SO4, 2.0g KH2PO4, 0.3g urea, 0.5mL Tween-80 to each 1L culture medium, and adjust the pH to 5.0 with anhydrous citric acid. Before use, add 0.6g / L MgSO4, 0.4g / L CaCl2, trace elements (final concentration 1×, mother solution 1000×), 10mM uridine; 1000× trace element mother solution formula: FeSO4·7H2O 0.5g, MnSO4·H2O 0.16g, ZnSO4·7H2O 0.14g, CoC l2 2H2O 0.2g, dilute to 100mL with ddH2O, and filter sterilize.

[0116] (1) Detection of cellulase exonuclease activity in fermentation broth

[0117] The Ctcbh1 gene driven by the bidirectional promoter BDP1 encodes a cellulase exonuclease, and its expression can be determined by detecting the exonuclease activity in the fermentation broth. The exonuclease activity analysis method is as follows: 50 μL of fermentation broth and 150 μL of pNPC substrate dissolved in pH 4.8 acetic acid-sodium acetate buffer are mixed evenly, reacted at 50°C for 30 minutes, and then 50 μL of 10% sodium carbonate is added to terminate the reaction. The product absorbance is read at a wavelength of 420 nm. One unit of exonuclease activity is defined as the amount of enzyme that releases 1 μmol of pNP per minute under standard conditions.

[0118] Test results such as Figure 8 As shown in A, the control strains OEypr1-Δsor1 and OEypr1-Δpyr4 could not produce extracellular cellulase under glucose conditions, while the expression strain driven by the bidirectional promoter BDP1 showed obvious extracellular cellulase activity, indicating that the Ctcbh1 gene was successfully expressed.

[0119] (2) Detection of cellulase endoenzyme activity in fermentation broth

[0120] The eg1 gene driven by the bidirectional promoter BDP1 encodes a cellulase endonuclease, and its expression is determined by detecting the endonuclease activity in the fermentation broth. The endonuclease activity analysis method is as follows: 50 μL of fermentation broth and 150 μL of CMC substrate dissolved in pH 4.8 acetic acid-sodium acetate buffer are mixed evenly, reacted at 50°C for 30 minutes, and then 50 μL of DNS solution is added to terminate the reaction, boiled for 10 minutes, and the product absorbance is read at a wavelength of 560 nm. One unit of endonuclease activity is defined as the amount of enzyme that releases 1 μmol of glucose per minute under standard conditions.

[0121] Test results such as Figure 8 As shown in B, the control strains OEypr1-Δsor1 and OEypr1-Δpyr4 could not produce extracellular cellulase under glucose conditions, while the expression strain driven by the bidirectional promoter BDP1 showed obvious extracellular cellulase activity, indicating that the eg1 gene was successfully expressed.

[0122] In summary, the bidirectional genes Ct-cbh1 and eg1 driven by the bidirectional promoter BDP1 were successfully expressed simultaneously in Trichoderma reesei.

[0123] Example 6: Bidirectional promoter BDP1 drives the expression of two cellulases to increase the activity of extracellular enzymes under cellulose culture conditions

[0124] Equal amounts of spores of the bidirectional cellulase expression strain constructed in Example 4 and the control strain OEypr1-Δsor1 were inoculated into MA liquid culture medium containing 1% glycerol (v / v) and 0.2% peptone (w / v), and cultured at 28°C and 180 rpm for 36 h. Then equal amounts of wet weight of mycelium were collected and transferred to MA liquid culture medium containing 1% Avicel (w / v), and cultured at 28°C and 180 rpm for 120 h.

[0125] Wherein, the formula of MA liquid culture medium and the method for detecting the activities of cellulase exonuclease and endoenuclease in fermentation broth are as described in Example 5.

[0126] Test results such as Fig. 9 As shown in Figure 2, under cellulose induction conditions, the extracellular exonuclease activity of the bidirectional cellulase expression strain increased by about 37% compared with the control strain ( Fig. 9 A), the endonuclease activity increased by about 17% compared with the control strain ( Fig. 9 B) showed that the bidirectional expression of the two cellulase genes ctcbh1 and eg1 increased the extracellular cellulase production and enzyme activity of Trichoderma reesei strains under BDP1 bidirectional drive expression. SEQUENCE LISTING <110> Shandong University <120> A bidirectional promoter BDP1 of Trichoderma reesei and its application <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 767 <212> DNA <213> Trichoderma reesei <400> 1 cttccgtcgt tgagggtggt cgataaagaa ggttgaaggg ggctgagtgt ttgcgtctgg 60 120 ttttacatat gcgactgctt cgaagcccag cttgagggcc gtcagcttca tagacgcgtc 180 ttgccatgac gaacctttc tataactcga tcagcagatc ctgtaagcgg cttgcgagtg 240 atacggacat actatcgtca ggattcgttt gagaaaca aattcctgac ggacgcggtc 300 ggagagcgg gaattcgg agtaagcaat taaagagag agaaaaagaa gcagaaaaag 360 tccgccttgg cagagtccga aggagtcgtc aagtgcgatc cgcatcggag tatctcaact 420 ccggagttca atgttggcga tcgtcactga gggcagaaac tcgaaactcc atgtcagcag 480 tatcaggcaa acgtttgaat gacggcggcg ggcaatctgt ctgtcagcat cgtcagcttc 540 tggcggacg area ggcgaatctc gcaaagatgc taaaagcatg cgtgcagtcc 600 acgacattct gtgtcttggt gactgcatgc atctaccatc ttgaaggcat cacgtttcgt 660 tcaccttgaa ggcttgtggt actcaacatt accacacaag ggggattcca gggcaacttc 720 aacagcagaa gcgcatcaac ctcgttcgcc agcttgcgca gacagtc 767

Claims

1. A Trichoderma reesei bidirectional promoter BDP1, characterized in that The nucleotide sequence is shown in SEQ ID NO.

1.

2. Use of the Trichoderma reesei bidirectional promoter BDP1 according to claim 1 in improving the extracellular cellulase production and enzyme activity of Trichoderma reesei strains.

3. A recombinant vector, characterized in that: The recombinant plasmid vector contains the gene expression cassette constructed by the Trichoderma reesei bidirectional promoter BDP1 described in claim 1.

4. An engineered strain of Trichoderma reesei with high cellulase production, characterized in that: The high-yield cellulase-producing Trichoderma reesei engineered bacteria is based on Trichoderma reesei QM9414-OE ypr1- Δ sor1 As the starting strain, Trichoderma reesei chromatin pyr4 The gene position region is replaced with a gene expression cassette containing the Trichoderma reesei bidirectional promoter BDP1 as described in claim 1, thereby constructing; Among them, the OE ypr1 Overexpression of transcriptional regulators ypr1 .

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