Trichoderma reesei expression site H2 and application thereof as well as recombinant trichoderma reesei strain for producing thaumatin II protein as well as construction method and application of recombinant trichoderma reesei strain

Abstract

The invention belongs to the technical field of biology, and particularly relates to a trichoderma reesei expression site H2 and application thereof as well as a recombinant trichoderma reesei strain for producing thaumatin II protein as well as a construction method and application of the recombinant trichoderma reesei strain. The Trichoderma reesei expression site H2 provided by the invention is located at the 210th site in a gene segment as shown in SEQ ID NO.6 in a Trichoderma reesei genome, and an expression level equivalent to that of a cbh1 site can be obtained by inserting an exogenous gene into the site. The recombinant trichoderma reesei strain for producing the thaumatin II protein provided by the invention contains a gene for coding the thaumatin II protein, which has a nucleotide sequence shown as SEQ ID NO.1, and the gene has high translation efficiency in the trichoderma reesei, so that the recombinant trichoderma reesei strain can efficiently produce the thaumatin II protein, and the proportion of the thaumatin II protein in the obtained total protein is high; the method has important significance in reducing the production cost of the thaumatin II protein and promoting the application of the thaumatin II protein in the food industry.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a Trichoderma reesei expression site H2 and an application thereof, as well as a recombinant Trichoderma reesei strain for producing thaumatin II protein and a construction method and application thereof. Background Art

[0002] Thaumatin is a natural sweetening protein extracted from the fruit of the African arrowroot. It consists of two main components: Thaumatin I and Thaumatin II. Thaumatin is characterized by high sweetness, low calories, and safety and non-toxicity. Its sweetness is 2,000 to 3,000 times that of an equal weight of sucrose. It also exhibits excellent thermal and acid stability. When combined with other sweeteners, it can enhance sweetness and improve flavor, making it valuable in the development of low-sugar, low-calorie foods.

[0003] Due to the African arrowroot's demanding growing environment and limited yield, traditional extraction methods using it as a raw material have struggled to meet market demand. Currently, research on exogenous thaumatin expression primarily focuses on recombinant protein production using microbial hosts (such as yeast or bacteria). Genetic engineering techniques, introducing the thaumatin gene into host cells and optimizing expression conditions, have the potential to enable large-scale industrial production. However, exogenous thaumatin expression still faces challenges, such as protein folding and post-production modifications, which can affect its sweetness and stability. Furthermore, while current exogenous expression technologies have achieved some progress, the expression levels of thaumatin remain limited, making it difficult to achieve the high efficiency required for industrial production. Consequently, the application of thaumatin in the food industry is somewhat limited.

[0004] The filamentous fungus Trichoderma reesei is a mesophilic, saprophytic filamentous fungus that naturally secretes large amounts of endogenous proteins and possesses a glycosylation system similar to that of higher mammals. Therefore, due to its diverse post-translational modifications, T. reesei is an ideal host for recombinant protein expression, expressing both homologous and heterologous proteins. It has been successfully used for the expression and production of a wide range of drugs, chemical reagents, and enzymes. In T. reesei, foreign DNA fragments often integrate randomly into the genome via non-homologous end joining (NHEJ). This random insertion pattern means that the expression level of recombinant proteins can vary significantly depending on the insertion site. For example, if a foreign gene is inserted into a silencer regulatory region, it may not be transcribed and expression may fail. The site of the cellobiohydrolase gene (cbh1) is generally considered an ideal candidate for foreign gene expression due to its high expression efficiency and is widely used in T. reesei. However, synthetic biology requires the coordinated integration of multiple genes. Inserting multiple copies simultaneously at existing sites will exponentially decrease recombination efficiency, while new sites can improve the efficiency of multi-gene integration by optimizing spacing. Therefore, developing new insertion sites for exogenous genes has positive significance for improving the ability of recombinant Trichoderma reesei strains to express target products (such as thaumatin). Summary of the Invention

[0005] To address the above issues, the present invention provides the Trichoderma reesei expression site H2 and its applications, as well as a recombinant Trichoderma reesei strain capable of producing thaumatin II protein, its construction method, and its applications. This invention provides a new site suitable for efficient exogenous gene expression, further enriching and improving the technology system for efficient recombinant protein expression using Trichoderma reesei as a host. The present invention also provides a recombinant Trichoderma reesei strain capable of efficiently producing thaumatin II protein.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a Trichoderma reesei expression site H2, wherein the Trichoderma reesei expression site H2 is located at position 210 in the gene fragment shown in SEQ ID NO. 6 in the Trichoderma reesei genome.

[0007] Through experimental research, the present invention has found that inserting genes encoding thaumatin II protein or other target products (such as alkaline lipase) into this site can achieve expression levels comparable to those achieved at the cbh1 site. Therefore, this site can be used to enhance the ability of recombinant Trichoderma reesei strains to express target products such as thaumatin II protein, providing a new and efficient expression strategy for efficient protein production.

[0008] A second aspect of the present invention provides the use of the above-mentioned Trichoderma reesei expression site H2 in constructing a recombinant Trichoderma reesei strain that highly expresses a target product.

[0009] The third aspect of the present invention provides a gene encoding thaumatin II protein, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0010] Compared to the original gene encoding thaumatin II protein, the gene with the nucleotide sequence shown in SEQ ID NO. 1 has a higher translation efficiency in Trichoderma reesei. Inserting this gene into Trichoderma reesei can significantly increase the level of thaumatin II protein expressed by the recombinant Trichoderma reesei strain.

[0011] In a fourth aspect, the present invention provides a recombinant plasmid pPcbh1-tTHAⅡ for heterologously expressing thaumatin II protein. The recombinant plasmid pPcbh1-tTHAⅡ contains the gene encoding the thaumatin II protein, and contains a cbh1 promoter and a cbh1 terminator.

[0012] Preferably, the backbone vector of the recombinant plasmid pPcbh1-tTHAⅡ is pBluescript II SK(+) plasmid.

[0013] In a fifth aspect, the present invention provides a recombinant Trichoderma reesei strain producing thaumatin II protein. The recombinant Trichoderma reesei strain is constructed by transferring the above-mentioned recombinant plasmid pPcbh1-tTHAⅡ into Trichoderma reesei protoplasts.

[0014] Preferably, the insertion site of the gene with the nucleotide sequence shown in SEQ ID NO. 1 in the recombinant plasmid pPcbh1-tTHAⅡ is the above-mentioned Trichoderma reesei expression site H2.

[0015] Preferably, the starting strain of the recombinant Trichoderma reesei strain is Trichoderma reesei QM6aΔpyr4. QM6aΔpyr4 is a wild type Trichoderma reesei strain transformed by the cas12a method. QM6a The uracil auxotrophic strain, constructed by functionally inactivating the pyr4 gene, has the genotype ATCC13631Δpyr4. The exogenous thaumatin II gene was transformed into the genome of the QM6aΔpyr4 strain via protoplasts. During transformation, pyr4 served as a selection marker to ensure integration of the gene and thus the stable expression of the exogenous thaumatin II protein.

[0016] Compared with the recombinant Trichoderma reesei strain DF230103 that has been disclosed for producing thaumatin II protein (deposit number CGMCC No. 41003, disclosed in the patent application number CN202311842705.3, and patent name "A recombinant Trichoderma reesei strain producing thaumatin II protein, its construction method and application"), the level of thaumatin II protein expressed by this recombinant Trichoderma reesei strain is significantly improved.

[0017] A sixth aspect of the present invention provides the use of the above-mentioned recombinant Trichoderma reesei strain in the production of thaumatin II protein.

[0018] Preferably, after the recombinant Trichoderma reesei strain is cultured to produce spores, the spores are made into a suspension, expanded in a liquid culture medium, and then fermented in the culture medium. The fermentation culture temperature is 28° C., the ventilation ratio is 0.8 vvm, and the pH is 5.

[0019] Further preferably, the liquid culture medium is MM+G liquid culture medium, whose components are: 1 mL each of 60 g / L CaCl2·2H2O, 60 g / L MgSO4·7H2O, 100 μL each of 3.7 g / L CoCl2·6H2O, 5 g / L FeSO4·7H2O, 1.4 g / L ZnSO4·7H2O, and 1.6 g / L MnSO4·H2O, 1.5 g of KH2PO4, 0.5 g of (NH4)2SO4, and 2 g of glucose, and the volume is adjusted to 100 mL.

[0020] Further preferably, the fermentation medium is MM + bagasse + peptone fermentation medium, whose components are: 1 mL each of 60 g / L CaCl2·2H2O, 60 g / L MgSO4·7H2O, 100 μL each of 3.7 g / L CoCl2·6H2O, 5 g / L FeSO4·7H2O, 1.4 g / L ZnSO4·7H2O, and 1.6 g / L MnSO4·H2O, 1.5 g KH2PO4, 0.5 g (NH4)2SO4, 2 g bagasse, and 0.5 g peptone, and the volume is fixed to 100 mL.

[0021] The beneficial effects of the present invention are as follows: the present invention provides a new site suitable for the efficient expression of exogenous genes in Trichoderma reesei, enriching and improving the technical system for the efficient expression of recombinant proteins using Trichoderma reesei as a host. Utilizing this new site, the ability of recombinant Trichoderma reesei strains to express target products is expected to be further enhanced. The present invention also performs codon optimization on the thaumatin II gene. The optimized gene has a higher translation efficiency in Trichoderma reesei, and its insertion into the Trichoderma reesei strain can significantly increase the level of thaumatin II protein expressed by the recombinant Trichoderma reesei strain. Based on the above-mentioned optimized thaumatin II gene, the present invention also provides a recombinant Trichoderma reesei strain that produces thaumatin II protein. This strain can efficiently produce thaumatin II protein, and the proportion of thaumatin II protein in the total protein obtained is high, which is of great significance for reducing the production cost of thaumatin II protein and promoting its application in the food industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1This is the plasmid map of the recombinant expression vector pPcbh1-tTHAⅡ in Example 1 of the present invention; Figure 2 This is the screening result of the high-yielding strain of Thaumatin II in Example 2 of the present invention; Figure 3 This is the plasmid map of the plasmid vector pH2-Pcbh1-tTHAⅡ in Example 2 of the present invention; Figure 4 The recombinant Trichoderma reesei strain H2-tThaⅡ, strain H2 and wild type strain in Example 2 of the present invention QM6a The secretion of thaumatin II protein in the fermentation supernatant; Figure 5 This is an SDS-PAGE verification image of the recombinant Trichoderma reesei strain H2 fermented in a 10 L fermentor in Example 2 of the present invention; Figure 6 This is the plasmid map of the recombinant expression vector pH2-Pcbh1-THAⅡ in Example 2 of the present invention; Figure 7 This is the expression verification result of the thaumatin II protein at the insertion site H2 in Example 2 of the present invention; Figure 8 This is the plasmid map of the recombinant expression vector pH2-pelc in Example 3 of the present invention; Figure 9 This is the result of detecting lipase activity on agar diffusion plate in Example 3 of the present invention. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0024] The production of thaumatin using microbial hosts has several drawbacks, such as poor sweetness and low expression levels, which hinder its application in the food industry. The filamentous fungus Trichoderma reesei naturally secretes large amounts of endogenous proteins into the cell's interior. However, exogenous DNA fragments often integrate randomly into the genome via non-homologous end joining, which does not necessarily result in target protein expression. The cbh1 locus is generally considered ideal for exogenous gene expression, but the simultaneous insertion of multiple copies at a single locus results in an exponential decrease in recombination efficiency. Consequently, the expression of exogenous genes in Trichoderma reesei presents a production bottleneck.

[0025] The present invention provides a Trichoderma reesei expression site H2, which is located at position 210 in the gene fragment of the Trichoderma reesei genome as shown in SEQ ID NO. 6. Insertion of foreign proteins into this site can achieve expression levels comparable to those at the cbh1 site.

[0026] The embodiments of the present invention also provide the use of the above-mentioned Trichoderma reesei expression site H2 in constructing a recombinant Trichoderma reesei strain that highly expresses a target product.

[0027] The present invention also provides a gene encoding thaumatin II protein, the nucleotide sequence of which is shown in SEQ ID NO. 1. This gene has a higher translation efficiency in Trichoderma reesei and can achieve a higher expression level of thaumatin II protein.

[0028] Based on this gene, an embodiment of the present invention further provides a recombinant plasmid pPcbh1-tTHAⅡ for heterologously expressing thaumatinⅡ protein.

[0029] The embodiment of the present invention also provides a recombinant Trichoderma reesei strain for producing thaumatin II protein. The recombinant Trichoderma reesei strain is constructed by transferring the above-mentioned recombinant plasmid pPcbh1-tTHAⅡ into Trichoderma reesei protoplasts.

[0030] The embodiments of the present invention also provide a method for constructing the above-mentioned recombinant Trichoderma reesei strain.

[0031] The embodiment of the present invention also provides the use of the recombinant Trichoderma reesei strain in producing thaumatin II protein.

[0032] The solutions of the present invention are described below through specific embodiments.

[0033] The primers used in the following examples are shown in Table 1: Table 1 Primers

[0034] The control strain DF230103 used in the following examples is a recombinant Trichoderma reesei strain with a deposit number of CGMCC No. 41003, which has been disclosed in the patent application number CN202311842705.3, entitled "A recombinant Trichoderma reesei strain producing thaumatin II protein, its construction method and application".

[0035] The original thaumatin II gene used in the following examples is the Thaumatin II fragment disclosed in the above-mentioned patent application number CN202311842705.3.

[0036] The control strains used in the following examples TPcdna1-pelc , pPcdna1-pelc plasmids have been disclosed in the thesis "Heterologous Expression of Alkaline Lipase in Trichoderma reesei and Analysis of Enzymatic Properties" (DOI: 10.27019 / d.cnki.gfjsu.2022.000859).

[0037] The wild type Trichoderma reesei used in the following examples QM6a It was purchased from the American Type Culture Collection, with the strain accession number being ATCC 13631.

[0038] The Trichoderma reesei QM6aΔpyr4 used in the following examples was obtained by converting the wild type Trichoderma reesei into QM6a The uracil auxotrophic strain was constructed by functionally inactivating the pyr4 gene of the strain, and the genotype is ATCC13631Δpyr4.

[0039] The components and preparation methods of the culture medium used in the following examples are: LB liquid medium: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract powder, sterilize at 121°C for 20 min, and store at room temperature.

[0040] LB+Amp solid medium: LB liquid medium plus 20 g / L agar, sterilize in an autoclave at 121°C for 20 min, then cool to about 45°C, add the prepared Amp solution to make the final concentration of 100 mg / L.

[0041] PDA solid culture medium: 200 g of peeled potatoes were boiled in tap water until muddy, filtered, and the supernatant was added with 20 g of glucose and 20 g of agar, and the volume was adjusted to 1 L with tap water. The solution was sterilized at 121°C for 20 min under natural pH.

[0042] MM+G liquid medium: 1 mL each of 60 g / L CaCl2·2H2O and 60 g / L MgSO4·7H2O, 100 μL each of 3.7 g / L CoCl2·6H2O, 5 g / L FeSO4·7H2O, 1.4 g / L ZnSO4·7H2O, and 1.6 g / L MnSO4·H2O, 1.5 g KH2PO4, 0.5 g (NH4)2SO4, and 2 g glucose. Make up to 100 mL and sterilize at 121°C for 20 min.

[0043] MM+G solid medium: 1 mL each of 60 g / L CaCl2·2H2O and 60 g / L MgSO4·7H2O, 100 μL each of 3.7 g / L CoCl2·6H2O, 5 g / L FeSO4·7H2O, 1.4 g / L ZnSO4·7H2O, and 1.6 g / L MnSO4·H2O, 1.5 g KH2PO4, 0.5 g (NH4)2SO4, 2 g glucose, and 2 g agar powder. Make up to 100 mL and sterilize at 121°C for 20 min.

[0044] MM + bagasse + peptone fermentation medium: 1 mL each of 60 g / L CaCl2·2H2O, 60 g / L MgSO4·7H2O, 100 μL each of 3.7 g / L CoCl2·6H2O, 5 g / L FeSO4·7H2O, 1.4 g / L ZnSO4·7H2O, and 1.6 g / L MnSO4·H2O, 1.5 g KH2PO4, 0.5 g (NH4)2SO4, 2 g bagasse, and 0.5 g peptone. The volume was adjusted to 100 mL and sterilized at 121°C for 20 min.

[0045] Unless otherwise specified, the materials and reagents used in the following examples are all commercially available products. Unless otherwise specified, the experimental methods used in the following examples are all conventional methods in the art.

[0046] Example 1 This example provides a recombinant plasmid pPcbh1-tTHAⅡ for heterologously expressing thaumatin II protein and a method for constructing the same.

[0047] 1. PCR amplification of the cbh1 promoter, cbh1 terminator, thaumatin II gene, and pyr4 gene expression cassette (1) Based on the nucleotide sequence of the Thaumatin II gene registered in Genebank (accession number AAA93095.1), the sequence was optimized according to the codon preference of Trichoderma reesei and synthesized by Beijing Qingke Biotechnology Co., Ltd. The optimized thaumatin nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0048] (2) Amplification of the promoter and terminator of the cellobiohydrolase I (CBH1) gene: PCR amplification was performed using the genomic DNA of Trichoderma reesei QM6a as a template and primers Gcbh1-UF and Gcbh1-UR to obtain the cbh1 promoter fragment Pcbh1 (nucleotide sequence shown in SEQ ID NO. 3). PCR amplification was performed using the genomic DNA of QM6a as a template and primers Gcbh1-DF and Gcbh1-DR to obtain the cbh1 terminator fragment Tcbh1 (nucleotide sequence shown in SEQ ID NO. 4).

[0049] (3) Amplification of the codon-optimized thaumatin II gene sequence: Using the gene synthesis plasmid as a template and GtThaⅡ-F and GtThaⅡ-R as primers, PCR amplification was performed to obtain the codon-optimized thaumatin II gene tThaⅡ fragment.

[0050] (4) Amplification of the plasmid vector backbone: Using the pBluescript II SK (+) vector as a template and Gbackbone-F and Gbackbone-R as primers, PCR amplification was performed to obtain a vector backbone fragment containing the Amp selection marker.

[0051] (5) Amplification of the screening marker pyr4 expression cassette: Using the genomic DNA of Trichoderma reesei QM6a as a template and pyr4-F and pyr4-R as primers, PCR amplification was performed to obtain the nucleotide sequence of the Trichoderma reesei orotidine-5ʹ-phosphate decarboxylase gene pyr4 as shown in SEQ ID NO.5.

[0052] The reaction system of the above PCR amplification is shown in Table 2, and the reaction procedure is shown in Table 3.

[0053] Table 2 PCR reaction system

[0054] Table 3 PCR reaction procedure

[0055] After PCR amplification of the above gene fragments, electrophoresis was performed on 1% agarose gel, and the DNA fragments were recovered, dissolved in 20 μL deionized water, and stored at -20°C.

[0056] 2. Using Gibson ligation, Pcbh1, tThaⅡ, Tcbh1, and vector backbone fragments were ligated using Vazyme 2x clonExpress Mix ligase to obtain the recombinant expression vector pPcbh1-tTHAⅡ. The plasmid map is shown in the figure. Figure 1 shown.

[0057] 3. Use the ligated vector product to transform competent E. coli T1 cells, spread onto LB+Amp plates, and culture overnight at 37°C to produce single colonies. Use primers VPcbh1-F and VtThaⅡ-R to perform colony PCR and identify transformants. After verifying that the ligation is correct, extract the plasmid from the transformant and send it for sequencing. Once sequencing is correct, the recombinant expression vector pPcbh1-tTHAⅡ containing the thaumatin II gene is obtained.

[0058] Example 2 This example provides a recombinant strain for heterologously expressing thaumatin II protein and a method for constructing the same.

[0059] 1. Preparation and Transformation of Trichoderma reesei QM6aΔpyr4 Protoplasts (1) Take freshly cultured spores of Trichoderma reesei QM6aΔpyr4 from a slant or plate, wash the spores with an appropriate amount of sterile water to prepare a spore suspension, and filter through a 200-mesh sieve to remove residual hyphae. Inoculate the filtered spore suspension into a 500-mL Erlenmeyer flask containing 100 mL of MM liquid medium and incubate at 28°C for 13-14 hours until the hyphae are extended.

[0060] (2) Filter the culture medium through a 200-mesh sieve, collect the cells, wash them 2-3 times with sterile water, and finally wash them once with 1.2 M MgSO4 solution, allowing the solution to drain naturally.

[0061] (3) Rinse the cells on the sieve into a flask containing 15 mL of lysis solution (1.2 M MgSO4 containing 150 mg of lysing enzyme and 15 mg of cellulase). Incubate at 30°C for 1.5 h. Observe the protoplast formation under a microscope. After 1 h, take samples every 10 min for observation.

[0062] (4) When a large number of protoplasts are produced and a large number of hyphae are still present, add an equal volume of 0.6 M sorbitol solution to terminate the reaction. Filter through a 200-mesh sieve to remove the remaining hyphae, and collect the protoplast precipitate by centrifugation at 3000 rpm for 10 min at room temperature.

[0063] (5) Pour off the supernatant along one side of the pellet, resuspend the protoplast pellet in 1.0 M sorbitol solution, and centrifuge at 3000 rpm for 10 min at room temperature.

[0064] (6) Repeat step (5), discard the supernatant and suspend the protoplasts in 200 μL of 1.0 M sorbitol solution. Observe and count using a hemocytometer.

[0065] (7) The pyr4 expression cassette obtained in step (5) of Example 1 was mixed with 3 μg of the thaumatin II expression cassette (amplified from the recombinant expression vector pPcbh1-tTHAⅡ in Example 1) at a molar ratio of 1:3 in a volume not exceeding 20 μL. The DNA mixture was added to the protoplasts prepared above and gently mixed. 50 μL of PEG4000 was then added to each of the protoplasts, mixed again, and placed on ice for 30 min. A control was set up in which an equal volume of sterile water was used instead of the DNA mixture.

[0066] (8) Add 1 mL of PEG4000 to each of the above tubes, mix well, and let it stand at room temperature for 20 min.

[0067] (9) Finally, add 1 mL of 1.0 M sorbitol to each plate, mix well, and then mix with 6 mL of MM solid culture medium for plating.

[0068] (10) Culture at 28°C for 3-4 days.

[0069] 2. Screening of high-yielding strains of Thaumatin II (1) The transformants grown on the protoplast transformation plate were picked onto a PDA solid plate and cultured at 28°C for 5-7 days. After the strain grew and produced spores, genomic DNA was extracted for verification. PCR verification was performed using genomic DNA as a template and VtThaⅡ-F and VtThaⅡ-R as primers to obtain a positive strain producing thaumatinⅡ.

[0070] (2) A large number of positive strains screened and the control strain DF230103 were inoculated on PDA solid plates and cultured at 28°C for 7 days. Fermentation was carried out after the strains grew and produced spores.

[0071] (3) Wash the Trichoderma reesei spores with sterile water, filter the mycelium with a magic filter cloth, collect the spore suspension in a 15 mL centrifuge tube and count.

[0072] (4) Inoculate the spore suspension into 50 mL of MM + bagasse + peptone fermentation medium in a 250 mL triangular flask with an inoculum volume of 10 6 The cells were cultured at 28°C and 220 rpm for 7 days.

[0073] (5) After the fermentation is completed, the fermentation broth was centrifuged at 13,000 rpm for 10 min, and the supernatant was used for sample preparation and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) verification.

[0074] (6) SDS-PAGE electrophoresis verification revealed that not all positive transformants could stably express the target protein. Figure 2As shown, among the seven positive transformants verified by fermentation, only strains H1, H2, and H3 showed clear thaumatin II protein bands, while no protein expression was detected in strains H4-H7. This result indicates that the insertion site significantly affects the expression level of the exogenous protein, and different insertion sites lead to different biological properties. Fermentation performance screening identified strain H2 as a high-yielding strain, with protein expression levels significantly superior to other positive clones, providing a reliable foundation for subsequent functional verification and process optimization.

[0075] (7) Thaumatin II protein performance testing: The sweetness characteristics of the fermented protein were verified by combining sensory evaluation with instrumental testing. Based on the international standard sensory evaluation method (referring to GB / T 12310-2008), a professionally trained sensory evaluation panel conducted a double-blind crossover test in a controlled environment at a temperature of 25±1°C and a humidity of 50%±5%. The sweetness intensity scale (0-100 points) was scored for protein solutions at different dilution gradients and 10% sucrose solution. The results showed that the protein expressed by the H2 strain had a sweetness perception equivalent to that of a 10% sucrose solution at a concentration of 0.00278% (i.e., 1 / 3600 of the sweetness of a 10% sucrose solution), and there was no significant aftertaste bitterness or off-flavor residue.

[0076] 3. Chromosome walking PCR was used to analyze the insertion site of H2 strain. The specific operation process is as follows: Using chromosome walking PCR, three nested specific primers (sp1, sp2, and sp3) were designed at the cbh1 terminator, along with one random degenerate primer (AD). The target fragment was selectively amplified through three consecutive PCR reactions using different annealing temperatures. TA cloning was used to connect the target fragments, and the plasmids were sequenced and analyzed.

[0077] (1) DNA from the recombinant protein high-producing strain H2 was extracted and the first PCR amplification was performed using degenerate primers AD and specific nested primer sp1. After the amplified product was detected by gel electrophoresis, the specific band was cut, recovered, and purified.

[0078] (2) Using the first purified PCR product as a template, replace the nested primer sp2 and perform a second PCR amplification with the degenerate primers AD, gradually "moving" to the unknown region of the genome. After the amplified product is detected by gel electrophoresis, the specific band is cut, recovered, and purified.

[0079] (3) Using the PCR product purified for the second time as a template, replace the nested primer sp3 and perform a third PCR amplification with the degenerate primers AD. After the amplified product is detected by gel electrophoresis, the specific band is excised, recovered and purified, and the third purified fragment is ligated and transformed using TA cloning, and the plasmid is sequenced and analyzed.

[0080] (4) Sequencing obtains the complete sequence of the unknown region of the genome, and by comparing the sequencing results with the host genome database, the insertion site H2 of the exogenous gene is determined.

[0081] Sequencing results revealed that the insertion site of the recombinant gene in strain H2 (referred to as the H2 site) was located in an unknown region of the Trichoderma reesei QM6aΔpyr4 genome (shown in SEQ ID NO. 6). Specifically, the recombinant gene in strain H2 was inserted at position 210 of the gene fragment shown in SEQ ID NO. 6. The coding gene closest upstream to the insertion site is ID 75027, and the coding gene closest downstream to the insertion site is ID 120260.

[0082] 4. Verification of high-yield strains: 4.1 Construction of recombinant Trichoderma reesei strain H2-tThaⅡ (1) Amplification Figure 3 Component fragments of the plasmid vector pH2-Pcbh1-tTHAⅡ: Using strain H2 genomic DNA as a template and primers GH2-UF and GH2-UR, PCR amplification was performed to obtain the homology arm sequence upstream of the insertion site H2. Using strain H2 genomic DNA as a template and primers GH2-DF and GH2-DR, PCR amplification was performed to obtain the homology arm sequence downstream of the insertion site H2. Using the pPcbh1-tTHAⅡ plasmid of Example 1 as a template and primers GH2-THAⅡ-UF and GH2-THAⅡ-UR, PCR amplification was performed to obtain a complete expression cassette containing the gene with the nucleotide sequence shown in SEQ ID NO. 1 (hereinafter referred to as the thaumatin II optimized gene). The expression cassette includes the cbh1 promoter, the thaumatin II optimized gene, and the cbh1 terminator. Using Trichoderma reesei QM6a genomic DNA as a template and primers pyr4-F and pyr4-R, PCR amplification was performed to obtain the selection marker gene pyr4. Using the pBluescriptII SK(+) vector as a template and primers Gbackbone-H2-F and Gbackbone-H2-R, PCR amplification was performed to obtain a vector backbone fragment containing the Amp selection marker. After PCR amplification, the gene fragment was subjected to electrophoresis on a 1% agarose gel, and the DNA fragment was recovered, dissolved in 20 µL of deionized water, and stored at -20°C.

[0083] (2) Construction of pH2-Pcbh1-tTHAⅡ plasmid vector: Using Gibson ligation, the upstream homology arm of the insertion site H2, the downstream homology arm of the insertion site H2, the complete expression cassette with the optimized thaumatin II gene, and the vector backbone fragment were connected. The competent E. coli T1 was transformed and plated on LB+Amp plates. After culturing at 37°C overnight, single colonies grew. Primers VH2-F and VtThaⅡ-R were designed for colony PCR to identify transformants. The plasmids of the transformants with correct ligation were extracted and sent for sequencing. After sequencing was correct, the recombinant expression vector pH2-Pcbh1-tTHAⅡ containing the optimized thaumatin II gene was obtained.

[0084] (3) Obtaining a recombinant strain of Trichoderma reesei: PCR amplification was performed to obtain a component fragment containing upstream and downstream homology arms of the insertion site H2 and a complete expression cassette containing the optimized thaumatin II gene. The component fragment and the selection marker pyr4 were transformed into Trichoderma reesei QM6aΔpyr4 by protoplast transformation, using the same method as step 1 of Example 2. PCR was performed using the transformant genomic DNA as a template, VH2-UF and VtThaⅡ-R as external primers, and VH2-F and VH2-R as internal primers to identify whether the optimized thaumatin II gene was integrated into the H2 site. The positive transformants obtained by screening were the recombinant Trichoderma reesei strain H2-tThaⅡ.

[0085] (4) Fermentation test: The recombinant Trichoderma reesei strain H2-tThaⅡ, strain H2 and wild-type strain QM6a The strains were inoculated on PDA plates and fermented after spore formation. The steps were the same as steps (2) to (4) in step 2 of this example. After 168 h of fermentation, the secretion of thaumatin II protein in the supernatant of the fermentation broth was detected by SDS-PAGE electrophoresis. Figure 4 The results showed that the recombinant Trichoderma reesei strain H2-tThaⅡ and strain H2 produced the same level of thaumatinⅡ protein. This construction method inserted the optimized thaumatinⅡ gene into the H2 site, and obtained a positive strain that highly expressed thaumatinⅡ protein.

[0086] 4.2 Fermentation of recombinant Trichoderma reesei strain H2-tThaⅡ in a 10 L tank (1) Inoculate the strain H2-tThaⅡ on a PDA solid plate and culture it at 28℃ for 7 days. Ferment it after the strain grows and produces spores.

[0087] (2) Wash the conidia from the PDA plate with sterile water in a clean bench, filter the mycelium with a magic filter cloth, and then count the spore suspension using a hemocytometer.

[0088] (3) The spore suspension was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of MM+G liquid medium. After 48 h of culture, the bacteria in the seed bottle were transferred to a 10 L fermenter. The volume of the medium in the fermenter (MM+bagasse+peptone fermentation medium) was 7 L. The culture was continuously fed at a rate of 1000 mL / 48 h. The culture conditions were as follows: temperature 28°C, 300 rpm, ventilation ratio controlled at about 0.8 vvm, and pH maintained at 5±1.

[0089] (4) Fermentation was carried out for 7 days from the start of inoculation. Samples were collected every 24 hours during the culture process. The fermentation supernatant was collected and centrifuged at 13,000 rpm for 10 minutes. The supernatant was stored at 4°C. The supernatant was used for sample preparation and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) verification.

[0090] The results of SDS-PAGE electrophoresis are as follows Figure 5 As shown in the figure, after the fermentation process was adjusted and optimized, the high-yield strain H2-tThaⅡ constructed could reach 12.11 g / L after fermentation in a 10 L fermenter, and the target protein thaumatin II accounted for 92.7% of the total protein. Its excellent purity characteristics significantly simplified the downstream purification process.

[0091] 5. Expression Verification of Insertion Site Using the Trichoderma reesei strain QM6aΔpyr4 as the starting strain, the original thaumatin II gene was integrated into the specific insertion site H2 identified in this study. Fermentation assays were performed to measure the expression level of the sweet protein thaumatin II in this strain, and the expression levels were compared with those of the strain H2 screened in this study and the control strain DF230103. The results showed that inserting the original thaumatin II gene into the H2 site resulted in expression levels comparable to those at the cbh1 site. The specific steps are as follows: (1) Amplification Figure 6Component fragments of the plasmid vector pH2-Pcbh1-THAⅡ: PCR amplification using strain H2 genomic DNA as a template and primers GH2-UF and GH2-UR obtained the homology arm sequence upstream of the insertion site H2. PCR amplification using strain H2 genomic DNA as a template and primers GH2-DF and GH2-DR obtained the homology arm sequence downstream of the insertion site H2. PCR amplification using the pPcbh1-THAⅡ plasmid as a template and primers GH2-THAⅡ-UF and GH2-THAⅡ-UR obtained the complete expression cassette containing the original thaumatin II gene, including the cbh1 promoter, the original thaumatin II gene, and the cbh1 terminator. PCR amplification using Trichoderma reesei QM6a genomic DNA as a template and primers pyr4-F and pyr4-R obtained the selectable marker gene pyr4. Using the pBluescript II SK(+) vector as a template and primers Gbackbone-H2-F and Gbackbone-H2-R, PCR amplification was performed to obtain a vector backbone fragment containing the Amp selection marker. The PCR amplification reaction system is shown in Table 2 of Example 1, and the reaction procedure is shown in Table 3 of Example 1. After PCR amplification of the gene fragment, the DNA fragment was recovered by electrophoresis on a 1% agarose gel, dissolved in 20 µL of deionized water, and stored at -20°C.

[0092] (2) Construction of pH2-Pcbh1-THAⅡ plasmid vector: Using Gibson ligation, the upstream homology arm of the insertion site H2, the downstream homology arm of the insertion site H2, the expression cassette containing the original thaumatin II gene, and the vector backbone fragment were connected. The competent E. coli T1 was transformed and plated on LB+Amp plates. After culturing at 37°C overnight, single colonies grew. Primers VH2-F and VthaⅡ-R were designed for colony PCR to identify transformants. The plasmids of the transformants with correct ligation were extracted and sent for sequencing. After sequencing was correct, the recombinant expression vector pH2-Pcbh1-THAⅡ containing the original thaumatin II gene was obtained.

[0093] (3) Obtaining a recombinant strain of Trichoderma reesei: PCR amplification was performed to obtain a component fragment containing the upstream and downstream homology arms of the insertion site H2 and the original thaumatin II gene expression cassette. This component fragment and the selection marker pyr4 were transformed into Trichoderma reesei QM6aΔpyr4 by protoplast transformation, using the same method as step 1. PCR was performed using the transformant genomic DNA as a template, VH2-UF and VthaⅡ-R as external primers, and VH2-F and VH2-R as internal primers to identify whether the original thaumatin II gene was integrated into the H2 site. The positive transformants identified were named H2-ThaⅡ.

[0094] (4) Fermentation detection: The positive transformants H2-ThaⅡ, strain H2, control strain DF230103, and wild-type strain QM6a were inoculated on PDA plates and fermented after the strains grew and produced spores. The steps were the same as steps (2) to (4) in step 2 of this example. After 168 hours of fermentation, the secretion of thaumatinⅡ in the supernatant of their fermentation broth was detected by SDS-PAGE electrophoresis. Figure 7 The results showed that the original thaumatin II gene inserted into the H2 site could achieve an expression level comparable to that of the cbh1 site under shake flask conditions.

[0095] Example 3 This example provides the use of the Trichoderma reesei expression site H2 in constructing a recombinant Trichoderma reesei strain that highly expresses alkaline lipase.

[0096] The alkaline lipase gene was integrated into the specific insertion site H2 identified in Example 2 using the Trichoderma reesei strain QM6aΔpyr4 as the starting strain. The expression level of lipase in the strain was detected by fermentation and compared with the control strain. TPcdna1-pelc By comparison, it was found that inserting the recombinant gene into the H2 site can achieve expression levels comparable to those at the cbh1 site. The specific steps are as follows: 1. Amplification Figure 8 The components of the plasmid vector pH2-pelc are shown: QM6a Using genomic DNA as a template and primers GH2-U-pyr4-F and GH2-U-pyr4-R, PCR amplification was performed to obtain the homology arm sequence upstream of the insertion site H2. Using QM6a genomic DNA as a template and primers GH2-DF and GH2-DR, PCR amplification was performed to obtain the homology arm sequence downstream of the insertion site H2. Using the pPcdna1-pelc plasmid as a template and primers GH2-pelc-UF and GH2-pelc-UR, PCR amplification was performed to obtain the complete expression cassette containing the alkaline lipase gene, including the selection marker pyr4, the cdna1 promoter, the alkaline lipase gene, and the cbh1 terminator. Using the pBluescript II SK(+) vector as a template and primers Gbackbone-H2-F and Gbackbone-H2-R, PCR amplification was performed to obtain the vector backbone fragment containing the Amp selection marker. After PCR amplification of the above gene fragments, electrophoresis was performed on 1% agarose gel, and the DNA fragments were recovered, dissolved in 20 μL deionized water, and stored at -20°C.

[0097] 2. Construction of the pH2-pelc plasmid vector: Using Gibson ligation, the upstream homology arm of insertion site H2, the downstream homology arm of insertion site H2, the alkaline lipase gene expression cassette, and the vector backbone fragment were ligated. The fragments were transformed into competent E. coli T1 cells, plated onto LB+Amp solid plates, and cultured overnight at 37°C to produce single colonies. Colony PCR using primers VH2-F and Vpelc-R was designed to identify transformants. Plasmids from transformants that confirmed correct ligation were extracted and sent for sequencing. Once sequencing was complete, the recombinant expression vector pH2-pelc containing the lipase gene was obtained.

[0098] 3. Obtaining a recombinant strain of Trichoderma reesei: PCR amplification was performed to obtain a component fragment with the upstream and downstream homology arms of the insertion site H2 and the lipase gene expression cassette. The component fragment was transformed into Trichoderma reesei QM6aΔpyr4 by protoplast transformation, and the steps were the same as those in step 1 of Example 2. PCR was performed using the transformant genomic DNA as a template, VH2-UF and Vpelc-R as external primers, and VH2-F and VH2-R as internal primers to identify whether the lipase gene expression cassette was integrated into the H2 site. The identified positive transformants were named H2-pelc .

[0099] 4. Fermentation test: The positive transformants screened H2-pelc , control strain TPcdna1-pelc Wild-type strain QM6a The strains were inoculated on PDA plates and fermented after spore formation, following the same steps as step 4 of Example 2. After 168 h of fermentation, the lipase secretion in the fermentation supernatant was detected by SDS-PAGE electrophoresis.

[0100] 5. Determination of alkaline lipase activity: (1) NaOH titration a. In a 100 mL beaker, add 5 mL of the emulsion, 4 mL of glycine-NaOH buffer, and 100 µL of 0.4 MCaCl2. Gently shake to mix, then preheat in a water bath at 34°C, 110 rpm for 20 min. b. Add 1 mL of glycine-NaOH buffer to the blank control and 1 mL of diluted fermentation supernatant to the sample to be measured. Incubate at 34°C and 110 rpm for 10 min. c. Add 20 mL of 95% ethanol to terminate the reaction; d. Titrate with 0.05 M NaOH solution to a final pH of 9.2, and record the volume of 0.05 M NaOH solution consumed; e. Lipase activity is expressed in lipase activity units, which is defined as the amount of 1 μmol of titratable fatty acid produced by 1 mL of liquid enzyme hydrolyzing the substrate in 1 minute under certain temperature and pH conditions. This is one enzyme activity unit, expressed as U / mL. The formula for calculating lipase activity is:

[0101] Where: X1 – enzyme activity of the sample U / mL; A – the volume of 0.05 M NaOH standard solution consumed when titrating the sample, in milliliters (mL); B – the volume of 0.05 M NaOH standard solution consumed in the blank titration, in milliliters (mL); 1 mL of 50–0.05 M NaOH solution is equivalent to 50 µmol of lipase; f – enzyme solution dilution multiple; T – reaction time, calculated as 10 min; M – The volume of the enzyme sample used in the assay, calculated per 1 mL.

[0102] The enzyme activity of the fermentation broth supernatant was measured by NaOH titration and is shown in Table 4. TPcdna1-pelc The enzyme activity was 432.5U / mL, and the experimental strain H2-pelc The enzyme activity was 457.5U / mL.

[0103] Table 4 Lipase activity determination

[0104] (2) Agar diffusion method Prepare a 1% olive oil emulsion test plate (1% olive oil emulsion and 2% agar powder dissolved in pH 9.2 Gly-NaOH buffer), punch holes on the plate with a hole puncher and add 20 μL of fermentation broth supernatant to the wells. Leave at room temperature at 22°C for 24 h before observation.

[0105] The test ends with the enzymatic hydrolysis cycle. Figure 9 As shown, strains were grown on agar diffusion plates. H2-pelc The enzymatic hydrolysis circle and TPcdna1- pelc The enzymatic hydrolysis circles are almost the same.

[0106] The above examples show that the contribution of this site to the expression levels of recombinant genes thaumatin II and alkaline lipase is comparable to that of the cbh1 site, and the H2 site has the potential to become a new heterologous protein expression site in Trichoderma reesei.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Trichoderma reesei expression site H2, characterized in that The Trichoderma reesei expression site H2 is located at position 210 in the gene fragment shown in SEQ ID NO. 6 in the Trichoderma reesei genome.

2. Use of the Trichoderma reesei expression site H2 according to claim 1 in constructing a recombinant Trichoderma reesei strain that highly expresses a target product.

3. A gene encoding thaumatin II protein, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

4. A recombinant plasmid pPcbh1-tTHAⅡ for heterologous expression of thaumatinⅡ protein, characterized in that: The recombinant plasmid pPcbh1-tTHAⅡ contains the gene encoding thaumatinⅡ protein, and contains a cbh1 promoter and a cbh1 terminator.

5. The recombinant plasmid pPcbh1-tTHAⅡ according to claim 4, characterized in that The backbone vector of the recombinant plasmid pPcbh1-tTHAⅡ is pBluescript II SK (+) plasmid.

6. A recombinant Trichoderma reesei strain producing thaumatin II protein, characterized in that: The recombinant Trichoderma reesei strain is constructed by transferring the recombinant plasmid pPcbh1-tTHAⅡ described in claim 5 into Trichoderma reesei protoplasts.

7. The recombinant Trichoderma reesei strain according to claim 6, characterized in that The insertion site of the gene with the nucleotide sequence shown in SEQ ID NO. 1 in the recombinant plasmid pPcbh1-tTHAⅡ is the above-mentioned Trichoderma reesei expression site H2.

8. The recombinant Trichoderma reesei strain according to claim 6, characterized in that The starting strain of the recombinant Trichoderma reesei strain is Trichoderma reesei QM6aΔpyr4.

9. Use of the recombinant Trichoderma reesei strain according to any one of claims 6 to 8 in producing thaumatin II protein.

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