Method for screening high-yield recombinant human lactoferrin through nucleic acid aptamer-assisted mutagenesis of trichoderma reesei strain and application

Abstract

The invention discloses a method for screening high-yield recombinant human lactoferrin through nucleic acid aptamer-assisted mutagenesis of a trichoderma reesei strain and application, and belongs to the technical field of microorganisms. The nucleic acid aptamer Lac-6a-FAM-BHQ1 capable of changing the fluorescence intensity according to the content of lactoferrin is constructed on the basis of the aptamer Lac-6a, and the nucleic acid aptamer can be used for rapidly selecting a mutant strain for producing high-titer recombinant human lactoferrin. The ARTP mutagenesis technology is combined with a nucleic acid aptamer auxiliary screening system, the mutant strain with the improved recombinant human lactoferrin yield is rapidly obtained from a large number of mutant strains, and the yield reaches 274.57 mg / L and is improved by 24.8% compared with that of an original strain. After the genes 111681 and 62716 are over-expressed in T. reesei, compared with a control strain, the yields of the recombinant human lactoferrin of the recombinant strain are respectively increased by 6.9% and 22.1%.

Description

Technical Field

[0001] This invention relates to a method and its application for screening high-yield recombinant human lactoferrin by nucleic acid aptamer-assisted mutagenesis of Trichoderma reesei strains, belonging to the field of microbial technology. Background Technology

[0002] Aptamers are oligonucleotide sequences composed of DNA or RNA that bind to specific target molecules with high specificity and high affinity. Aptamers are typically obtained from nucleic acid molecular libraries through a method called SELEX (Spiritual Enrichment of Ligand Systematic Evolution). RNA aptamers have become one of the most effective and widely used intracellular / intracellular biosensors. Small-molecule biosensors based on RNA aptamers can achieve specific recognition and binding of the neurotransmitter precursors 5-hydroxytryptophan and 3,4-dihydroxyphenylalanine, enabling aptamer diversity and more flexible use (published in the paper "Recurrent RNA motifs as scaffolds for geneticallyencodable small-molecule biosensors").

[0003] Lactoferrin is widely used in the food and pharmaceutical industries due to its unique biological functions (such as antibacterial, antiviral, and anti-inflammatory effects), especially in dairy product processing. As a representative of health foods, lactoferrin not only provides a source of nutrition but also offers multiple layers of protection for human health. Lactoferrin added to infant formula can enhance the immunity of infants and young children. Because of its safety and immunocompatibility with the host, lactoferrin also holds great potential in the development of new drugs and vaccines.

[0004] Constructing chassis cells for efficient protein expression is one of the core tasks in the fields of synthetic biology and biomanufacturing. Previous reports have demonstrated the successful use of *Trichoderma reesei* as chassis cells for recombinant human lactoferrin expression (disclosed in patent publication number CN117487675A). However, further modifications to *Trichoderma reesei* are needed to achieve even higher levels of recombinant human lactoferrin production. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides a method and application for screening high-yield recombinant human lactoferrin from Trichoderma reesei strains using nucleic acid aptamer-assisted mutagenesis. The aim is to increase the yield of recombinant human lactoferrin in Trichoderma reesei strains by combining mutagenesis technology with a highly efficient screening system assisted by nucleic acid aptamers, thereby promoting its application in the fields of medicine and food.

[0006] The first technical solution provided by the present invention is a method for screening Trichoderma reesei that produces high levels of recombinant human lactoferrin. The method involves characterizing the yield of human lactoferrin by using the fluorescence intensity of the nucleic acid aptamer Lac-6a-FAM-BHQ1 after it binds to the fermentation broth of Trichoderma reesei, and screening out Trichoderma reesei that produces high levels of recombinant human lactoferrin.

[0007] In one embodiment, the structure of the nucleic acid aptamer Lac-6a-FAM-BHQ1 is “5′FAM+Lac-6a+3′-BHQ1”.

[0008] In one embodiment, the nucleotide sequence of the nucleic acid aptamer Lac-6a is shown in SEQ ID NO.1.

[0009] In one embodiment, the Trichoderma reesei is subjected to ARTP mutagenesis.

[0010] In one implementation, the ARTP mutagenesis time is selected as 180s, and the lethality rate can reach 98%.

[0011] In one embodiment, Trichoderma reesei CJ2095 is used as the starting strain for mutagenesis. Trichoderma reesei CJ2095 is disclosed in patent publication number CN117487675A, corresponding to Trichoderma reesei CJ2095 in patent publication number CN117487675A.

[0012] The second technical solution provided by the present invention is the application of gene 111681 and / or gene 62716 in increasing the yield of recombinant human lactoferrin from Trichoderma reesei, wherein the nucleotide sequences of gene 111681 and gene 62716 are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0013] In one embodiment, the application is the overexpression of gene 111681 and / or gene 62716 in Trichoderma reesei.

[0014] The third technical solution provided by the present invention is a Trichoderma reesei engineered strain that produces recombinant human lactoferrin. The engineered strain is Trichoderma reesei as the starting strain, and the overexpression of gene 111681 and / or gene 62716 in the starting strain is shown in SEQ ID NO.2 and SEQ ID NO.3.

[0015] In one embodiment, the starting strain also knocks out the DNA repair protein gene ku80 and integrates and expresses the genes 111681 and 62716 at the ku80 site.

[0016] In one embodiment, the gene ku80The nucleotide sequence is NCBI numbered NW_006711151.1.

[0017] In one embodiment, Trichoderma reesei CJ2095 is used as the starting strain, which is disclosed in patent publication number CN117487675A, corresponding to Trichoderma reesei CJ2095 in patent publication number CN117487675A.

[0018] The fourth technical solution provided by the present invention is a method for improving lactoferrin production in Trichoderma reesei. The method involves overexpressing gene 111681 and / or knocking out gene 62716 in Trichoderma reesei. The nucleotide sequences of gene 111681 and gene 62716 are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0019] In one embodiment, the Trichoderma reesei also knocked out the DNA repair protein gene ku80 and integrated and expressed the genes 111681 and 62716 at the ku80 site.

[0020] In one embodiment, Trichoderma reesei CJ2095 is used as the starting strain, which is disclosed in patent publication number CN117487675A, corresponding to Trichoderma reesei CJ2095 in patent publication number CN117487675A.

[0021] The fifth technical solution provided by this invention is the application of the engineered Trichoderma reesei strain described in the third technical solution or the method described in the fourth technical solution in the preparation of lactoferrin or lactoferrin-containing products.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Based on the aptamer Lac-6a, the present invention constructs a nucleic acid aptamer Lac-6a-FAM-BHQ1 that can change the fluorescence intensity according to the lactoferrin content, which can be used to quickly select mutagenic strains that produce high titers of recombinant human lactoferrin.

[0023] (2) This invention uses ARTP mutagenesis technology combined with a nucleic acid aptamer-assisted screening system to quickly obtain mutagenesis strains with increased recombinant human lactoferrin production from a large number of mutagenesis strains. The production reached 274.57 mg / L, which is 24.8% higher than that of the starting strain.

[0024] (3) In T. reesei overexpressed genes 111681 and 62716 Afterwards, compared with the control strain, the recombinant human lactoferrin production of the recombinant strain increased by 6.9% and 22.1%, respectively. Attached Figure Description

[0025] Figure 1 A: Schematic diagram of a functionalized nucleic acid aptamer; B: Specificity and functional verification of the nucleic acid aptamer for detecting HLf; C: Functional verification of the nucleic acid aptamer for detecting HLf in fermentation broth.

[0026] Figure 2 The relationship between ARTP mutagenesis time and lethality of Trichoderma reesei spores.

[0027] Figure 3 This shows the 96-well plate culture status of the mutant strain.

[0028] Figure 4 The fluorescence intensity of the fermentation broth of the mutagenic strain in a 96-well plate was detected using the nucleic acid aptamer Lac-6a-FAM-BHQ.

[0029] Figure 5 This is a comparison of the recombinant human lactoferrin production of the mutagenized strain and the original strain during shake-flask fermentation.

[0030] Figure 6 This represents the number of genes that have undergone significant changes in the mutagenic strain.

[0031] Figure 7 To compare transcriptomic analyses, GO enrichment was used to classify differentially expressed genes.

[0032] Figure 8 For molecular modification T . reesei The effect of synthetic recombinant human lactoferrin content. Detailed Implementation

[0033] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0034] Culture medium: LB medium (1 L): 10 g peptone, 10 g NaCl, 5 g yeast extract.

[0035] PDA medium (1 L): 37 g potato dextrose agar medium.

[0036] Malt extract agar medium (1 L): 30 g malt extract, 20 g agar powder.

[0037] Fermentation medium (1 L): 4 g KH2PO4, 2.8 g (NH4)2SO4, 0.6 g MgSO4·7H2O, 0.5 g CaCl2, 0.6 g urea, 3 g peptone, 1 g Tween-80, 5 g CaCO3, trace elements (0.005 g FeSO4·7H2O, 0.0016 g MnSO4·H2O, 0.0014 g ZnSO4·7H2O, 0.002 g CoCl2·6H2O), 20 g wheat bran, 30 g microcrystalline cellulose.

[0038] Seed culture medium (1 L): 20 g glucose, 15 g K2HPO4, 5 g (NH4)2SO4, 0.6 g MgSO4·7H2O, 0.6 g CaCl2, 2 g peptone, trace elements (0.005 g FeSO4·7H2O, 0.0016 g MnSO4·H2O, 0.0014 g ZnSO4·7H2O, 0.002 g CoCl2·6H2O).

[0039] IM medium (1 L): 1.45 g KH2PO4, 2.05 g K2HPO4, 0.5 g (NH4)2SO4, 0.5 g MgSO4·7H2O, 0.15 g NaCl, 5 g glycerol, 0.06 g CaCl2, 0.0025 g FeSO4·7H2O, 1.8 g glucose, 8.54 g 2-morpholinoethanesulfonic acid, 200 μM acetylsyleugenone.

[0040] E. coli and A. tumefaciens Cultured in LB medium. T. reesei Cultured in PDA medium. Malt extract agar medium was used for culture. T. reesei Spore production. Co-culture medium (IM) is used. T. reesei The conversion. When the above culture medium needs to be prepared as a solid medium, add 15 g·L⁻¹. -1 Agar powder, 1 g·L⁻¹, is added when culturing uracil-deficient strains. -1 Uracil.

[0041] T. reesei Transformation: pass A. tumefaciens Mediated transformation method T. reesei Gene editing was performed. Genes containing recombinant plasmids were then... A. tumefaciens Inoculated into LB tubes containing kanamycin and rifampin and cultured for 24 days 36 h. Collect 400 μL of bacterial cells, centrifuge, discard the supernatant, resuspend the bacterial pellet in 5 mL of IM tube containing MES and AS, and pre-incubate at 30°C and 200 rpm until OD. 600 Reaching 0.6 Between 0.8 and 0.8, protected from light. Take an equal volume of... T. reesei Spores and pre-cultured A. tumefaciens Spread onto IM plates containing cellophane and incubate at 24°C in the dark for 48 h. After incubation, transfer the cellophane to screening plates and incubate at 30°C for 2 hours. Wait 7 days for transformants to grow. Pick transformants and culture them on a secondary screening plate for 1 day. Two days later, the hyphae were picked and cultured in 2 mL centrifuge tubes containing MM medium until hyphae emerged. The genome of the strain that had grown hyphae was extracted for verification. The verified strain was streaked on a PDA plate containing 0.1% (v / v) Triton-X100 to isolate single spores, and the single spores were selected and cultured on PDA medium for sporulation. Finally, the culture was preserved with 40% glycerol.

[0042] T. reesei Genome extraction T. reesei The genome extraction method was based on Meng Qingshan's paper. Mycelia or spores were inoculated into seed culture medium and cultured at 30℃ and 200 rpm for 2 days. 3 days. Then, 12,000 × g Centrifuge for 3 min, discard the supernatant, add 500 μL of chromosome extraction buffer, then add an appropriate amount of glass beads, and vortex for 20 min. Then incubate at 65℃ for 30 min. Next, add a phenol-chloroform-isoamyl alcohol mixture (25:24:1), vortex for 10 min, and centrifuge at 12,000 × 10⁻⁶. g Centrifuge for 3 min. Transfer the supernatant to a new 2 mL centrifuge tube, add 50 μL of 3 M sodium acetate and 600 μL of isopropanol, mix, and incubate on ice for 15 min. Finally, incubate at 4 °C and 12,000 × 10⁻⁶. g Centrifuge for 10 min, discard the supernatant, wash the DNA with 75% ethanol, and after the ethanol evaporates, dissolve it in an appropriate amount of sterile water before proceeding with subsequent PCR verification.

[0043] Detection of recombinant human lactoferrin content Take an appropriate amount of fermentation broth, centrifuge at 12,000 × g and 4℃ for 3 min, and remove the supernatant. Take 100 μL of the supernatant and detect the recombinant human lactoferrin content according to the lactoferrin detection kit (disclosed in patent publication number CN119985996A).

[0044] ARTP-induced mutagenesis of Trichoderma reesei Trichoderma reesei spores were diluted with spore eluent to a final concentration of 1 × 10⁻⁶. 6 10 µL of spore suspension was taken and spread onto a completely flaked sterile carrier. The carrier was then irradiated in an ARTP machine for 0, 30, 60, 90, 120, 150, 180, 210, 240, 270, and 300 s. After treatment, the EP tube was continuously shaken to ensure complete spore suspension. An appropriate amount of the mutagenized spores was spread onto a PDA (0.1% Triton X-100) plate and incubated at 30°C for 3 days. Finally, the colony count was calculated to determine the lethality rate. A represents the single colony count of the untreated control strain on the PDA plate, and B represents the single colony count of the mutant strain on the plate.

[0045]

[0046] Nucleic acid aptamer screening for mutagenic strains Lac-6a-FAM-BHQ (10 μL, 250 nM) was added separately to 100 μL of lactoferrin standard solution (or bovine serum albumin standard solution, fermentation broth) and incubated at 37 °C for 1 h. The plate was then directly scanned and data were collected using a multi-mode microplate reader (excitation: 485 / 20, emission: 528 / 20).

[0047] Transcriptomic analysis of changes in gene expression levels in mutant strains Samples were collected for transcriptome analysis 40 hours after fermentation. Collected cells were rapidly frozen in liquid nitrogen and stored at -80°C until use. Transcriptome sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd.

[0048] The primer sequences involved in the following examples are shown in Table 1.

[0049] Table 1 Primers used in the examples

[0050] Example 1 Construction of nucleic acid aptamers Lac-6a is a nucleic acid aptamer that specifically binds to lactoferrin, obtained through the SELEX process (published in the paper "Selection of aptamers based on a protein microarray integrated with a microfluidic chip"). Although Lac-6a can specifically bind to human lactoferrin, it lacks simple and easy-to-use screening features such as fluorescence signal. Therefore, we added a fluorescent group (FAM) to its 5′ end and a quencher group (BHQ1) to its 3′ end to construct a nucleic acid aptamer with a fluorescent signal, Lac-6a-FAM-BHQ1. Figure 1 A) FAM is commonly used as a fluorescent reporter group in TaqMan probes, molecular beacons, and scorpion primers, and is widely applied in real-time PCR; in this application, FAM is usually paired with the quencher group BHQ1. When a nucleic acid aptamer specifically binds to a target substance, its conformation changes accordingly. In the absence of lactoferrin, the nucleic acid aptamer is in a disordered state, with a large distance between its 5′ and 3′ ends, resulting in high fluorescence intensity; when lactoferrin is present, the nucleic acid aptamer binds tightly to lactoferrin, resulting in a smaller distance between its 5′ and 3′ ends, and lower fluorescence intensity. Therefore, in this study, a mutagenic strain with low fluorescence intensity was selected as the strain for subsequent shake-flask fermentation validation.

[0051] Example 2: Functional Validation of Nucleic Acid Aptamers To verify the accuracy of the nucleic acid aptamer Lac-6a-FAM-BHQ1 constructed in Example 1 in screening for human lactoferrin, five different concentrations (0 μg / mL, 5 μg / mL, 15 μg / mL, 25 μg / mL, and 50 μg / mL) of human lactoferrin and bovine serum albumin were incubated with Lac-6a-FAM-BHQ1. As the concentration of lactoferrin increased, the fluorescence intensity gradually decreased; while the fluorescence produced by bovine serum albumin and Lac-6a-FAM-BHQ1 remained relatively stable. Figure 1 B). Therefore, using nucleic acid aptamers to assist in the mutagenesis screening of *Trichoderma reesei* is feasible. Next, to verify whether Lac-6a-FAM-BHQ1 could play a practical role in the fermentation broth, the nucleic acid aptamer was added to... T . reesei CJ2095 and T . reesei RUT-C30 fermentation broth (one containing HLf, one without HLf) Figure 1C). The results showed that the fluorescence intensity was significantly reduced in the *Trichoderma reesei* fermentation broth containing HLf compared to the HLf-free broth. Microbial fermentation broths are complex, containing various substances including proteins, sugars, lipids, and other complex compounds. Lac-6a-FAM-BHQ1 remained functional in the fermentation broth, demonstrating its excellent stability and specificity.

[0052] Example 3: ARTP-induced mutagenesis of Trichoderma reesei Mutagenesis techniques are widely used to improve fungal strains. ARTP, as a type of physical mutagenesis, has gradually become a mainstream mutagenesis method due to its advantages such as simplicity, safety, and high mutation rate. Using ARTP technology to mutagenesis of *Trichoderma reesei* spores, the lethality rate reached 98% when the irradiation time reached 180 s. Figure 2 In general mutagenesis operations, the lethality rate needs to reach more than 90%, because the higher the lethality rate, the higher the possibility of strain mutation. Therefore, the subsequent irradiation time was chosen to be 180 seconds.

[0053] Example 4: Nucleic Acid Aptamer-Assisted Mutagenesis Screening System for Trichoderma reesei because T . reesei Because CJ2095 can produce recombinant human lactoferrin, the fermentation broth appears light pink. Figure 3 However, visually screening for mutagenic strains may lead to inaccurate results due to the inability to distinguish between strains with similar colors. Therefore, the nucleic acid aptamer Lac-6a-FAM-BHQ1 constructed in Example 1 was used to assist in the mutagenesis screening of Trichoderma reesei. Trichoderma reesei spores, after ARTP mutagenesis according to Example 3, were spread onto PDA solid medium containing 0.1% Triton X-100 and cultured at 30°C for approximately 3 days. Then, they were inoculated into 96-well plates containing fermentation medium and cultured at 30°C for approximately 48 hours. First, strains producing red color were initially screened visually. Then, Lac-6a-FAM-BHQ1 was combined with the fermentation broth, and the fluorescence intensity was detected using a microplate reader.

[0054] During the mutagenesis process, approximately 191 mutant strains were selected from 1500 mutant strains by visual inspection. Figure 4 Then, through nucleic acid aptamer-assisted screening, three mutant strains (L42, M3, and J52) with the lowest fluorescence intensity were obtained for shake-flask fermentation verification. Figure 5 High-titer strains were obtained through this mutagenesis screening system. T . reesei The yield of CJ2095M3 reached 274.57 mg / L, which is 24.8% higher than that of the starting strain.

[0055] Example 5: Transcriptomic analysis of changes in gene expression levels after mutagenesis To investigate the reasons for the changes in yield in *Trichoderma reesei* strains after ARTP mutagenesis, transcriptomic analysis was performed on the strains before and after mutagenesis. Transcriptomic data revealed significant changes in the expression levels of 271 genes. Figure 6 Of these, 43 genes were upregulated and 228 genes were downregulated. The main purpose of GO analysis is to describe the function of gene products using standardized terminology to facilitate comparison and communication between different studies. It is often used to annotate the function of differentially expressed genes and understand their potential roles in biological processes. According to the GO analysis results, these 271 significantly differentially expressed genes are mainly involved in metabolic processes, cellular processes, localization, and cell component organization or biogenesis. Figure 7 To further verify the effects of significantly altered genes... T . reesei To investigate the effects of recombinant human lactoferrin production, we selected samples from the Trichoderma reesei genome database (https: / / mycocosm.jgi.doe.gov / Trire2 / Trire2.home.html) numbered as follows: 62716 and 111681 Two genes, whose nucleotide sequences are shown in SEQ ID NO.2 and SEQ ID NO.3, were downregulated and upregulated, respectively, in transcriptomic analysis. Therefore, the knockout and overexpression of these two genes were performed to verify their effects.

[0056] Example 6 Construction of recombinant plasmids For genes 111681 Construction of overexpression plasmids: T . reesei Using the CJ2095 genome as a template, with P gpd1 ( 111681 Using primers )-FW and Pgpd1-RS, the gene was obtained by PCR amplification. gpd1 promoter P gpd1 .by T . reesei The genome serves as a template. 111681 -FW and 111681 -RS are primers used for PCR amplification to obtain the gene. 111681 and its termination sub-fragment T 11168 1. The PCR amplification conditions were: 95℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s; 55℃ annealing for 30 s, 72℃ extension (1 min extension per 1 kb according to fragment length), 30 cycles; 72℃ extension for 10 min. T . reeseiUsing the genome as a template, pyr4 ( 111681 )-FW and pyr4 -RS are primers used for PCR amplification to select marker genes. pyr 4. With T . reesei Using the genome as a template, gda410 (111681)-FW / RS was used as primers for PCR amplification. pyr 4. Helper deletion sequences for screening marker genes gda410 ( 111681 This is used to display subsequent filter tags. Next, [the following will be done / will be done]. pyr 4. Screening marker genes and gda410 ( 111681 Two gene fragments are fused together using fusion PCR to form... pyr4 cassette. T. reesei Using the CJ2095 genome as a template, respectively with Δ ku80 -up-FW / RS and Δ ku80 Using -down-FW / RS primers, PCR amplification was performed to obtain the gene. ku80 upstream and downstream homologous arms UPku80 (1.15 kb) and DOWNku80 (1.2 kb). Using plasmid pCAMBIA1303-TrpC- Hygro -gpdA- GFP Using backbone-FW and backbone-RS as templates, and the PCR-amplified gene fragment pCAMBIA1303-LB-RB as the vector backbone, the above-mentioned PCR-amplified gene fragments are: UP ku80 P gpd1 , 111681、 T 111681 , pyr4 cassette, DOWN ku80、 pCAMBIA1303 -LB-RB Ligation was performed using the ClonExpress MultiS One Step Cloning Kit, and the ligation solution was converted into... E. coliJM109 competent cells were cultured in solid LB medium containing a final concentration of 25 μg / mL kanamycin at 37°C for 10 h. PCR amplification and verification were performed using OE universal validation 2-FW and OE universal validation 3-RS primers, followed by gene sequencing verification to screen for positive clones. The recombinant plasmid pCAMBIA1303-LB-RB-UP was obtained using the EndoFree Plasmid Mini Kit. ku80- P gpd1 - 111681 -T 111681 - pyr4 cassette-DOWN ku80 .

[0057] For genes 62716 Construction of knockout plasmids: T . reesei Using the genome as a template, respectively with Δ 62716 -up-FW / RS and Δ 62716 Using -down-FW / RS primers, PCR amplification was performed to obtain the gene. 62716 Upstream and downstream homologous arms UP 62716 (1.2 kb) and DOWN 62716 (1.2 kb). PCR amplification conditions were: 95℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s; 55℃ annealing for 30 s, 72℃ extension (1 min extension per 1 kb according to fragment length), 30 cycles; 72℃ extension for 10 min. T . reesei The genome serves as a template. pyr4 (Δ 62716 )-FW and pyr4- RS was used as a primer for PCR amplification of selection marker genes. pyr4 .by T . reesei Using the genome as a template, gda410 (62716)-FW / RS was used as primers for PCR amplification. pyr 4. Helper deletion sequences for screening marker genes gda410 ( 62716 This is used to display subsequent filter tags. Next, [the following will be done / will be done]. pyr 4. Screening marker genes and gda410 ( 62716 Two gene fragments are fused together using fusion PCR to form... pyr4 cassette. Using plasmid pCAMBIA1303-TrpC- Hygro -gpdA- GFPUsing backbone-FW and backbone-RS as primers, the PCR-amplified gene fragment pCAMBIA1303-LB-RB served as the vector backbone. All PCR-amplified gene fragments UP 62716 DOWN 62716 , pyr4 Cassette and pCAMBIA1303-LB-RB were connected using the ClonExpress MultiS One Step Cloning Kit, and the ligation fluid was converted into... E. coli JM109 competent cells were cultured in solid LB medium containing a final concentration of 25 μg / mL kanamycin at 37°C for 10 h. PCR amplification and verification were performed using primers Δuniversal validation 1-FW and Δuniversal validation 4-RS, followed by gene sequencing to screen for positive clones. Finally, the recombinant plasmid pCAMBIA1303-LB-RB-UP was obtained using an EndoFree Plasmid Mini Kit. 62716 - pyr4 casssette-DOWN 62716 .

[0058] Example 7 Recombination T . reesei Construction pass A . tumefaciens Mediated transformation method T . reesei Molecular modification. The recombinant plasmid constructed in Example 6 will be used. A . tumefaciens and T. reesei CJ2095M3Δ ku80 Spread onto solid IM medium covered with cellophane and co-incubate at 24°C in the dark for 48 h. Transfer the cellophane to fresh PDA solid selection medium (containing 20 mg / L hygromycin and 200 mg / L cefotaxime sodium) and incubate at 30°C for 2 hours. 7 days. The resulting single colonies were picked and cultured on a secondary screening solid medium for 2 days. After 3 days, mycelia were picked and transferred to EP tubes containing 1 mL of seed culture medium. After 2 days of culture, genomic DNA was extracted and verified. Positive clones were streaked onto PDA solid medium containing 0.1% (v / v) Triton-X 100 and cultured for 2 days. Day 3: Isolate single spores and inoculate them into PDA medium for sporulation culture. The bacterial strain was preserved for 7 days. The engineered strain was finally obtained. T. reesei CJ2095M3Δ ku80 :: 111681 , T. reesei CJ2095M3Δ ku80 Δ 62716 .

[0059] Example 8: Engineering Modification T . reesei Fermentation production of recombinant human lactoferrin The engineered strain constructed in Example 7 T. reesei CJ2095M3Δ ku80 :: 111681 , T. reesei CJ2095M3Δ ku80 Δ 62716 Compared with control strain T. reesei CJ2095M3Δ ku80 After culturing on malt extract agar for approximately 7 days, mature spores were eluted from the agar using spore elution buffer (physiological saline containing 0.05% Tween-80) and inoculated into seed culture medium, with a final spore concentration of 1 × 10⁻⁶. 6 Cells / mL, incubated at 30℃ and 200 rpm for 48 h.

[0060] The seed culture was transferred to 100 mL of fermentation medium with microcrystalline cellulose as the carbon source at an inoculum of 10%, and cultured for 7 days at 30°C and 200 rpm. T. reesei overexpressed genes 111681 and 62716 Subsequently, compared with the control strain, the recombinant human lactoferrin yield of the recombinant strain increased by 6.9% and 22.1%, respectively. Figure 8 ).

[0061] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for screening Trichoderma reesei that produces high levels of recombinant human lactoferrin, characterized in that, The method involves characterizing the production of human lactoferrin by using the fluorescence intensity of the nucleic acid aptamer Lac-6a-FAM-BHQ1 after binding with the fermentation broth of Trichoderma reesei, and screening out high-yielding Trichoderma reesei producers of recombinant human lactoferrin. The nucleotide sequence of Lac-6a in the nucleic acid aptamer Lac-6a-FAM-BHQ1 is shown in SEQ ID NO.

1.

2. The method according to claim 1, characterized in that, The Trichoderma reesei strain used was Trichoderma reesei CJ2095 as the starting strain and underwent ARTP mutagenesis. The ARTP mutagenesis time was selected as 180s, and the lethality rate reached 98%.

3. The application of gene 111681 and / or gene 62716 in increasing the yield of recombinant human lactoferrin from Trichoderma reesei, characterized in that, The nucleotide sequences of genes 111681 and 62716 are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

4. The application according to claim 3, characterized in that, The application refers to the overexpression of gene 111681 and / or gene 62716 in Trichoderma reesei.

5. An engineered Trichoderma reesei strain that produces human lactoferrin, characterized in that, The engineered bacteria is Trichoderma reesei as the starting strain, and the overexpressed genes 111681 and / or 62716 in the starting strain are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

6. The engineered Trichoderma reesei strain according to claim 5, characterized in that, The starting strain also knocked out the DNA repair protein gene ku80 and integrated and expressed the genes 111681 and 62716 at the ku80 site.

7. The engineered Trichoderma reesei strain according to claim 5, characterized in that, Trichoderma reesei CJ2095 was used as the starting strain.

8. A method for improving lactoferrin production by Trichoderma reesei, characterized in that, The method involves overexpressing gene 111681 and / or gene 62716 in Trichoderma reesei CJ2095, and the nucleotide sequences of gene 111681 and gene 62716 are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

9. The method according to claim 8, characterized in that, The Trichoderma reesei also knocked out the DNA repair protein gene ku80 and integrated and expressed the genes 111681 and 62716 at the ku80 site.

10. The use of the engineered Trichoderma reesei strain according to any one of claims 5 to 7 or the method according to any one of claims 8 to 9 in the preparation of lactoferrin or lactoferrin-containing products.

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