Recombinant trichoderma reesei for producing lactoferrin and application of recombinant trichoderma reesei

Abstract

The invention provides recombinant trichoderma reesei for producing lactoferrin and application of the recombinant trichoderma reesei, and belongs to the technical field of genetic engineering. Trichoderma reesei CJ2095 is used as a starting strain, disulfide bond isomerase is over-expressed in the trichoderma reesei CJ2095, a protein activating enzyme gene is knocked out, and the lactoferrin yield of the obtained recombinant trichoderma reesei reaches 306.31 mg / L. In addition, fermentation conditions of the recombinant trichoderma reesei are also explored, the recombinant trichoderma reesei can produce lactoferrin at high yield in a culture medium taking microcrystalline cellulose as a carbon source, and the concentration of bran and the concentration of Tween-80 can influence the yield of the lactoferrin. Therefore, in a culture medium which takes microcrystalline cellulose as a carbon source and contains bran and Tween-80, the recombinant trichoderma reesei disclosed by the invention can be fermented to generate 1349.5 mg / L of lactoferrin.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant Trichoderma reesei producing lactoferrin and application thereof. Background Art

[0003] Lactoferrin is mainly extracted from milk by cation exchange chromatography. However, due to the high cost of raw materials and extraction, lactoferrin has a high market price. In recent years, many research results have been achieved by using Pichia pastoris as a chassis cell to produce lactoferrin through various synthetic biology methods. Blanca et al. successfully expressed the porcine lactoferrin gene in Pichia pastoris under the regulation of the AOX1 promoter, and its expression level reached 3.5g / L (published in the paper "High-level expression of recombinant bovine lactoferrin in Pichiapastoris with antimicrobial activity"). Lv et al. studied and modified the rate-limiting step of heterologous protein expression in P. pastoris, and the yield of recombinant human lactoferrin reached 5.4g / L, which is the highest lactoferrin yield reported so far (published in the paper "Cascaded de novo biosynthesis of lacto-proteins from CO2 by engineered Pichia pastoris").

[0004] Trichoderma reesei is a safe filamentous fungus that produces high levels of cellulase. Li et al. overexpressed the genes of four key enzymes, phytoene synthase / lycopene cyclase (CarRP), phytoene dehydrogenase (CarB), geranyl diphosphate synthase (GGS1 / CrtE), and hydroxymethylglutaryl-CoA reductase (HMG1) in T. reesei, and the cellulase activity reached 22.33 IU / mL, and the β-carotene production reached 286.63 mg / L (published in the paper "Simultaneous Production of Cellulase and β-Carotene in the Filamentous Fungus Trichoderma reesei"), which reflects the great potential of T. reesei as a chassis cell for producing proteins and terpene compounds. However, in the prior art, when T. reesei is used as chassis cells to produce lactoferrin, the yield is about 2 mg / L, which is relatively low. Therefore, it is urgent to construct a method for high-yield lactoferrin in T. reesei. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problem in the prior art of lacking a method for high-yield lactoferrin based on T. reesei.

[0006] To solve the above technical problems, the present invention provides a recombinant Trichoderma reesei producing lactoferrin and its application. The present invention uses Trichoderma reesei CJ2095 as the starting strain, overexpresses disulfide bond isomerase in Trichoderma reesei CJ2095 and knocks out the protein activating enzyme gene, and the lactoferrin yield of the obtained recombinant Trichoderma reesei reaches 306.31mg / L after fermentation and cultivation. In addition, the present invention also explores the fermentation conditions of recombinant Trichoderma reesei, and recombinant Trichoderma reesei can produce high lactoferrin in a culture medium with microcrystalline cellulose as a carbon source, and the concentrations of bran and Tween-80 can affect the yield of lactoferrin. Therefore, in a culture medium with microcrystalline cellulose as a carbon source and containing bran and Tween-80, the recombinant Trichoderma reesei of the present invention can ferment and produce 1349.5mg / L of lactoferrin.

[0007] The first objective of the present invention is to provide a lactoferrin-producing recombinant Trichoderma reesei, wherein the recombinant Trichoderma reesei is transformed using Trichoderma reesei as a host, and the transformation includes: overexpressing disulfide isomerase and knocking out protein activating enzyme gene.

[0008] Furthermore, the NCBI number of the gene pdi1 encoding the disulfide isomerase is NW_006711148.1, and the NCBI number of the gene Pea1 encoding the protein activating enzyme is NW_006711165.1.

[0009] Further, the Trichoderma reesei includes Trichoderma reesei CJ2095. The Trichoderma reesei strain CJ2095 has been deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China, and its classification name is Trichoderma CJ2095 Trichoderma sp. CJ2095, the deposit date is August 14, 2023, and the deposit number is CCTCC NO: M 20231481.

[0010] The second object of the present invention is to provide a method for producing lactoferrin, wherein the method comprises inoculating the above-mentioned recombinant Trichoderma reesei into a culture medium for fermentation to obtain the lactoferrin.

[0011] Furthermore, the culture medium includes a carbon source, a nitrogen source, an inorganic salt and metal ions.

[0012] Furthermore, the carbon source is selected from one or more of glucose, xylan, cellobiose, and microcrystalline cellulose.

[0013] Furthermore, the culture medium also includes bran.

[0014] Furthermore, the added amount of the bran is 0-2% (W / V).

[0015] Furthermore, the culture medium also includes a surfactant.

[0016] Furthermore, the surfactant includes Tween-80, and the added amount of Tween-80 is 0-2 g / L.

[0017] Beneficial effects of the present invention:

[0018] The present invention provides a recombinant Trichoderma reesei producing lactoferrin and its application. The present invention uses Trichoderma reesei CJ2095 as a starting strain, overexpresses disulfide bond isomerase in Trichoderma reesei CJ2095 and knocks out the protein activating enzyme gene, and the yield of lactoferrin obtained by fermenting the recombinant Trichoderma reesei reaches 306.31 mg / L. In addition, the present invention also explores the fermentation conditions of recombinant Trichoderma reesei, and the recombinant Trichoderma reesei can produce high lactoferrin in a culture medium with microcrystalline cellulose as a carbon source, and the concentrations of bran and Tween-80 can affect the yield of lactoferrin. Therefore, in a culture medium with microcrystalline cellulose as a carbon source and containing bran and Tween-80, the recombinant Trichoderma reesei of the present invention can ferment and produce 1349.5 mg / L of lactoferrin. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0020] Figure 1 The difference of human lactoferrin production induced by different carbon sources in T.reesei; A: The yield and biomass change of human lactoferrin after different carbon sources induced T.reesei; B: SDS-PAGE of extracellular protein after different carbon sources induced T.reesei, 1, 2, 3, 4, 5 represent glucose, xylan, microcrystalline cellulose, cellobiose, lactose respectively; C: The growth status of T.reesei in different carbon source plates;

[0021] Figure 2 Molecular modification of T.reesei; A: Effects of overexpression of key genes on the content and biomass of human lactoferrin synthesized by T.reesei; B: Effects of pea1 gene knockout on the content and biomass of human lactoferrin synthesized by T.reesei; C: SDS-PAGE of extracellular proteins of T.reesei after molecular modification, 1, 2, 3, 4, 5, 6 represent strains T.reeseiCJ2095, T.reesei CJ2095::bip1, T.reesei CJ2095::pdi1, T.reesei CJ2095::ero1, T.reesei CJ2095::xyr1, T.reesei CJ2095Δpea1, respectively; D: Comparison of the content and biomass of human lactoferrin synthesized by the engineered strains T.reesei CJ2095Δpea1::pdi1 and T.reesei CJ2095Δpea1;

[0022] Figure 3 The effects of different concentrations of Tween-80 and bran addition on the synthesis of recombinant human lactoferrin by T.reesei; A: The effect of bran addition on the synthesis of human lactoferrin by T.reesei; B: SDS-PAGE of T.reesei extracellular protein under different bran contents, 1, 2, 3 represent 0%, 1%, 2% bran addition, respectively; C: The effect of Tween-80 on the synthesis of human lactoferrin by T.reesei; D: SDS-PAGE of T.reesei extracellular protein under different concentrations of Tween-80. 1, 2, 3, 4, 5 and 6 represent 2g / L, 1.5g / L, 1g / L, 0.5g / L, 0.25g / L and 0g / L Tween-80 addition, respectively;

[0023] Figure 4This is a 5L tank fermentation of the engineered strain T.reesei CJ2095Δpea1::pdi1; A: Production of recombinant human lactoferrin during fed-batch fermentation of the engineered strain T.reeseiCJ2095Δpea1::pdi1; B: Growth status of the strain during fed-batch fermentation of the engineered strain T.reeseiCJ2095Δpea1::pdi1.

[0024] Standard deviation (SD) ± mean was calculated from the data of three parallel experiments. Data were statistically analyzed using Student t test. Statistical significance was set at p value < 0.05. *, **, ***, ns represent p value < 0.05, p value < 0.01, p value < 0.001, and p value > 0.05, respectively. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0026] Culture medium:

[0027] LB medium (1 L): 10 g peptone, 10 g NaCl, 5 g yeast powder.

[0028] PDA medium (1 L): 37 g potato dextrose agar medium.

[0029] Malt extract agar medium (1L): 30g malt extract, 20g agar powder.

[0030] Fermentation medium (1L): 4g KH2PO4, 2.8g (NH4)2SO4, 0.6g MgSO4·7H2O, 0.5g CaCl2, 0.6g urea, 3g peptone, 1g Tween-80, 5g CaCO3, trace elements (0.005g FeSO4·7H2O, 0.0016g MnSO4·H2O, 0.0014g ZnSO4·7H2O, 0.002gCoCl2·6H2O), 20g bran, 30g microcrystalline cellulose.

[0031] Seed culture medium (1L): 20g glucose, 15g K2HPO4, 5g (NH4)2SO4, 0.6g MgSO4·7H2O, 0.6g CaCl2, 2g peptone, trace elements (0.005g FeSO4·7H2O, 0.0016g MnSO4·H2O, 0.0014g ZnSO4·7H2O, 0.002g CoCl2·6H2O).

[0032] IM medium (1L): 1.45g KH2PO4, 2.05g K2HPO4, 0.5g (NH4)2SO4, 0.5g MgSO4·7H2O, 0.15g NaCl, 5g glycerol, 0.06g CaCl2, 0.0025g FeSO4·7H2O, 1.8g glucose, 8.54g 2-morpholineethanesulfonic acid, 200μM acetosyringone.

[0033] Determination of T. reesei biomass

[0034] The changes in biomass of T. reesei were indirectly calibrated by measuring the DNA content in the cells. 1 mL of bacterial solution was placed in a 2 mL EP centrifuge tube, centrifuged at 12,000 × g and 4°C for 3 min, the precipitate was collected, washed three times with 1 mL of deionized water, and centrifuged at 12,000 × g and 4°C for 3 min to remove the supernatant. 1 mL of 10% trichloroacetic acid solution was added to the washed mycelium, shaken to mix, placed in an ice bath for 3 min, and centrifuged at 12,000 × g and 4°C for 3 min to remove the supernatant. 1 mL of 10% trichloroacetic acid solution was added, shaken to mix, and placed in an ice bath for 3 min. Place in a boiling water bath for 30 min, cool, and centrifuge at 12,000 × g for 10 min, take the supernatant, and use a NanoDrop spectrophotometer (Hangzhou Aosheng Instrument Co., Ltd., Hangzhou, China) to measure the absorbance value A at 260 nm. 260 .

[0035] Detection of recombinant human lactoferrin content

[0036] Take an appropriate amount of fermentation broth, centrifuge at 12,000×g, 4°C for 3 min, remove the supernatant, take 100 μL of the supernatant, and detect the content of recombinant human lactoferrin according to the lactoferrin ELISA kit (Shanghai Sangon Biotechnology Co., Ltd., Shanghai, China).

[0037] SDS-PAGE analysis:

[0038] Take 30 μL of fermentation supernatant, add 10 μL of 4× loading buffer, and boil in water for 3 min. Take 5 μL of sample for SDS-PAGE analysis.

[0039] Example 1: Construction of recombinant plasmid

[0040] For the construction of gene overexpression plasmid, the construction of endoplasmic reticulum chaperone binding protein gene bip1 overexpression plasmid pCAMBIA1303-LB-RB-Pgpd1-bip1-Tbip1-hyg cassette was taken as an example: the T.reesei CJ2095 genome was used as a template, Pgpd1(bip1)-FW and Pgpd1-RS were used as primers, and the promoter Pgpd1 of the gene gpd1 was obtained by PCR amplification. The T.reesei genome was used as a template, bip1-FW and bip1-RS were used as primers, and the gene bip1 and its terminator fragment Tbip1 were obtained by PCR amplification. The conditions for PCR amplification were: 95℃ pre-denaturation for 5min, 94℃ denaturation for 30s; 55℃ annealing for 30s, 72℃ extension (extend 1min according to the fragment length according to 1kb), 30 cycles; 72℃ extension for 10min. The plasmid pCAMBIA1303-TrpC-Hygro-gpdA-GFP preserved in this laboratory was used as a template, and hyg(bip1)-FW and hyg-RS were used as primers to PCR amplify the hygromycin resistance gene expression cassette hyg cassette. The plasmid pCAMBIA1303-TrpC-Hygro-gpdA-GFP was used as a template, and backbone-FW and backbone-RS were used as primers. The gene fragment pCAMBIA1303-LB-RB amplified by PCR was used as the vector backbone. All the above PCR amplified gene fragments: Pgpd1, bip1, Tbip1, hyg cassette, pCAMBIA1303-LB-RB were connected by the one-step cloning kit ClonExpress MultiS One Step Cloning Kit, and the connection solution was transformed into E.coli JM109 competent cells and cultured at 37°C for 10 hours on solid LB medium containing kanamycin at a final concentration of 25μg / mL. OE universal validation 2-FW and OE universal validation 3-RS were used as primers for PCR amplification verification, gene sequencing verification, and screening of positive clones. The recombinant plasmid pCAMBIA1303-LB-RB-Pgpd1-bip1-Tbip1-hyg cassette was obtained by EndoFree Plasmid MiniKit. The construction process of the recombinant plasmid overexpressing the disulfide isomerase gene pdi1, the oxidoreductase gene ero1, and the xylanase regulatory factor gene xyr1 was the same as above.

[0041] For the construction of gene knockout plasmid, the construction of knockout plasmid pCAMBIA1303-LB-RB-UPpea1-hyg casssette-DOWNpea1 of protease regulatory factor gene was taken as an example: T.reesei genome was used as template, Δpea1-up-FW / RS and Δpea1-down-FW / RS were used as primers, and PCR amplification was performed to obtain the upstream and downstream homologous arms UPpea1 (1.2kb) and DOWNpea1 (1.2kb) of gene pea1. The conditions of PCR amplification were: 95℃ pre-denaturation for 5min, 94℃ denaturation for 30s; 55℃ annealing for 30s, 72℃ extension (extend 1min according to the fragment length according to 1kb), 30 cycles; 72℃ extension for 10min. The plasmid pCAMBIA1303-TrpC-Hygro-gpdA-GFP was used as a template, and hyg(Δpea1)-FW and hyg(Δpea1)-RS were used as primers to PCR amplify the hygromycin resistance gene expression cassette hyg cassette. The plasmid pCAMBIA1303-TrpC-Hygro-gpdA-GFP was used as a template, and backbone-FW and backbone-RS were used as primers. The gene fragment pCAMBIA1303-LB-RB amplified by PCR was used as the vector backbone. All PCR amplified gene fragments UPpea1, DOWNpea1, hyg cassette, and pCAMBIA1303-LB-RB were connected using ClonExpress MultiS One Step Cloning Kit, and the connection solution was transformed into E.coli JM109 competent cells and cultured at 37°C for 10 hours on solid LB medium containing kanamycin at a final concentration of 25μg / mL. PCR amplification was performed using Δuniversal validation 1-FW and Δuniversal validation 4-RS as primers, and gene sequencing was performed to screen positive clones. Finally, the recombinant plasmid pCAMBIA1303-LB-RB-UPpea1-hyg casssette-DOWNpea1 was obtained using the EndoFree Plasmid Mini Kit.

[0042] Table 1 Primers and their sequences involved in Example 1

[0043]

[0044]

[0045] Example 2: Construction of recombinant T. reesei

[0046] The molecular modification of T.reesei was performed by A.tumefaciens-mediated transformation. A.tumefaciens and T.reesei containing the recombinant plasmid constructed in Example 1 were spread on a solid IM medium covered with cellophane and co-cultured at 24°C in the dark for 48 hours. The cellophane was transferred to a new PDA solid screening medium (containing 20 mg / L hygromycin and 200 mg / L ceftriaxone sodium) and cultured at 30°C for 2-7 days. The grown single clone was picked and cultured on a rescreening solid medium for 2-3 days, and the mycelium was picked into an EP tube containing 1 mL of seed culture medium and cultured for 2 days, and its genomic DNA was extracted and verified. The positive clone was streaked and cultured on a PDA solid medium containing 0.1% (v / v) Triton-X 100 for 2-3 days, and the single spores were isolated and inoculated into a PDA medium for spore culture for 5-7 days, and the strain was preserved. Finally, the engineered strains T.reesei CJ2095::bip1, T.reesei CJ2095::pdi1, T.reesei CJ2095::ero1, T.reesei CJ2095::xyr1, and T.reeseiCJ2095Δpea1 were obtained.

[0047] Example 3: Effects of different inducers on the synthesis of recombinant human lactoferrin by T. reesei

[0048] The mature spores on the malt extract agar medium were washed with spore washing solution (physiological saline containing 0.05% Tween-80) and inoculated into the seed medium. The final spore concentration was 1×10 6 / mL, and cultured at 30°C, 200 rpm for 48 h.

[0049] The seed solution was transferred to 100 mL of fermentation medium at a 10% inoculation rate and cultured at 30°C and 200 rpm for 7 days. The fermentation medium used 30 g / L of different carbon sources (glucose, lactose, xylan, cellobiose, microcrystalline cellulose) for T. reesei fermentation, and the optimal carbon source for recombinant human lactoferrin gene expression in T. reesei CJ2095 was optimized. Figure 1As shown in A, microcrystalline cellulose has the best effect on inducing T.reesei CJ2095 to produce recombinant human lactoferrin, and its recombinant human lactoferrin production can reach 184.76 mg / L. When glucose, xylan, and cellobiose are used as carbon sources, the production of recombinant human lactoferrin reaches 0.07 mg / L, 0.77 mg / L, and 0.36 mg / L, respectively. However, when lactose is used as the carbon source, the expression of human lactoferrin is not detected. The SDS-PAGE results are consistent with the results of the measured recombinant human lactoferrin production. When microcrystalline cellulose is used as the carbon source, the recombinant human lactoferrin content produced in T.reesei CJ2095 is the highest ( Figure 1 B). In addition, due to the presence of a large amount of insoluble matter (calcium carbonate, bran, microcrystalline cellulose) in the fermentation medium, the dry weight method cannot be used to detect the biomass of T.reesei. Therefore, in this study, the growth status of T.reesei was indirectly characterized by detecting the intracellular nucleic acid content. As can be seen from the figure, when glucose, microcrystalline cellulose, cellobiose, xylan, and lactose were used as carbon sources, the biomass of T.reesei CJ2095 gradually decreased, which were 19.56, 18.49, 16.20, 15.38, and 13.75, respectively. It can be determined from the growth status of T.reesei on solid seed culture media with different carbon sources that the colony diameter of the recombinant T.reesei when xylan is used as the carbon source is larger than the colony diameter when glucose and microcrystalline cellulose are used as the carbon source, but it is significantly larger than the colony diameter when cellobiose and lactose are used as carbon sources ( Figure 1 C).

[0050] Example 4: Fermentation of recombinant human lactoferrin by recombinant T. reesei after key gene enhancement

[0051] The engineered strains T. reesei CJ2095::bip1, T. reesei CJ2095::pdi1, T. reesei CJ2095::ero1, and T. reesei CJ2095::xyr1 constructed in Example 2 were cultured on malt extract agar medium for about 7 days. The mature spores on the malt extract agar medium were washed with spore washing solution (physiological saline containing 0.05% Tween-80) and inoculated into seed medium. The final spore concentration was 1×10 6 / mL, and cultured at 30°C, 200 rpm for 48 h.

[0052] The seed solution was transferred to 100 mL of fermentation medium with microcrystalline cellulose as the carbon source at an inoculum of 10%, and cultured at 30°C and 200 rpm for 7 days. After overexpressing the genes pdi1 and xyr1 in T. reesei, the production of recombinant human lactoferrin in the recombinant bacteria increased by 25.13% and 22.21% respectively compared with the control strain, and the production was 240.26 mg / L and 234.64 mg / L respectively ( Figure 2 A and 2C).

[0053] Example 5: Fermentation production of recombinant human lactoferrin by recombinant T. reesei strain with knockout protein activating enzyme gene pea1

[0054] The engineered strain T. reesei CJ2095Δpea1 constructed in Example 2 was cultured on malt extract agar medium for about 7 days, and the mature spores on the malt extract agar medium were eluted with spore elution solution (physiological saline containing 0.05% Tween-80) and inoculated into seed medium. The final spore concentration was 1×10 6 / mL, and cultured at 30°C, 200 rpm for 48 h.

[0055] The production of recombinant human lactoferrin in the engineered strain T.reesei CJ2095Δpea1 reached 250.97 mg / L, which was 30.71% higher than that in the original strain T.reesei CJ2095 ( Figure 2 B and Figure 2 C). In order to further improve the ability of T. reesei to synthesize recombinant human lactoferrin, the disulfide bond isomerase gene pdi1 was further overexpressed on the basis of the engineered strain T. reesei CJ2095Δpea1 to obtain the engineered strain T. reesei CJ2095Δpea1::pdi1. Figure 2 D shows that compared with the engineered strain T.reeseiΔpea1, the recombinant human lactoferrin production of the engineered strain T.reesei CJ2095Δpea1::pdi1 reached 306.31 mg / L, an increase of 7.23%.

[0056] Example 6: Effects of different bran concentrations on the synthesis of recombinant human lactoferrin by T. reesei

[0057] The mature spores on the malt extract agar medium were washed with spore washing solution (physiological saline containing 0.05% Tween-80) and inoculated into the seed medium. The final spore concentration was 1×10 6 / mL, and cultured at 30°C, 200 rpm for 48 h.

[0058] The seed solution was transferred to 100 mL of fermentation medium with microcrystalline cellulose as the carbon source at an inoculum of 10%, and cultured at 30° C. and 200 rpm for 7 days. The amount of bran added to the fermentation medium was adjusted to 0%, 1%, and 2%.

[0059] The study optimized the effect of bran addition on the production of recombinant human lactoferrin by T.reesei CJ2095Δpea1::pdi1. When the bran addition was 1%, the yield of recombinant human lactoferrin produced by T.reesei CJ2095Δpea1::pdi1 was the highest, reaching 313.42 mg / L. When the bran addition increased from 1% to 2%, there was no significant increase in the yield of recombinant human lactoferrin. When the bran addition was 0%, the yield of recombinant human lactoferrin decreased significantly to 0.23 mg / L, which severely inhibited the level of recombinant human lactoferrin produced by T.reesei CJ2095Δpea1::pdi1 ( Figure 3 A and Figure 3 B). At the same time, when the bran content was 0%, 1%, and 2%, the biomass of T. reesei CJ2095Δpea1::pdi1 was 1.32, 15.43, and 18.49 ( Figure 3 A). The results showed that bran addition was essential for the fermentation of recombinant human lactoferrin by T. reesei CJ2095Δpea1::pdi1. Lower bran addition was not conducive to the growth of T. reesei CJ2095Δpea1::pdi1 and also inhibited the synthesis of recombinant human lactoferrin.

[0060] Example 7: Effect of different concentrations of Tween-80 on the synthesis of recombinant human lactoferrin by T. reesei

[0061] The mature spores on the malt extract agar medium were washed with spore washing solution (physiological saline containing 0.05% Tween-80) and inoculated into the seed medium. The final spore concentration was 1×10 6 / mL, and cultured at 30°C, 200 rpm for 48 h.

[0062] The seed solution was transferred to 100 mL of fermentation medium with microcrystalline cellulose as the carbon source at an inoculum of 10%, and cultured at 30°C and 200 rpm for 7 days. The amount of Tween-80 added to the fermentation medium was adjusted to 0 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, 1.5 g / L, and 2 g / L.

[0063] The study optimized the effect of Tween-80 addition on the production of recombinant human lactoferrin by T.reesei CJ2095Δpea1::pdi1. As Tween-80 increased, the content of recombinant human lactoferrin also increased. When the addition of Tween-80 reached 1.5g / L, the recombinant human lactoferrin production of T.reesei CJ2095Δpea1::pdi1 was the highest, reaching 368.47mg / L. When the addition of Tween-80 was increased further, the content of recombinant human lactoferrin began to decrease ( Figure 3 C and Figure 3 D).

[0064] Example 8: 5-L tank fermentation

[0065] The engineered strain T. reesei CJ2095Δpea1::pdi1 constructed in Example 4 was cultured on malt extract agar medium for about 7 days, and the mature spores on the malt extract agar medium were eluted with spore elution solution (physiological saline containing 0.05% Tween-80) and inoculated into seed medium at a final spore concentration of 1×10 6 / mL, and cultured at 30°C, 200 rpm for 48 h.

[0066] The optimal carbon source, bran addition amount, and Tween-80 addition amount in Examples 1, 2, and 3 were used to prepare the fermentation medium. A 5L fermenter was used for batch feeding fermentation, with an inoculation amount of 10%, a fermentation medium liquid volume of 2.5L, a culture temperature of 30°C, a rotation speed of 400rpm, a ventilation volume of 2vvm, a pH value of 5.0, and a fermentation period of 7d.

[0067] We started feeding at the 48th hour of fermentation, and fed every 24 hours, with 250 mL (25 g microcrystalline cellulose, 2.5 g peptone, 4 g bran) each time. In the first 5 days of fermentation, the strain rapidly accumulated recombinant human lactoferrin. When the fermentation reached the 6th day, the recombinant human lactoferrin content reached the highest concentration of 1349.5 mg / L ( Figure 4 A and Figure 4 B).

[0068] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A recombinant Trichoderma reesei producing lactoferrin, characterized in that: The recombinant Trichoderma reesei is obtained by transformation using Trichoderma reesei as a host, and the transformation includes: overexpressing disulfide bond isomerase and knocking out protein activating enzyme gene.

2. The recombinant Trichoderma reesei according to claim 1, characterized in that The NCBI number of the gene pdi1 encoding the disulfide isomerase is NW_006711148.1, and the NCBI number of the gene Pea1 encoding the protein activating enzyme is NW_006711165.

1.

3. The recombinant Trichoderma reesei according to claim 1, characterized in that The Trichoderma reesei includes Trichoderma reesei CJ2095.

4. A method for producing lactoferrin, characterized in that: The method comprises inoculating the recombinant Trichoderma reesei described in any one of claims 1 to 3 into a culture medium for fermentation to obtain the lactoferrin.

5. The method according to claim 4, characterized in that The culture medium comprises a carbon source, a nitrogen source, inorganic salts and metal ions.

6. The method according to claim 5, characterized in that The carbon source is selected from one or more of glucose, xylan, cellobiose and microcrystalline cellulose.

7. The method according to claim 5, characterized in that The culture medium also includes bran.

8. The method according to claim 7, characterized in that The added amount of the bran is 0-2% (W / V).

9. The method according to claim 5, characterized in that The culture medium also includes a surfactant.

10. The method according to claim 9, characterized in that The surfactant includes Tween-80, and the added amount of the Tween-80 is 0-2 g / L.

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