A method for improving the secretion capacity of filamentous fungal proteins

Abstract

The application discloses a method for improving the protein secretion capacity of Aspergillus and a recombinant strain obtained from the method. An16g07795 The method comprises performing genetic operation on Aspergillus to delete or reduce the activity of specific genes An16g07795 , so as to improve the protein secretion capacity of saccharifying enzyme, alpha-amylase, other proteins participating in starch hydrolysis and the like. The application utilizes the CRISPR-Cas9 genome editing technology to knock out the genes of Aspergillus niger, obtains a gene editing deletion mutant strain, and significantly improves the production level of starch hydrolysis enzymes, including the protein secretion level and the activity of saccharifying enzyme, so the application has great application value.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and biotechnology. Specifically, this invention relates to a method for improving the protein secretion capacity of Aspergillus, specifically involving genetic modification of Aspergillus, and more specifically involving the deletion and editing of specific genes in the Aspergillus genome. Background Technology

[0002] Glucoamylase (EC 3.2.1.3), also known as glucoamylase, is an exoglycolytic enzyme that uses starch or other related polysaccharides as substrates to hydrolyze α-1,4 glycosidic bonds from their non-reducing ends, releasing individual β-D-glucose molecules. It can also hydrolyze α-1,6 and α-1,3 glycosidic bonds. Glucoamylase is the main enzyme responsible for hydrolyzing starch to produce glucose and is one of the most important enzymes in industrial enzyme preparations. It is widely used in industries requiring the maximum conversion of starch to glucose, such as beer, monosodium glutamate (MSG), starch sugars, and antibiotics.

[0003] Filamentous fungi such as Aspergillus niger ( Aspergillus niger ) and Aspergillus oryzae ( Aspergillus oryzae ) and Trichoderma reesei ( Trichoderma reesei Glucosamine enzymes are one of the most important hosts for industrial protein production, and are widely used in the production of various enzyme preparations due to their strong protein secretion capacity and good safety profile. The main producing strains of glycation enzymes are filamentous fungi, derived from the genus *Aspergillus*. Aspergillus ) and Rhizopus ( Rhizopus In my country, saccharifying enzymes are mainly refined from superior strains of Aspergillus niger through deep fermentation. Through decades of unremitting efforts and continuous development by researchers, my country's saccharifying enzyme industry is now capable of independently producing large-volume enzyme preparations such as saccharifying enzymes. For example, Chinese patent CN 102827817 B discloses a thermostable saccharifying enzyme GAI, its gene, and its applications. By screening for thermostable saccharifying enzymes or cloning and expressing thermostable saccharifying enzyme genes, increasing the operating temperature of the saccharifying enzyme allows the starch saccharification process to be completed in a short time, similar to starch liquefaction. Invention patent CN 113061539 A discloses a method for improving the saccharifying enzyme production capacity of Aspergillus niger and a recombinant Aspergillus niger strain, which significantly improves the saccharifying enzyme production capacity of Aspergillus niger through the regulation of fatty acid metabolism pathways. CN105255741A discloses a strain of Aspergillus niger CGMCC 10788, which is a high-saccharifying enzyme produced by multiple rounds of protoplast electrofusion breeding and selection, using Aspergillus niger G131 and F285 as the starting strains. The average fermentation enzyme activity of this strain can reach 87,000-90,000 u / mL, which is 99.3% higher than that of the starting strain.

[0004] Although these studies have reached a high level, current research mainly focuses on using traditional methods for screening and cloning expression of thermostable saccharifying enzymes, or using traditional strain selection methods for breeding high-yielding saccharifying enzyme strains, or on transcriptional regulation to increase the expression level of saccharifying enzyme genes. Protein synthesis and secretion involve the coordinated action of multiple systems, including transcriptional expression regulation, post-translational folding modification, and transport and secretion out of the cell membrane, making it a highly complex process. Therefore, we still need to further understand the protein secretion pathway, reduce or even eliminate bottlenecks in the secretion pathway, and further enhance the protein secretion capacity of filamentous fungi.

[0005] High-secreting filamentous fungi have great potential in protein production. Therefore, it is necessary to develop a method to enhance the protein secretion capacity of filamentous fungi by modifying the secretion pathway, and to use gene editing technology to obtain recombinant strains with significantly increased secretion levels of starch hydrolases such as saccharifying enzymes. This would reduce the time and labor costs required by traditional screening and breeding methods, and meet the needs of industrial fermentation applications of saccharifying enzymes in my country, representing a significant innovation. Summary of the Invention

[0006] Through extensive gene screening and in-depth research, the inventors discovered that the gene in Aspergillus... An16g07795 Knockout editing can significantly increase protein secretion levels. Therefore, the purpose of this invention is to provide a strain modification method that can improve the protein secretion capacity and amylase production capacity of filamentous fungi, by genetically modifying genes related to the protein secretion pathway of filamentous fungi to improve their protein secretion capacity and amylase production.

[0007] Therefore, the present invention adopts the following technical solution:

[0008] This invention provides a method to improve the efficiency of Aspergillus ( Aspergillus A method for constructing recombinant strains with high protein secretion levels, characterized in that it involves using genes from Aspergillus... An16g07795 Inactivation or reduction can enhance its ability to secrete proteins.

[0009] Specifically, the gene An16g07795 The amino acid sequence is shown in SEQ ID NO.1; the gene An16g07795 The nucleotide sequence is shown in SEQ ID No. 2.

[0010] In a specific implementation, the... An16g07795 Gene inactivation or reduction is achieved by completely or partially deleting its gene expression cassette, by insertional inactivation, or by rendering the gene nonfunctional relative to its intended purpose to prevent the expression of a functional protein, or by reducing the expression of a functional protein; preferably, by genetic manipulation of the gene in Aspergillus cells. An16g07795 It was knocked out.

[0011] Preferably, the Aspergillus is Aspergillus niger (Aspergillus niger). Aspergillus niger ).

[0012] In a specific embodiment, the enhanced secretory protein refers to a protein involved in starch hydrolysis. More specifically, the protein is a saccharifying enzyme and / or α-amylase.

[0013] The present invention therefore also provides recombinant strains obtained by the method.

[0014] The present invention further provides a method for expressing starch-hydrolyzed proteins using the recombinant strain, characterized in that the recombinant strain is cultured in the presence of starch raw material or starch inducer to obtain starch-hydrolyzed proteins, preferably saccharifying enzymes and / or α-amylases; further comprising the step of isolating the starch-hydrolyzed proteins.

[0015] The present invention also provides a method for hydrolyzing starch using the recombinant strain, characterized in that the recombinant strain is cultured in the presence of starch raw material or starch inducer, thereby hydrolyzing starch.

[0016] In a specific embodiment, the present invention obtains gene-edited mutant bacteria by gene knockout of two Aspergillus niger strains (CBS513.88 and saccharifying enzyme industrial production strain N1). Specifically, the construction method includes the following steps: constructing two gene-edited mutants targeting specific genes. An16g07795 The guide RNA (sgRNA) expression element, with nucleotide sequences shown in SEQ ID NO. 3 (AnU6p-An16g07795-sgRNA-1) and SEQ ID NO. 4 (AnU6p-An16g07795-sgRNA-2), was constructed. A homologous donor DNA sequence containing the G418 resistance marker gene An16g07795 was constructed, with nucleotide sequences shown in SEQ ID NO. 5 (donor-An16g07795). The Cas9 protein expression cassette, AnU6p-An16g07795-sgRNA-1, AnU6p-An16g07795-sgRNA-2, and donor DNA donor-An16g07795 were co-transformed into protoplasts of two host strains. Gene-edited mutant strains were obtained through homologous recombination. These recombinant strains were named CBS-Δ. An16g07795 and N1-Δ An16g07795 Genetically modified strains, which are those with gene knockouts An16g07795 Aspergillus strains.

[0017] In this invention, the meanings of the terms used are explained.

[0018] When "recombinant" is used to refer to a strain, cell, nucleic acid, protein, or vector, it indicates that the strain, cell, nucleic acid, protein, or vector has been modified by introducing a foreign nucleic acid or protein or by altering the native nucleic acid or protein. For example, a recombinant strain expresses a gene not found in the native (non-recombinant) form of the strain, or expresses a native gene.

[0019] "Host strain" or "host cell" refers to a suitable host for the expression vector or DNA construct. Specifically, the host strain is preferably Aspergillus, and most preferably Aspergillus niger. The host cell can be an Aspergillus host cell or a genetically modified host cell.

[0020] "Genome editing" refers to the deletion, insertion, or replacement of genomic DNA in an organism to modify the target sequence.

[0021] "CRISPR-Cas9 editing technology" refers to the ability of the Cas9 protein, guided by sgRNA, to cut specific DNA sequences in the genome of an organism, creating DNA double-strand breaks. This triggers the cell's own repair mechanisms to perform non-homologous end joining (NHEJ) or homologous recombination (HR) repair at the DSB site, thereby achieving gene knockout, gene inactivation, gene knock-in, and gene replacement, and realizing the goal of efficient genome editing.

[0022] The "target site" or "protospacer" refers to a portion of the nucleic acid sequence that defines the target DNA. Specifically, it refers to the 20-base sequence at the 5' end of the guide RNA (sgRNA). This sequence is identical to the target DNA sequence. Under sufficient conditions for binding, the sgRNA requires this sequence to bind to the target DNA. The Cas9-sgRNA complex then cleaves the target DNA.

[0023] A “sequence” refers to a nucleotide sequence of any suitable length, which can be DNA or RNA; it can be linear, circular, or branched, and it can be single-stranded or double-stranded. The term “donor DNA sequence” refers to the nucleotide sequence that is inserted into the genome. The donor sequence can be of any length, for example, preferably between about 500 and 3,000 nucleotides (or any integer value between them).

[0024] This invention achieves the following beneficial effects: genetic manipulation of Aspergillus cells to delete or reduce the activity of the An16g07795 gene significantly enhances the production level of starch hydrolase. In a specific experiment, this invention utilizes CRISPR-Cas9 editing technology to knock out the CBS513.88 gene in Aspergillus niger strains.An16g07795 Gene-editing deletion mutant strain CBS-Δ was obtained. An16g07795 This mutant strain significantly improved the production level of starch hydrolases, with protein secretion increasing by approximately 27% compared to the original strain, saccharifying enzyme activity increasing by approximately 23%, and α-amylase activity increasing by approximately 18%. In another specific experiment, the gene was knocked out in the Aspergillus niger strain N1 used for industrial production of saccharifying enzymes. An16g07795 The deletion mutant strain N1-Δ was obtained. An16g07795 This mutant strain can significantly improve the production level of starch hydrolase, with protein secretion level increasing by about 18% compared to the original strain, and saccharifying enzyme activity increasing by about 16% compared to the original strain, which has great application value. Attached Figure Description

[0025] Figure 1 To knock out target genes using CRISPR-Cas9 editing technology An16g07795 A schematic diagram.

[0026] Figure 2 To knock out target genes using CRISPR-Cas9 editing technology An16g07795 PCR identification of nucleic acid electrophoresis image.

[0027] Figure 3 Aspergillus niger gene An16g07795 Deletion mutant strain CBS-Δ An16g07795 SDS-PAGE electrophoresis analysis of proteins from the supernatant of the starting strain CBS513.88 fermented under starch-induced conditions.

[0028] Figure 4 Aspergillus niger gene An16g07795 Deletion mutant strain CBS-Δ An16g07795 Protein secretion from the starting strain CBS513.88 under starch-induced fermentation conditions.

[0029] Figure 5 Aspergillus niger gene An16g07795 Deletion mutant strain CBS-Δ An16g07795 The saccharifying enzyme activity of the supernatant from the starting strain CBS513.88 fermentation under starch-induced conditions.

[0030] Figure 6 Aspergillus niger gene An16g07795 Deletion mutant strain CBS-Δ An16g07795 α-Amylase activity of the supernatant from the fermentation of the starting strain CBS513.88 under starch-induced conditions.

[0031] Figure 7 The gene deletion in Aspergillus niger strain N1 for industrial production of saccharifying enzymes An16g07795The protein secretion level and saccharifying enzyme activity were then observed during fermentation under starch-induced conditions. Detailed Implementation

[0032] To further illustrate the technical means and effects of this invention, the following description, in conjunction with preferred embodiments, further explains the technical solution of this invention. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the technical solution of this invention can be made without departing from the spirit and scope of this invention, but all such modifications or substitutions fall within the protection scope of this invention.

[0033] Unless otherwise specified, the methods used in the following examples are conventional methods. For specific steps, please refer to: "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold SpringHarbor).

[0034] The methods for obtaining various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials used in this invention. In fact, the sources of biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted and used according to the suggestions in the embodiments.

[0035] The original starting strain Aspergillus niger CBS513.88 used in the examples was purchased from the Central Bureauvoor Schimmelcultures CBS Fungal Biodiversity Centre and was obtained through commercial channels; the Aspergillus niger strain N1 used to produce the saccharifying enzyme was obtained from Shandong Longket Enzyme Preparation Co., Ltd., with accession number CGMCC No. 10788.

[0036] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels. Unless otherwise specified, all percentage concentrations are mass percentages. All primers and nucleic acid sequencing were performed by GENEWIZ, a Suzhou-based biotechnology company. An16g07795 "This is the gene locus number for Aspergillus niger".

[0037] Example 1: Construction of CRISPR-Cas9-mediated Aspergillus niger gene An16g07795 Editing medium

[0038] Previous research involved analyzing the dynamic changes of 266 transcription factors in *Neurospora crassa* under endoplasmic reticulum stress at the whole-genome level. This revealed 33 significantly upregulated transcription factors. Systematic screening of the cellulase secretion phenotypes of these transcription factors identified a novel transcription factor (a homolog of An16g07795) that influences lignocellulase secretion. Therefore, this invention knocked out the gene An16g07795 in two *Aspergillus niger* strains, CBS513.88, and an industrial strain of saccharifying enzyme, N1, to investigate whether this could promote saccharifying enzyme secretion.

[0039] (1) Gene An16g07795 Construction of sgRNA expression cassette vector

[0040] Design target genes using the sgRNACas9 tool software. An16g07795 The target site was determined. The Aspergillus niger AnU6p promoter, target site, and sgRNA backbone were linked together using fusion PCR, and the sgRNA expression cassette vector was constructed using the gene overlap extension (SOE) method. The primer sequences required for the sgRNA expression cassette vector are shown in Table 1.

[0041] The PCR reaction system consisted of: 10 μL of 5×Phusion HF buffer, 1 μL of 10 mM dNTPs, 2.5 μL of upstream / downstream primers, 1 μL of template DNA, 0.5 μL of Phusion DNA polymerase, and 32.5 μL of ddH2O.

[0042] The PCR reaction conditions were as follows: first 98℃ for 30s; then 98℃ for 10s, 65℃ for 30s, 72℃ for 1min, for 34 cycles; finally 72℃ for 10min, 4℃ for 10min.

[0043] SOE-PCR amplification was used to form sgRNA expression plasmids AnU6p-An16g07795-sgRNA-1 and AnU6p-An16g07795-sgRNA-2, whose sequences are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively.

[0044] (2) Construction of homologous donor DNA vector

[0045] In this embodiment, the homologous donor DNA fragments are respectively derived from the target gene. An16g07795Upstream and downstream homologous fragments (944 bp and 936 bp), the PtrpC-neo fragment of the G418 resistance gene expression cassette, were ligated into plasmid pUC118 linearized with restriction endonucleases BamHI and HindIII using Gibson Assembly to construct the donor DNA fragment donor-An16g07795, the nucleic acid sequence of which is shown in SEQ ID No. 5. The PCR primer sequences required for constructing the donor DNA fragment are shown in Table 1.

[0046] Table 1: Primers used in Example 1

[0047] SEQ ID NO. Primer Sequence (5'-3') 6 AnU6p-F CAGTTACTTATAAGCTTGGAGCTTG 7 gRNA-R AAAAAGCACCGACTCGGTGC 8 An16g07795-T1-R GCTCTAAAACAAGCCATTCGTTGTCTGGACTTTGAATTATATAGGAATAATGGTT 9 An16g07795-T1-F AATTCAAAGTCCAGACAACGAATGGCTTGTTTTAGAGCTAGAAATAGCAAGTT 10 An16g07795-T2-R GCTCTAAAACGTCATAAGAGGAAGCACGTCTTTGAATTATATAGGAATAATGGTT 11 An16g07795-T2-F AATTCAAAGACGTGCTTCCTCTTATGACGTTTTAGAGCTAGAAATAGCAAGTT 12 An16g07795-up-F ATTACGAATTCGAGCTCGGTACCCGGGGATCCTCTTCCCTCTGCTACTTGCGGTGTT 13 An16g07795-up-R CTCCTTCAATATCAGTTAACGTCGGTTTAAACTGGACCCAGTCTTCCGAGATAGCAT 14 AnNeo-F ATGCTATCTCGGAAGACTGGGTCCAGTTTAAACCGACGTTAACTGATATTGAAGGAGCAT 15 AnNeo-R GGGACTGAACCGAGGAGACCGATGAGGTACCTCAGAAGAACTCGTCAAGAAGGCGATA 16 An16g07795-down-F CGCCTTCTTGACGAGTTCTTCTGAGGTACCTCATCGGTCTCCTCGGTTCAGTCCC 17 An16g07795-down-R CGTTGTAAAACGACGGCCAGTGCCAAGCTTCTGCCCATCAAAGACACATAGGACA 18 An16g07795-ko-F ACATTAACCCTCGCATCCTCCCACC 19 An16g07795-ko-R GCAGGTGACGATGGGGCACTTTTCG

[0048] Example 2: CRISPR-Cas9 system for the detection of Aspergillus niger gene An16g07795 Gene knockout

[0049] (1) Protoplast transformation of Aspergillus niger

[0050] A. Mycelial preparation

[0051] Mature Aspergillus niger spores were collected with 0.05% Tween-80 sterile water, and after filtering out the hyphae with lens paper, they were inoculated into MM liquid culture and cultured at 30°C and 200 rpm for 16 h.

[0052] B. Protoplast preparation

[0053] After collecting the hyphae by filtration, place them in 30 mL of lysis buffer (formula: 0.15 g lysin, 30 mL solution A, filtered and sterilized; Solution A: 1.0361 g potassium dihydrogen phosphate, 21.864 g sorbitol, dissolved in 90 mL deionized water, pH adjusted to 5.6, volumetrically adjusted to 100 mL, and sterilized at high temperature). Lyse at 30°C for 2 h, gently shaking every 30 min. Then filter through cellophane, centrifuge at 2000 rpm for 10 min at 4°C, discard the supernatant, add 4 mL of solution B (formula: 0.735 g calcium chloride, 18.22 g sorbitol, 1 mL Tris-HCl (1 M, pH 7.5), dissolved in 90 mL deionized water, pH adjusted to 7.6, volumetrically adjusted to 100 mL, and sterilized at high temperature), centrifuge at 2000 rpm at 4°C for 10 min; discard the supernatant, and add a certain volume of solution B at 200 μL / plasmid.

[0054] C. Protoplast transformation

[0055] In a pre-chilled 15 mL centrifuge tube, add 50 μL of pre-chilled PEG (12.5 g PEG6000, 0.368 g calcium chloride, 500 μL Tris HCl (1 M pH 7.5)) sequentially. Mix the DNA fragment to be transformed and add it to 200 μL of protoplasts. Place on ice for 20 min, then add 2 mL of pre-chilled PEG solution and incubate at room temperature for 5 min. Then add 4 mL of solution B and mix gently. Take 3 mL of the above solution and add it to 12 mL of thawed MM medium containing 1.5 mg / mL FOA. Spread the solution evenly on a plate and incubate at 35°C. After 3 days, pick individual mycelia and grow them on the corresponding antibiotic plates.

[0056] (2) CRISPR-Cas9 system for Aspergillus niger gene An16g07795 Gene knockout at loci

[0057] The Cas9 protein expression element p0380-Ptef1-Cas9, along with the AnU6p-An16g07795-sgRNA-1 and AnU6p-An16g07795-sgRNA-2 expression elements and the homologous donor DNA donor-An16g07795, were mixed in equal proportions and co-transformed into Aspergillus niger strains CBS513.88 and N1 protoplast cells, respectively. Figure 1 As shown, Cas9, mediated by sgRNA, identifies and cuts target sites by pairing the target sequence with the DNA strand of the target gene on the host cell genome. Subsequently, homologous recombination occurs between the donor DNA fragment and the sequences flanking the target site, thereby achieving the purpose of genome editing. Transformants are selected by adding G418 to the plate.

[0058] (3) Verification of Aspergillus niger transformants

[0059] A. Extraction of Aspergillus niger genome

[0060] Genomic DNA was extracted from transformants selected during the above transformation process using the phenol-chloroform method, specifically including the following steps:

[0061] 1) Add 200 mg of zirconium beads and 1 mL of lysis buffer (formula: 0.2 M Tris•HCl (pH 7.5), 0.5 M NaCl, 10 mM EDTA, 1% SDS (w / v)) to a 2.0 mL sterile DNA extraction tube, and pick the Aspergillus niger hyphae growing on the plate into the DNA extraction tube.

[0062] 2) Place all DNA extraction tubes on the grinding aid and oscillate at maximum speed for 30 seconds. Repeat twice.

[0063] 3) Bathe in a 65℃ water bath for 30 minutes, vortexing every few minutes during the water bath process;

[0064] 4) After the water bath, remove the tubes and add 80 μL of 1M Tris•HCl (pH 7.5) to each tube to neutralize it;

[0065] 5) Add 400 μL of phenol:chloroform (1:1) and centrifuge at 13000 rpm for 5 minutes;

[0066] 6) Take 300 μL of supernatant into a new 1.5 mL EP tube and add 600 μL of 95% ethanol (DNA grade).

[0067] 7) Incubate on ice for one hour, then centrifuge at 4°C and 13,000 rpm. You will see white DNA precipitate at the bottom of the EP tube.

[0068] 8) Wash with 400 μL of 75% alcohol (DNA grade), centrifuge at 13000 rpm at 4°C, and gently remove the supernatant;

[0069] 9) Place the EP tube in a vacuum concentrator to remove residual alcohol;

[0070] 10) Add 50 μL ddH2O to dissolve the DNA, measure the DNA concentration using NanoDrop, and store the extracted DNA at -20℃ for PCR verification in the next step.

[0071] B. PCR verification of transformants

[0072] Using the extracted genomic DNA as a template, the transformants were validated by gene PCR using primers An16g07795-ko-F and An16g07795-ko-R. PCR reagents were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0073] The primer sequences are as follows:

[0074] An16g07795-ko-F:ACATTAACCCTCGCATCCTCCCACC (SEQ ID No. 18)

[0075] An16g07795-ko-R: GCAGGTGACGATGGGGCACTTTTCG (SEQ ID No. 19)

[0076] The PCR reaction system consisted of: 2 μL of 10×Taq Buffer, 0.2 μL of 10 mM dNTP Mix, 0.4 μL each of upstream and downstream primers, 1 μL of DNA template, 0.2 μL of Taq DNA Polymerase, and 15.8 μL of water.

[0077] The PCR reaction conditions were as follows: first, 94℃ for 5 min; then 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1.2 min, for 30 cycles; finally, 72℃ for 7 min, 4℃ for 10 min.

[0078] Experimental results: PCR amplification products were subjected to 1% agarose gel electrophoresis (120V, 30 minutes). Clear gene amplification bands were observed under the gel imaging system. The PCR amplification band of the mutant was 1880 bp, and the target band of the wild-type strain was 1148 bp. The results are as follows: Figure 2 As shown, homologous recombination occurred between the donor DNA fragment and the sequences flanking the target site, resulting in a gene-edited mutant. Of the 20 transformants obtained from *Aspergillus niger* strain CBS513.88, 7 were successfully knocked out, with a knockout efficiency of 35%. Of the 20 transformants obtained from *Aspergillus niger* strain N1, 4 were successfully knocked out, with a knockout efficiency of 20%.

[0079] Example 3: Gene-editing deletion strain CBS-Δ An16g07795 Analysis of protein secretion levels under starch-induced conditions

[0080] (1) Cultivation of the deletion mutant strain in starch-inducible medium: The gene obtained above was cultured in starch-inducible medium. An16g07795 Deletion mutant strain (CBS-Δ) An16g07795 -1, CBS-Δ An16g07795 -2 and CBS-Δ An16g07795 -3) Both the original strain CBS513.88 and the starting strain were inoculated into 50 mL of culture medium in 250 mL Erlenmeyer flasks. The culture medium formula was: 30 g maltose, 10 g peptone extract, 5 g yeast extract, 1 g KH2PO4, 0.5 g MgSO4, 0.03 g ZnCl2, 0.02 g CaCl2, 0.0076 g MnSO4, 0.3 g FeSO4, 3 mL Tween 80 (adjusted to a final volume of 1000 mL, autoclaved), with an inoculum size of 2.5 x 10⁻⁶. 5 The samples were cultured at 30℃ and 200 rpm for 6 days, and the supernatant was collected by centrifugation. The protein concentration was analyzed by SDS-PAGE electrophoresis.

[0081] (2) Determination of secretory protein concentration: The protein concentration in the supernatant was detected using the Bradford Protein Rapid Test Kit. The results are as follows: Figure 3 As shown, compared with the original strain CBS513.88, the protein yield of all mutant strains was significantly increased. Under starch culture conditions, the protein yield on day 5 and day 6 was increased by ~24% and ~27% respectively compared with the original strain.

[0082] (3) SDS-PAGE electrophoresis detection of supernatant: Based on the concentration of protein 6, take an equal volume of supernatant and perform 4-12% SDS-PAGE electrophoresis detection. The results are as follows: Figure 4 As shown. Compared to the starting strain CBS513.88, the corresponding bands of starch hydrolases such as saccharifying enzymes were significantly darker, and the secreted protein level was significantly higher than that of the starting strain CBS513.88. The starting strain CBS513.88 and the gene... An16g07795 The protein yields of the deletion mutant strains were ~0.34 g / L and ~0.43 g / L, respectively.

[0083] Example 4: Gene-editing deletion strain CBS-Δ An16g07795 Phenotypic analysis of saccharifying enzymes and α-amylases in production

[0084] The above-obtained gene An16g07795 deletion mutant strain (CBS-Δ) An16g07795 -1, CBS-Δ An16g07795 -2 and CBS-Δ An16g07795 -3) Both the original strain CBS513.88 and the starting strain were inoculated into 50 mL of culture medium in 250 mL Erlenmeyer flasks. The culture medium formula was: 30 g maltose, 10 g peptone extract, 5 g yeast extract, 1 g KH2PO4, 0.5 g MgSO4, 0.03 g ZnCl2, 0.02 g CaCl2, 0.0076 g MnSO4, 0.3 g FeSO4, 3 mL Tween 80 (adjusted to a final volume of 1000 mL, autoclaved), with an inoculum size of 2.0 x 10⁻⁶. 5 The samples were cultured at 30℃ and 200 rpm for 6 days, and the supernatant was collected by centrifugation to determine the activities of saccharifying enzyme and α-amylase.

[0085] (1) Assay of glucoamylase activity: The crude enzyme solution was diluted with 0.05 M pH 4.6 sodium acetate buffer to a suitable ratio, with a final volume of 0.25 mL. It was preheated in a 40 °C water bath, removed, and 0.25 mL of 1% soluble starch substrate solution preheated in a 40 °C water bath was added. The mixture was mixed and reacted at 40 °C for 10 min. The reaction was terminated with 0.5 mL of DNS solution, boiled for 10 min, cooled on ice, and distilled water was added to make up to 2.5 mL. The mixture was shaken well and the amount of glucose released was determined by the DNS method. The OD value was measured at a wavelength of 540 nm. The blank group used inactivated enzyme solution as a control.

[0086] The definition of saccharifying enzyme activity is: the amount of enzyme that hydrolyzes soluble starch to produce 1 μmol of glucose per minute in 1 mL of enzyme solution at 40 °C and pH 4.6, which is one unit of enzyme activity (U).

[0087] The results are as follows Figure 5As shown, compared with the starting strain, the genes An16g07795 The saccharifying enzyme activity of the deletion mutant strain was significantly increased, with an increase of ~23% compared to the original strain. The original strain CBS513.88 and the gene... An16g07795 The glucoamylase activities of the deletion mutant strains were ~427.6 U / mL and ~524.1 U / mL, respectively.

[0088] (2) α-Amylase activity assay: The activity of α-amylase was determined using Solarbio's amylase activity assay kit (catalog number: BC0610). The crude enzyme solution was diluted with 0.05M pH 6.0 phosphate buffer to a suitable ratio, with a final volume of 0.25 mL. It was placed in a 70 °C water bath for 15 min to passivate. After that, 0.25 mL of starch substrate reagent preheated in a 40 °C water bath was added, mixed well, and reacted at 40 °C for 5 min. The reaction was terminated with 0.5 mL of DNS solution, boiled for 10 min, cooled on ice, and then diluted to 2.5 mL with distilled water. The mixture was shaken well, and the amount of reducing sugar released was determined using the DNS method. The OD value was measured at a wavelength of 540 nm. The blank group used inactivated enzyme solution as a control.

[0089] α-Amylase activity is defined as the amount of enzyme that hydrolyzes soluble starch to produce 1 mg of reducing sugar per minute in 1 mL of enzyme solution at 40 °C and pH 6.0, which is one unit of enzyme activity (U).

[0090] The results are as follows Figure 6 As shown, compared with the original strain, the α-amylase activity of the gene An16g07795 deletion mutant strain was significantly increased, with an α-amylase activity that was ~18.6% higher than that of the original strain. The α-amylase activities of the original strain CBS513.88 and the gene An16g07795 deletion mutant strain were ~10.8 U / mL and ~13.1 U / mL, respectively.

[0091] Example 5: Gene-editing deletion strain N1-Δ An16g07795 Phenotypic analysis of protein secretion levels and production of glucoamylase

[0092] The above-obtained gene An16g07795 deletion mutant strain (N1-Δ) was used to... An16g07795 -1, N1-Δ An16g07795 -2 and N1-Δ An16g07795 -3) Both strain N1 and the starting strain were inoculated into 50 mL of culture medium in 250 mL Erlenmeyer flasks. Culture medium formula: 10 g glucose, 1.5 g corn steep liquor, 1.5 g soybean meal powder, adjusted to 50 mL, autoclaved, inoculation amount 2.0 x 10⁻⁶. 5The samples were cultured at 30℃ and 200 rpm for 6 days, and the supernatant was collected by centrifugation to determine the protein concentration and saccharifying enzyme activity.

[0093] (1) Determination of secretory protein concentration: The protein concentration in the supernatant was detected using the Bradford Protein Rapid Test Kit. The results are as follows: Figure 7 As shown, compared with the wild type, the protein yield of the mutant strains was significantly increased. Under starch culture conditions, the protein yield on day 6 was ~18% higher than that of the original strain N1 and the deletion mutant strain N1-Δ. An16g07795 The protein yields were ~4.0 g / L and ~4.8 g / L, respectively.

[0094] (3) Assay of glucoamylase activity: Dilute the crude enzyme solution with 0.05 M pH 4.6 sodium acetate buffer to a suitable ratio, with a final volume of 0.25 mL. Preheat the solution in a 40 °C water bath, remove it, add 0.25 mL of 1% soluble starch substrate solution preheated in a 40 °C water bath, mix well, react at 40 °C for 10 min, terminate the reaction with 0.5 mL DNS solution, boil for 10 min, cool on ice, add distilled water to make up to 2.5 mL, shake well, and determine the amount of glucose released using the DNS method. Measure the OD value at a wavelength of 540 nm. Use inactivated enzyme solution as a control for the blank group.

[0095] The definition of saccharifying enzyme activity is: the amount of enzyme that hydrolyzes soluble starch to produce 1 μmol of glucose per minute in 1 mL of enzyme solution at 40 °C and pH 4.6, which is one unit of enzyme activity (U).

[0096] The results are as follows Figure 7 As shown, compared with the starting strain, the genes An16g07795 The saccharifying enzyme activity of the deletion mutant strain was significantly increased, with an increase of ~16% compared to the original strain. The original strain CBS513.88 and the gene... An16g07795 The glucoamylase activities of the deletion mutant strains were ~22200 U / mL and ~25733 U / mL, respectively. <110> Shandong Longket Enzyme Preparations Co., Ltd.; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> A method to enhance the protein secretion capacity of filamentous fungi <160> 19 <210> 1 <211> 681 <212> PRT <213> Aspergillus niger <400> 1 MQSHADTSDFVLFPTHFTGDNKMLALDSSRQQHPPYFQSYPMDPSTFIDPLAFHVDDLGFSQTHDQSGVPQSSNYGTPPIYSESFSDANKAAGFPPMPATPPSLPYSSDHFIPGLSTASGPSVASASSSAIGSPNTGSAHAISEDWVQTTNGLGLPAAVMSDYFPNEYFGNTLDSEGFYQQKCPENFVGATSDPSVIQPMLQQHPINPPTISFPEQPDYVVSQSAFLPQSPDPSHLHPSESYAANQSFAQHPSLVPTSSPSMAPALPQSRRASSYDRRSSVSSVQSQRSHPSPAASNAESDDDTKEKGRCPFPECRRVFKDLKAHILTHQSERPEKCPIVTCEYHIKGFARKYDKNRHTLTHYKGTMVCGFCPGSGSPAEKSFNRADVFKRHLTSVHGVEQTPPNCRKRSPTAAASKGTSSYSPDATGKCSTCSITFSNAQDFYEHLDDCVLRVVQQEEPSEAINQKLLSEVDADEEVQKTMEKHNLNDTAGTVDIYNDEYDDDDDDAYEYSNLRSGKGPLKSTKGSGAVARPILGVNNAVTKGSSNANAKMRATTSKRRNNRDRYPQSWGCPSSSIKTKKRVLCVFDGQRRLWKDEMMLDNEFEVRVKLPGGAGDGTNREAYVTDLDVETLKRAEGVLSANDEERGPWVDNQSTQLIGQPAVLLPDTYRPHDAEPMDMTY 681 <210>2 <211>2033 <212>DNA <213> Aspergillus niger <400>2 <210>3 <211>625 <212>DNA <213>Artificial sequence <400>3 CAGTTACTTATAAGCTTGGAGCTTGGATCTCTTTGAGGTGGACCTTCCTTGAAGGGTTTCATCTCTGTACTATCATGCGAATGCTAAAGCAGAACTTTAACAGAACCACCAGTGTCTAATAAATTCGATCCGTATATTGTGCACCATTACTCATCTGTGTTTCCCCCAAACATGCAGTCTCCTGCGCAGATAGACTGTCAACTATAGTAATTCCCGTCCGCGAAGCCGCCCTATCCAAAAGTGTATTACCCTCTCTTGTATGCAACAAGAGTCGTTCTTTCTCGCGCTAATACCCATCCGTCTATCGCACAATTAAACCTTCTGATCCCTACAATTTGCCTGACAAAATAAATGAAGTTCAACGTGCAAACAAGCTAGAGCCAGTGTACATTGAGTATCATCTGCAGCTCTACTCAAGGTACTATAGTACCTCAGCCAATTTGATGTTCCTGCCTTCCCGCCCCTCGCTTAGCCGACCAATTAGAGTTCGTTAATTCTAACCATTATTCCTATATAATTCAAAGTCCAGACAACGAATGGCTTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT 625 <210>4 <211>625 <212>DNA <213>Artificial sequence <400>4 CAGTTACTTATAAGCTTGGAGCTTGGATCTCTTTGAGGTGGACCTTCCTTGAAGGGTTTCATCTCTGTACTATCATGCGAATGCTAAAGCAGAACTTTAACAGAACCACCAGTGTCTAATAAATTCGATCCGTATATTGTGCACCATTACTCATCTGTGTTTCCCCCAAACATGCAGTCTCCTGCGCAGATAGACTGTCAACTATAGTAATTCCCGTCCGCGAAGCCGCCCTATCCAAAAGTGTATTACCCTCTCTTGTATGCAACAAGAGTCGTTCTTTCTCGCGCTAATACCCATCCGTCTATCGCACAATTAAACCTTCTGATCCCTACAATTTGCCTGACAAAATAAATGAAGTTCAACGTGCAAACAAGCTAGAGCCAGTGTACATTGAGTATCATCTGCAGCTCTACTCAAGGTACTATAGTACCTCAGCCAATTTGATGTTCCTGCCTTCCCGCCCCTCGCTTAGCCGACCAATTAGAGTTCGTTAATTCTAACCATTATTCCTATATAATTCAAAGACGTGCTTCCTCTTATGACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT 625 <210>5 <211>3057 <212>DNA <213>Artificial sequence <400>5 <210> 6 <211> 25 <212> DNA <213> Artificial sequence <400> 6 CAGTTACTTATAAGCTTGGAGCTTG 25 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <400> 7 AAAAAGCACCGACTCGGTGC 20 <210> 8 <211> 55 <212> DNA <213> Artificial sequence <400> 8 GCTCTAAAACAAGCCATTCGTTGTCTGGACTTTGAATTATATAGGAATAATGGTT 55 <210> 9 <211> 53 <212> DNA <213> Artificial sequence <400> 9 AATTCAAAGTCCAGACAACGAATGGCTTGTTTTAGAGCTAGAAATAGCAAGTT 53 <210> 10 <211> 55 <212> DNA <213> Artificial sequence <400> 10 GCTCTAAAACGTCATAAGAGGAAGCACGTCTTTGAATTATATAGGAATAATGGTT 55 <210> 11 <211> 53 <212> DNA <213> Artificial sequence <400> 11 AATTCAAAGACGTGCTTCCTCTTATGACGTTTTAGAGCTAGAAATAGCAAGTT 53 <210> 12 <211> 57 <212> DNA <213> Artificial sequence <400> 12 ATTACGAATTCGAGCTCGGTACCCGGGGATCCTCTTCCCTCTGCTACTTGCGGTGTT 57 <210> 13 <211> 57 <212> DNA <213> Artificial sequence <400> 13 CTCCTTCAATATCAGTTAACGTCGGTTTAAACTGGACCCAGTCTTCCGAGATAGCAT 57 <210> 14 <211> 60 <212> DNA <213> Artificial sequence <400> 14 ATGCTATCTCGGAAGACTGGGTCCAGTTTAAACCGACGTTAACTGATATTGAAGGAGCAT 60 <210> 15 <211> 58 <212> DNA <213> Artificial sequence <400> 15 GGGACTGAACCGAGGAGACCGATGAGGTACCTCAGAAGAACTCGTCAAGAAGGCGATA 58 <210> 16 <211> 55 <212> DNA <213> Artificial sequence <400> 16 CGCCTTCTTGACGAGTTCTTCTGAGGTACCTCATCGGTCTCCTCGGTTCAGTCCC 55 <210> 17 <211> 55 <212> DNA <213> Artificial sequence <400> 17 CGTTGTAAAACGACGGCCAGTGCCAAGCTTTCTGCCCATCAAAGACACATAGGACA 55 <210> 18 <211> 25 <212> DNA <213> Artificial sequence <400> 18 ACATTAACCCTCGCATCCTCCCACC 25 <210> 19 <211> 25 <212> DNA <213> Artificial sequence <400> 19 GCAGGTGACGATGGGGCACTTTTCG 25

Claims

1. A method to improve the growth of Aspergillus niger ( Aspergillus niger A method for constructing recombinant strains with high protein secretion levels, characterized in that, The gene An16g07795 in Aspergillus niger is inactivated to enhance its ability to secrete proteins. The amino acid sequence encoded by the gene An16g07795 is shown in SEQ ID NO.1; The protein is a saccharifying enzyme and / or α-amylase.

2. The method as described in claim 1, characterized in that, Inactivating the gene An16g07795 is achieved by completely or partially deleting its gene expression cassette or by inserting an inactivation mechanism to prevent the gene from expressing a functional protein.

3. The method as described in claim 2, characterized in that, Genetic manipulation was used to knock out the gene An16g07795 in Aspergillus niger cells.

4. The recombinant strain obtained by the method according to any one of claims 1 to 3.

5. A method for expressing saccharifying enzymes and / or α-amylases using the recombinant strain as described in claim 4, characterized in that... The recombinant strain of claim 4 is cultured in the presence of starch raw material or starch inducer to obtain saccharifying enzyme and / or α-amylase; the process also includes the step of isolating the saccharifying enzyme and / or α-amylase.

6. The method for hydrolyzing starch using the recombinant strain as described in claim 4, characterized in that, The recombinant strain of claim 4 is cultured in the presence of starch raw material or starch inducer to hydrolyze starch.

Images