Aspergillus vacuole proton pump subunit AnVma1 gene and application thereof

Abstract

The invention discloses an aspergillus vacuole proton pump subunit AnVma1 gene and application thereof. The amino acid sequence of the gene is as shown in SEQ ID NO. 1. The aspergillus is subjected to genetic manipulation to increase or enhance the activity of a specific gene, so that the secretion ability of glucoamylase and other proteins participating in starch hydrolysis can be improved. In a specific experiment, a recombinant strain with high-level expression of the gene is obtained by utilizing an overexpressed aspergillus niger gene AnVma1, the production level of amylolytic enzyme can be remarkably improved by the recombinant strain, the protein secretion level after culture in a 5L fermentation tank is improved by about 44% compared with that of an original strain, the activity of saccharifying enzyme is improved by about 30% compared with that of the original strain, and the yield of the amylolytic enzyme is improved by about 30% compared with that of the original strain. And the method has a relatively high application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering and biotechnology. Specifically, the present application relates to a gene of vacuolar-type proton-ATPase subunit AnVma1 and its application, and particularly relates to improving the protein secretion level of Aspergillus by overexpressing the gene AnVma1 in the genome of Aspergillus niger. BACKGROUND

[0002] Aspergillus niger is a filamentous fungus of the genus Aspergillus, an important industrial fermentation microorganism, and an important component of the filamentous fungal protein expression system. It has high protein secretion capacity and post-translational modification capacity, and is therefore used to produce various industrial enzymes, including saccharifying enzymes, xylanases, amylases, proteases, pectinases, etc. Filamentous fungi secrete proteins to the extracellular space through a series of specific and efficient recognition, binding transport, processing and modification by key proteins from the polypeptide synthesis site, and finally transport the proteins to the extracellular space through membrane vesicles. This is a very complex process.

[0003] The vacuole is a cellular organelle with a bubble-like structure coated by a biological membrane, which is formed by the fusion of a series of small vesicles. It is functionally similar to a vesicle, a multifunctional organelle for the storage of nutrients and the maintenance of ion and pH homeostasis in cells, and a core organelle involved in protein synthesis and secretion in filamentous fungi. + Vacuolar-type proton ATPase (V-ATPase) is a highly conserved ATP-driven proton pump present in the membrane system of eukaryotic cells, which plays an important role in promoting endocytosis and maintaining ion and pH homeostasis in the vacuole lumen. V-ATPase is a multi-subunit complex, and VmaA / Vma1 is a key component subunit of V-ATPase. VmaA / Vma1 is a core marker protein for studying the dynamic development of fungal vacuoles, which is involved in proton channel formation and complex stability maintenance, and plays an important role in proton pump activity, enabling V-ATPase to generate the required proton gradient across the membrane.

[0004] Most of the research on vacuolar proton pump V-ATPase is in plants. It plays an important role in the process of plant resistance to abiotic stress. Under stress conditions, increasing the activity of vacuolar V-ATPase is beneficial to improving the adaptability of plants to stress, thereby reducing the adverse effects of stress on plant growth and development, and providing a research direction for accelerating the breeding of high-quality stress-resistant crops. However, most of the research on vacuolar proton pump V-ATPase in fungi is focused on model fungi, such as Saccharomyces cerevisiae, Neurospora crassa and Aspergillus nidulans, mainly on the growth and development of the strains and their sensitivity to pH and ion concentration.

[0005] Aspergillus niger is an important industrial protein production "cell factory", and the study of the mechanism of the vacuolar proton pump in the process of protein synthesis and secretion in Aspergillus niger industrial strain is of great significance to the improvement of industrial enzyme production. SUMMARY

[0006] At present, there are few reports on the vacuolar proton pump V-ATPase in Aspergillus niger. According to the research in model fungi, the protein secretion level and glucoamylase activity of the gene edited strain of the vacuolar proton pump V-ATPase subunit AnVma1 and the starting strain are detected in the industrial production of glucoamylase in Aspergillus niger, the influence of the vacuolar proton pump on the protein synthesis and secretion of filamentous fungi is studied, the mechanism of the key organelle vacuole in the secretion process of glucoamylase is further understood, the protein secretion capacity of filamentous fungi is further improved, and the recombinant strain with significantly improved yield of glucoamylase and other starch industrial enzymes is obtained to meet the fermentation application of glucoamylase in the industrial field, which has important innovation and application value.

[0007] The present inventors have conducted extensive and in-depth research, and the purpose is to provide an Aspergillus vacuolar proton pump subunit AnVma1 gene and its application, which provides technical support and theoretical basis for studying and improving the protein secretion level of filamentous fungi and constructing high-yield industrial strains of enzyme preparations. The present application improves the yield of filamentous fungal amylase and the protein secretion level of filamentous fungi by genetically modifying the related genes of the core organelle vacuole of filamentous fungi. By constructing the gene AnVma1 or AnVma1-GFP overexpression recombinant expression vector of the Aspergillus niger strain, the gene AnVma1 or AnVma1-GFP overexpression recombinant strain is obtained, which can significantly improve the protein secretion level and significantly improve the production capacity and enzyme activity level of glucoamylase.

[0008] To achieve this purpose, the technical scheme adopted by the present application is as follows:

[0009] The present application provides an Aspergillus vacuolar proton pump V-ATPase subunit gene AnVma1, and the amino acid sequence encoded by the gene AnVma1 is shown in SEQ ID NO. 1.

[0010] Preferably, the nucleotide sequence of the AnVma1 is shown in SEQ ID No. 2.

[0011] The present application provides the use of the vacuolar proton pump subunit gene AnVma1 in improving the protein secretion level of filamentous fungi. Preferably, the filamentous fungi is Aspergillus, and most preferably the Aspergillus is Aspergillus niger.

[0012] The present application further provides a method for improving the secretion level of protein in filamentous fungi, which is overexpressing or enhancing the expression of the vacuolar proton pump subunit gene AnVma1 in filamentous fungi, thereby improving the ability of filamentous fungi to secrete protein.

[0013] Specifically, the overexpression or enhancement of the vacuolar proton pump subunit gene AnVma1 is achieved by constructing an expression cassette of the gene AnVma1 and introducing it into filamentous fungi; preferably, the promoter of the expression cassette is the promoter Ptef1 of the translation elongation factor TEF1A (An18g04840) of Aspergillus niger, and the terminator of the expression cassette is the terminator TtrpC of the trpC gene of Aspergillus nidulans.

[0014] The filamentous fungi is Aspergillus, and most preferably the Aspergillus is Aspergillus niger.

[0015] Specifically, the nucleotide sequence of the expression cassette is shown in SEQ ID No. 3, and the backbone plasmid of the expression vector used is pAN52-neo.

[0016] In the specific embodiment, the recombinant expression vector is transformed into the host cell of filamentous fungi by genetic manipulation, so that the gene AnVma1 in the host cell is overexpressed.

[0017] More specifically, the improved secreted protein refers to a protein involved in starch hydrolysis. Further specifically, the protein is glucoamylase.

[0018] In the present application, the meanings of the terms used are explained.

[0019] "Recombinant" when used with reference to a strain, cell, nucleic acid, protein, or vector indicates that the strain, cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein. For example, a recombinant strain is one which expresses genes not found under natural (non-recombinant) conditions or expresses native genes.

[0020] "Host strain" or "host cell" refers to a suitable host for an expression vector or DNA construct, and 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.

[0021] "Genome editing" refers to the deletion, insertion, or replacement of genomic DNA of an organism, so as to achieve the purpose of modifying the target sequence.

[0022] "RNA interference (RNAi)" refers to the interference with gene expression at the transcriptional level, using an artificially or biosynthetically produced specific complementary DNA or RNA fragment (or a modified product thereof) to inhibit or block the expression of a gene of interest according to the principle of base complementarity. The RNA interference vector is integrated into the genome to encode double-stranded RNA complementary to the target gene. This linear RNA interference reduces the transcription of a target sequence containing an inverted repeat sequence, which forms a hairpin structure after folding. This hairpin structure of the vector will facilitate the increase of the level of gene silencing.

[0023] "CRISPR-Cas9 editing technology" refers to the Cas9 protein which can cut specific DNA sequences in the genome of an organism under the guidance of a guide sgRNA, making a double-stranded DNA break, thereby triggering the cell's own repair mechanism to repair the DSB site by non-homologous end joining (NHEJ) or homologous recombination (HR) repair, thereby performing gene knockout, gene inactivation, gene knock-in and gene replacement, and achieving the purpose of efficient genome editing.

[0024] "Target site" or "protospacer" refers to a nucleic acid sequence defining a portion of a nucleic acid, which is a 20-base sequence at the 5' end of a guide RNA (sgRNA), which is identical to the DNA sequence of interest. The sgRNA needs this sequence to bind to the DNA of interest under the condition of sufficient binding, and the complex of Cas9 and sgRNA performs cleavage on the DNA of interest.

[0025] "Sequence" refers to a nucleotide sequence of any suitable length, which can be DNA or RNA; can be linear, circular or branched, and can be single-stranded or double-stranded. The term "donor DNA sequence" refers to a 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 in length (or any integer value therebetween).

[0026] The present application obtains the following beneficial effects: the genetic operation is carried out to aspergillus cell to enhance or increase the activity of AnVma1 (An02g10440) gene, which can significantly improve the production level of starch hydrolytic enzyme. In a specific experiment, the present application obtains the overexpression recombinant strain of gene AnVma1 or AnVma1-GFP by using genetic modification technology, and the overexpression recombinant strain can significantly improve the production level of starch hydrolytic enzyme, wherein the protein secretion level is increased by about 41-46% than the starting strain, the glucoamylase activity is increased by about 24-27% than the starting strain, and has great application value. In another specific experiment, the present application reduces the gene AnVma1 of aspergillus niger by using CRISPR-Cas9 editing technology and RNA interference technology, and obtains the gene reduction mutant strain, and the gene reduction mutant can significantly reduce the production level of starch hydrolytic enzyme, wherein the protein secretion level and the glucoamylase activity are reduced by about 27-32% and about 26-33% respectively than the starting strain, and the results verify again that the gene AnVma1 (An02g10440) can positively regulate the protein secretion level and the yield of glucoamylase of aspergillus niger, and is a key element for improving the production level of glucoamylase of aspergillus niger. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is a schematic diagram of AnVma1 and AnVma1-GFP gene expression cassette (A) and PCR identification nucleic acid electrophoresis diagram of overexpression recombinant strain (B).

[0028] Figure 2 It is the influence of overexpression gene AnVma1 on the protein secretion level and the glucoamylase activity of aspergillus niger, wherein: (A) is the mycelium fluorescence microscopic graph of overexpression recombinant strain OE-AnVma1-GFP; (B) and (C) are respectively the supernatant extracellular protein level (B) and the glucoamylase enzyme activity (C) of overexpression strains OE-AnVma1 and OE-AnVma1-GFP and starting strain AnN1 at the 4th day and the 6th day of shake flask fermentation culture.

[0029] Figure 3 It is the secreted protein concentration and the glucoamylase activity of aspergillus niger gene AnVma1 overexpression strain and starting strain under 5L fed-batch fermentation conditions, wherein: (A) is the protein secretion level of fermentation; (B) is the glucoamylase activity of fermentation supernatant; (C) is the protein SDS-PAGE electrophoresis analysis graph of fermentation supernatant.

[0030] Figure 4 It is a schematic diagram of knocking out target gene AnVma1 by CRISPR-Cas9 editing technology (A) and PCR identification nucleic acid electrophoresis diagram of gene knockdown mutant strain (B).

[0031] Figure 5Schematic diagram for silencing target gene AnVma1 by RNA interference technology (A) and nucleic acid electrophoresis map for PCR identification of gene-silenced mutants (B).

[0032] Figure 6 Protein concentration (A) and saccharifying enzyme activity (B) of shake flask fermentation supernatant of gene AnVma1 knockdown strain KD-Anvma1 and silenced mutant S-Anvma1. DETAILED DESCRIPTION

[0033] In order to further illustrate the technical means adopted by the present application and its effects, the technical solutions of the present application will be further described below in combination with the preferred embodiments of the present application. It should be understood that these embodiments are only used to illustrate the present application and not used to limit the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications or replacements all fall within the protection scope of the present application.

[0034] In the following examples, the methods used are conventional methods unless otherwise specified, and the specific steps can be found in: Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0035] The obtaining routes of various biological materials described in the examples are only provided as a route for experimental obtaining to achieve the specific disclosed purposes, and should not be regarded as a limitation on the sources of biological materials of the present application. In fact, the sources of the biological materials used are extensive, and any biological material that can be obtained without violating laws and moral ethics can be replaced and used according to the prompts in the examples.

[0036] The Aspergillus niger starting strain AnN1 used in the examples is derived from Shandong Longketo Enzyme Preparation Co., Ltd.

[0037] The specific techniques or conditions not specified in the examples are carried out according to the techniques or conditions described in the literature in the art, or according to the product instructions. The reagents or instruments not specified by the manufacturer are all conventional products that can be commercially available through regular channels. Among them, the percentage concentrations appearing without specific instructions are all mass percentage concentrations. The primers and nucleic acid sequencing used are completed by Suzhou Genewiz Biotechnology Co., Ltd. GENEWIZ. Among them, "An02g10440" is the gene site number of Aspergillus niger.

[0038] Example 1, Aspergillus niger high-level recombinant expression of vacuolar proton pump subunit AnVma1

[0039] (1) Construction of recombinant expression vector of vacuolar proton pump subunit AnVma1 (An02g10440)

[0040] The expression vector was constructed using plasmid pAN52-neo (Gu SY, et al. Metabolic engineering of the thermophilic filamentous fungus Myceliophthora thermophila to produce fumaric acid. Biotechnology for Biofuels. 2018, 11:323.) as a backbone. Using the sequence of Neurospora crassa vacuolar protein Vma1 (NCU01207; Bowman BJ, Draskovic M, Freitag M, Bowman EJ. 2009. Structure and distribution of organelles and cellular location of calcium transporters in Neurospora crassa. Eukaryot Cell 8:1845-55.) as a reference, bioinformatics analysis and alignment were performed on the protein sequence encoded by Aspergillus niger, and the vacuolar proton pump subunit An02g10440 was found. The gene was named AnVma1, and the amino acid sequence and nucleotide sequence of AnVma1 are shown in SEQ ID NO. 1 and SEQ ID NO. 2 in the sequence listing, respectively.

[0041] In order to study the cellular localization of the proton pump subunit AnVma1 (An02g10440) in Aspergillus niger and its role in protein synthesis and secretion, the present application constructs a high-efficiency recombinant expression vector of AnVma1-GFP and AnVma1, fuses and expresses the proton pump subunit AnVma1 and green fluorescent protein GFP, places the fusion protein AnVma1-GFP or the single protein AnVma1 under the promoter Ptef1 of the translation elongation factor TEF1A (An18g04840) for transcription and expression, and selects Aspergillus nidulans TtrpC as a terminator. The cDNA of the industrial strain AnN1 of Aspergillus niger is used as a template to PCR-amplify the proton pump subunit AnVma1 (An02g10440), and the PCR primer pairs required for the expression cassette construction are Ptef1-F / Ptef1-R, AnVma1-F / AnVma1-R and GFP-F / GFP-R, the primer sequences are shown in Table 1, and Gibson Assembly technology system is used to rapidly assemble the above multiple PCR fragments to the backbone plasmid pAN52-neo double-digested by restriction endonucleases Bgl II and BamH I, transform into Trans-T1 competent cells, select positive clones for sequencing, thereby constructing the expression vector of the proton pump subunit AnVma1-GFP or AnVma1, and the schematic diagram is shown in Fig. A, wherein the nucleotide sequences of the AnVma1 and AnVma1-GFP expression cassettes are shown in SEQ ID No. 3 and SEQ ID No. 4. Figure 1

[0042] Table 1: Primers used

[0043]

[0044] (2) Recombinant expression of vacuolar proton pump subunit AnVma1 in Aspergillus niger

[0045] 10 μg of the recombinant expression vectors pAN52-AnVma1 and pAN52-AnVma1-GFP linearized by restriction endonuclease Hind III are respectively transformed into the cells of the glucoamylase industrial production strain Aspergillus niger AnN1 by the protoplast transformation method, and the transformants are selected by adding 200 μg / mL of G418 in the plate.

[0046] A, Protoplast transformation of Aspergillus niger

[0047] Mycelium preparation:

[0048] The mature Aspergillus niger spores are collected with 0.05% Tween-80 sterilized water, filtered out of the mycelium by lens paper, inoculated in MM liquid culture, and cultured at 30°C, 200 rpm for 16 h.

[0049] ​Protoplast preparation:

[0050] The mycelium was collected by filtration and placed in 30 mL lysis solution (formula: 0.15 g lyticase, 30 mL solution A, filtered to remove bacteria; solution A: 1.0361 g potassium dihydrogen phosphate, 21.864 g sorbitol, dissolved in 90 mL deionized water, pH adjusted to 5.6, quantified to 100 mL, high-temperature sterilization) for 2 h at 30°C with gentle shaking every 30 min. Then it was filtered with glass paper and centrifuged at 2000 rpm for 10 min at 4°C, the supernatant was discarded, and 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, quantified to 100 mL, high-temperature sterilization) was added, and centrifuged at 2000 rpm for 10 min at 4°C; the supernatant was discarded, and a certain volume of solution B was added according to 200 μL / plasmid.

[0051] Protoplast transformation:

[0052] In a pre-cooled 15 mL centrifuge tube, 50 μL of pre-cooled PEG (12.5 g PEG6000, 0.368 g calcium chloride, 500 μL Tris HCl (1 M pH 7.5) were added, and the DNA fragments to be transformed were mixed well and added to 200 μL of protoplasts. After being placed on ice for 20 min, 2 mL of pre-cooled PEG liquid was added, and it was placed at room temperature for 5 min, and then 4 mL of solution B was added, and it was mixed gently. Take 3 mL of the above solution and add it to 12 mL of melted MM medium containing G418, and plate it on a plate, and incubate at 35°C, and after 3 days, pick single mycelium on the corresponding resistant plate.

[0053] B. Verification of Aspergillus niger transformants

[0054] Extraction of Aspergillus niger genome:

[0055] The genomic DNA was extracted from the transformants selected in the above transformation process by the phenol-chloroform method, which included the following operations:

[0056] 1) 200 mg of zirconium beads and 1 mL of lysis solution (formula: 0.2 M Tris-HCl (pH 7.5), 0.5 M NaCl, 10 mM EDTA, 1% SDS (w / v)) were added to a 2.0 mL sterile DNA extraction tube, and Aspergillus niger mycelium growing on the plate was picked into the DNA extraction tube;

[0057] 2) Place all the DNA extraction tubes on a bead beater and shake at maximum speed for 30 s, repeat twice;

[0058] 3) 65°C water bath for 30 min, vortex every few minutes during the water bath;

[0059] 4) After the end of the water bath, remove each tube and add 80 μL of 1 M Tris-HCl pH 7.5 for neutralization;

[0060] 5) Add 400 μL of phenol:chloroform (1 : 1) and centrifuge at 13000 rpm for 5 minutes;

[0061] 6) Take 300 μL of the supernatant in a new 1.5 mL EP tube and add 600 μL of 95% ethanol (DNA grade);

[0062] 7) Incubate on ice for one hour, then centrifuge at 4°C at 13000 rpm. A white DNA pellet should be visible at the bottom of the EP tube;

[0063] 8) Wash the pellet with 400 μL of 75% ethanol (DNA grade) at 4°C at 13000 rpm. Gently remove the supernatant;

[0064] 9) Place the EP tube in a vacuum concentrator to remove the residual ethanol;

[0065] 10) Add 50 μL of ddH20 to dissolve the DNA. Measure the DNA concentration using a NanoDrop. After measuring the concentration, store the extracted DNA at -20°C until the next step for PCR verification.

[0066] PCR verification of transformants:

[0067] Using the extracted genomic DNA as a template, use primers Ptefl-SF and TtrpC-SR to perform gene PCR verification of the transformants. PCR reagents were purchased from Nanjing Novi Biological Technology Co., Ltd. Perform 1% agarose gel electrophoresis (120V voltage, 30 minutes) on the PCR amplification products. The gel imaging system shows obvious gene amplification bands. The PCR amplification bands of the OE-AnVma1 and OE-AnVma1-GFP recombinant strains are 2269 bp and 2955 bp, respectively. The starting strain cannot amplify the target band. The results are shown in Figure B, indicating that the recombinant expression cassette is transformed into the host cell, and the AnVma1 gene recombinant expression strain OE-AnVma1 and OE-AnVma1-GFP is obtained. Figure 1

[0068] Example 2, Analysis of protein secretion amount and saccharifying enzyme activity of the AnVma1 overexpression strain under shake flask fermentation conditions

[0069] ​(1) Overexpression strains were cultured in starch-induced medium: the obtained recombinant strains OE-AnVma1 and OE-AnVma1-GFP and the starting strain AnN1 were inoculated into 50 mL medium in 250 mL Erlenmeyer flasks. The medium formula: 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, constant volume to 1000 mL, autoclaved), inoculation amount was 2.5*10 5 6 days, the sample was centrifuged to obtain the supernatant, and the protein concentration and glucoamylase activity were determined.

[0070] (2) Fluorescence microscopic observation of the expression of green fluorescent protein by recombinant strains: on the 4th day of shake flask culture, green fluorescent signals were detected on the mycelium of the recombinant overexpression strains Figure 2 A). By fusing the GFP signal with the red fluorescence of the membrane-selective dye FM4-64, it was found that the GFP fluorescent signal had good stability and strong green fluorescence, and the fusion protein AnVma1-GFP had been successfully expressed in the recombinant strain.

[0071] (2) Determination of the concentration of secreted proteins: the protein concentration in the supernatant was detected using the Biorad Bradford protein rapid test kit, and the results are shown in Figure 2 B. Compared with the starting strain AnN1, the protein yield of the recombinant strains OE-AnVma1 and OE-AnVma1-GFP was significantly improved, and under starch culture conditions, the protein yield was increased by about 28-38% and about 24-27% on the 4th day and the 6th day, respectively.

[0072] (3) Glucoamylase activity detection: the crude enzyme solution was diluted by an appropriate multiple with 0.05M pH 4.8 sodium acetate buffer, the final volume was 0.25 mL, and it was preheated in a 50°C water bath, then it was taken out, 0.25 mL of 1% soluble starch (Difco) substrate solution preheated in a 50°C water bath was added, mixed well, and reacted at 50°C for 10 min. The reaction was terminated by adding 0.5 mL of DNS solution, boiled for 10 min, cooled on ice, and made up to 2.5 mL with distilled water, shaken well, and the amount of released glucose was determined by the DNS method. The OD value was measured at a wavelength of 540 nm, and the blank group used inactivated enzyme solution as a control. The glucoamylase activity was defined as follows: 1 mL of enzyme solution hydrolyzed 1 μmol of soluble starch to produce glucose per min under the conditions of 50°C and pH 4.8, which was defined as one enzyme activity unit (U).

[0073] The supernatant was diluted based on the protein concentration, and the enzyme activity of saccharifying enzymes was measured. The results are as follows: Figure 2 As shown in Figure C, the saccharifying enzyme activity of the recombinant strains was significantly improved. Compared with the original strain AnN1, the saccharifying enzyme activity increased by about 32-33% and about 23-28% on day 4 and day 6, respectively.

[0074] Example 3: Phenotypic analysis of saccharifying enzyme production by overexpressed recombinant strains under 5L fermenter culture conditions.

[0075] To further detect the yield of saccharifying enzyme in the fermenter, the recombinant expression strains OE-AnVma1 and OE-AnVma1-GFP and the starting strain AnN1 were inoculated into a 5L fermenter for fed-batch fermentation. The saccharifying enzyme production phenotype of the overexpressed recombinant strains and the starting strain was observed over a 9-day fermentation period. The fermentation samples were centrifuged, and the supernatant was collected to determine the protein concentration and saccharifying enzyme activity. SDS-PAGE electrophoresis analysis was then performed.

[0076] The results are as follows Figure 3 As shown, compared with the original strain AnN1, the protein secretion levels of the recombinant strains OE-AnVma1 and OE-AnVma1-GFP were significantly enhanced during the 9-day fermentation period, and were significantly increased by approximately 41-46% after the 9th day of fermentation compared with the AnN1 strain. Figure 3 (A), the activity of saccharifying enzyme was also increased accordingly, showing a significant increase of approximately 28-31% compared to the starting strain AnN1. Figure 3 (B), SDS-PAGE results of protein supernatant on day 9 of fermentation ( Figure 3 (C) This more intuitively demonstrates the phenotype of recombinant overexpression strains OE-AnVma1 and OE-AnVma1-GFP, which can secrete more proteins and have significantly improved glycan production levels.

[0077] Example 4: Gene degradation of the vacuolar proton pump subunit AnVma1 using the CRISPR-Cas9 system and RNA interference technology.

[0078] The present application uses CRISPR-Cas9 gene editing system to knockout Anvma1 gene in AnN1 strain for multiple attempts, and still cannot obtain a homozygous mutant with Anvma1 gene knocked out, which is consistent with the reports of other filamentous fungi, indicating that Anvma1 gene is an essential gene for the growth of filamentous fungi. Therefore, the present application constructs Anvma1 gene reduction mutants by replacing weak promoters or RNA interference (RNAi) technology, respectively. In order to obtain a mutant strain with reduced vacuolar proton pump subunit AnVma1 (An02g10440) gene, the present application uses CRISPR-Cas9 genome editing technology to knock down the gene of the weak promoter replacement strategy of Aspergillus niger strain, and obtains the gene reduction mutant KD-Anvma1, and at the same time, uses RNA interference technology to construct a gene silencing mutant strain S-Anvma1.

[0079] (1) Knocking down the gene of Aspergillus niger gene AnVma1 (An02g10440)

[0080] The target site of the target gene AnVma1 is designed by software sgRNACas9 tool, and the primer sequence is shown in Table 1. The Aspergillus niger AnU6p promoter, target site and sgRNA skeleton are connected by fusion PCR method, and the sgRNA expression plasmid AnU6p-AnVma1-sgRNA is constructed by gene overlap extension (SOE) method, and its sequence is shown as SEQ ID No. 5. In this embodiment, the homologous donor DNA fragment is composed of the upstream and downstream homologous fragments of the target gene AnVma1, the hph resistance gene expression frame PtrpC-hph fragment, and the weak promoter from An01g08590 gene. The donor DNA fragment donor-AnVma1 is connected to the plasmid pUC118 linearized by restriction endonuclease BamH I and Hind III by Gibson Assembly method. The expression frame of Cas9 protein, AnU6p-AnVma1-sgRNA and donor DNA donor-AnVma1 are co-transformed into host strain protoplast cell, and the gene editing mutant strain can be obtained by homologous recombination. The experimental results are shown in Figure 4 As shown in FIG. 2A, the PCR amplification band of the mutant is 3379 bp, and the target band of the host strain is 996 bp, indicating that the donor DNA fragment and the sequence on both sides of the target site have undergone homologous recombination, and then the gene knockout mutant KD-Anvma1 is obtained.

[0081] The plasmid pAN52-S1 / S2 (Liu Q, et al. Unveiling equal importance of two 14-3-3 proteins for morphogenesis, conidiation, stress tolerance and virulence of an insect pathogen. Environ Microbiol 2015. 17: 1444-62.) was used as the backbone to construct the RNA interference vector. The cDNA of AnN1 was used as a template to amplify the AnVma1 gene fragment by PCR, and the AnVma1 gene silencing vector pAN52-Silent-AnVma1 was constructed. After the gene interference vector pAN52-Silent-AnVma1 was transformed into the host strain protoplast cells, the AnVma1 gene silencing mutant strain was obtained. The experimental results are as follows Figure 4 As shown in FIG. 8A, the PCR amplification band of the mutant was 3124 bp, and the starting strain could not amplify the target band, indicating that the gene silencing mutant S-Anvma1 was successfully obtained.

[0082] (2) Phenotypic analysis of gene reduction mutant strain glucoamylase production

[0083] The above obtained gene knockdown mutant KD-Anvma1 and gene silencing mutant S-Anvma1 and the starting strain AnN1 were inoculated into 50 mL of enzyme production medium in a 250 mL flask. The enzyme production medium formula is as described in Example 2, the inoculation amount was 2.5*10 5 / mL, 30°C, 200 rpm for 6 days, the sample was centrifuged to obtain the supernatant, and the protein concentration and glucoamylase activity were determined.

[0084] The protein concentration in the supernatant was detected using the Bole Bradford protein rapid test kit, and the results are as shown in FIG. 8B. Figure 4 As shown in FIG. 8B, compared with the starting strain AnN1, the protein yield of the gene knockdown strain KD-Anvma1 and the silencing mutant S-Anvma1 was significantly reduced, and the protein yield was reduced by about 27-30% and about 28-32% on the 4th day and the 6th day, respectively.

[0085] The glucoamylase enzyme activity of the gene reduction strain KD-Anvma1 and S-Anvma1 after shake flask fermentation culture was determined. The supernatant was diluted according to the protein concentration for glucoamylase enzyme activity determination, and the results are as shown in FIG. 8C. Figure 4As shown in Figure B, the recombinant strains showed significantly improved glucoamylase activity. Compared with the starting strain AnN1, the glucoamylase activity of the recombinant strains was increased by about 26-28% and about 27-33% on the 4th day and the 6th day, respectively. The above results again verified that the gene AnVma1 can positively regulate the protein secretion level and the yield of glucoamylase of A. niger, and is the key to improve the production level of glucoamylase of A. niger.

Claims

1. A type of Aspergillus ( Aspergillus The vacuolar proton pump V-ATPase subunit gene AnVma1 is characterized by, The amino acid sequence of the gene AnVma1 is shown as SEQ ID NO.

1.

2. The tonoplast proton pump subunit gene AnVma1 according to claim 1, wherein, The nucleotide sequence of the AnVma1 is shown as SEQ ID No.

2.

3. Use of the vacuolar H+-ATPase subunit gene An Vma1 according to claims 1 and 2 for increasing the level of protein secretion in a filamentous fungus; preferably the filamentous fungus is an Aspergillus ( Aspergillus ), most preferably the Aspergillus is an Aspergillus niger ( Aspergillus niger ).

4. A method for increasing the level of protein secretion in a filamentous fungus, characterized in that, It is overexpression or enhancement of the vacuolar proton pump subunit gene AnVma1 in filamentous fungi as claimed in claim 1, which improves the ability of filamentous fungi to secrete proteins.

5. The method of claim 4, wherein, The overexpression or enhancement of the vacuolar proton pump subunit gene AnVma1 as claimed in claim 1 is achieved by constructing an expression cassette of the gene AnVma1 and introducing it into filamentous fungi; preferably, the promoter of the expression cassette is the promoter Ptef1 of the translation elongation factor TEF1A (An18g04840) of Aspergillus niger, and the terminator of the expression cassette is the terminator TtrpC of the trpC gene of Aspergillus nidulans. The filamentous fungus is an Aspergillus ( Aspergillus ), most preferably the Aspergillus is an Aspergillus niger ( Aspergillus niger ).

6. The method of claim 5, wherein, The nucleotide sequence of the expression cassette is shown as SEQ ID No.

3.

7. The method of claim 5, wherein, The backbone plasmid of the expression vector is pAN52-neo.

8. The method of claim 7, wherein, It is achieved by transforming the recombinant expression vector into the host cell of filamentous fungi by genetic manipulation, so that the gene AnVma1 in the host cell is overexpressed.

9. The method according to any one of claims 4 to 8, characterized in that, The improved secreted protein refers to a protein involved in starch hydrolysis.

10. The method of claim 9, wherein, The protein is glucoamylase.

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