Method for improving activity of aspergillus oryzae amylase through CreA C terminal dephosphorylation mutation
By using site-directed mutagenesis and homologous recombination at the C-terminal phosphorylation site of the CreA protein, the problem of carbon metabolism inhibition of amylase expression by the CreA protein was solved, and the activity of Aspergillus oryzae amylase was significantly improved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-16
- Publication Date
- 2026-04-10
AI Technical Summary
In the existing technology, the industrial production of amylase is affected by the carbon metabolism inhibitory factor of CreA protein, resulting in low enzyme activity. Existing methods are difficult to effectively relieve carbon source inhibition and improve amylase expression.
By site-directed mutagenesis of the C-terminal phosphorylation sites of the CreA protein, especially the S414, S415, T416, and S419 sites, mutating them to valine, a CreA dephosphorylation mutant strain of Aspergillus oryzae was constructed. The strain was then introduced into Aspergillus oryzae using homologous recombination to enhance amylase activity.
The mutant strain significantly improved the activity of Aspergillus oryzae amylase, with the amylase activity being approximately three times that of the control strain, without affecting the strain's growth and industrial stability.
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Figure CN121825940A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a method for improving the activity of Aspergillus oryzae amylase by CreA C-terminal dephosphorylation mutation. Background Technology
[0002] Amylases are key industrial enzymes that hydrolyze starch and its derivatives, directly converting uncooked raw starch into fermentable sugars such as glucose. Based on their mechanism of action, they can be classified into α-amylases and β-amylases. α-Amylases are widely distributed in animals (saliva, pancreas, etc.), plants (malt, hawthorn), and microorganisms. Amylases are diverse in type and characteristics, and have wide applications in the food, fermentation, textile, pulp and paper, and bioenergy industries. With the development of the biomanufacturing industry, the market demand for highly active and stable amylases is increasing. However, current industrial production of amylases usually relies on microbial fermentation, and their enzyme activity levels are still limited by various factors, including gene expression regulation inhibition, insufficient secretion pathway efficiency, protein folding capacity limitations, and carbon source regulation effects. Obtaining high-yield and stable amylase strains has become a core issue for the industrial utilization of amylases.
[0003] Currently, filamentous fungi, including Aspergillus oryzae, are widely used in industry to produce amylase. However, even though these fungi possess strong protein secretion capabilities, the expression of their target enzymes is still strictly limited by carbon source metabolism regulation. In filamentous fungi, amylase secretion is strongly influenced by the carbon metabolism repression mechanism (CCR). The core regulator of CCR is the transcriptional repressor CreA. CreA can bind to the promoter regions of multiple carbohydrate hydrolase genes (including various amylase genes), thereby inhibiting their transcription in the presence of preferential carbon sources such as glucose, leading to a significant reduction in the expression level of the target enzyme. Recent studies have also shown that the regulatory activity of CreA is closely related to its phosphorylation state. Phosphorylation at different sites affects nucleocytoplasmic transport, DNA binding capacity, and protein stability; therefore, phosphorylation modification is an important way to regulate the degree of CCR. However, current technologies lack effective strategies to utilize specific dephosphorylation mutations of CreA to remove or partially weaken CCR, thereby enhancing amylase expression.
[0004] Currently, increasing amylase production mainly relies on promoter enhancement or replacement, overexpression of key genes in the secretion pathway, and optimization of fermentation conditions. However, these methods have limited effectiveness in addressing controlled inhibition caused by CCR (carbon metabolism inhibition), while directly knocking out the CreA-encoding gene often leads to strain growth defects, metabolic disorders, and decreased industrial stability, making them unsuitable for modifying industrial production strains. Therefore, developing a strategy based on dephosphorylation mutations at specific sites of CreA to weaken its carbon metabolism inhibition without affecting other functions, and then precisely regulating carbon source inhibition to enhance amylase gene expression and production, is a pressing technological solution needed in the industry. Summary of the Invention
[0005] Purpose of the invention: To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for improving amylase activity expression. This method involves site-directed mutation of the C-terminal phosphorylation site of the CreA protein and its introduction into Aspergillus oryzae using homologous recombination to obtain a CreA dephosphorylated mutant strain of Aspergillus oryzae. Then, amylase expression can be performed using this strain to obtain amylase with high enzyme activity.
[0006] Technical solution: To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:
[0007] A method for improving the expression of Aspergillus oryzae amylase activity involves site-directed mutagenesis of the Aspergillus oryzae transcription repressor factor CreA to obtain a CreA dephosphorylated mutant strain, and then using the mutant strain for fermentation to produce amylase.
[0008] In some embodiments, the site-directed mutation is a dephosphorylation mutation targeting the phosphorylation modification site at the C-terminus of the CreA protein.
[0009] In some embodiments, the C-terminal phosphorylation modification site includes at least one of serine at position 414, serine at position 415, threonine at position 416, and serine at position 419.
[0010] In some embodiments, the site-directed mutagenesis involves simultaneously mutating S414, S415, T416, and S419 to valine.
[0011] An expression cassette for constructing any of the CreA dephosphorylated mutant strains, the expression cassette comprising the following components connected in sequence: a 5' homologous arm fragment creA-N of the Aspergillus oryzae CreA gene, a CreA gene fragment creA-C containing the site-directed mutation site as described in claim 3 or 4, a nutrient selection marker gene pyrG, and a 3' homologous arm fragment creA-ter of the CreA gene.
[0012] A CreA dephosphorylated mutant strain of Aspergillus oryzae, the strain being obtained by introducing the expression cassette into an Aspergillus oryzae strain and replacing the wild-type creA gene in its genome via homologous recombination.
[0013] In some embodiments, the mutant strain exhibits significantly increased amylase activity relative to the reference strain introduced with an empty vector under conditions where starch is the primary carbon source.
[0014] In some embodiments, the amylase activity of the mutant strain is three times that of the reference strain.
[0015] A method for producing amylase includes: culturing any of the Aspergillus oryzae CreA dephosphorylated mutant strains described herein, and harvesting amylase from its fermentation culture.
[0016] An amylase, prepared by the method for producing amylase described above.
[0017] Compared with the prior art, the beneficial effects of this application are as follows:
[0018] This invention introduces the CreA C-terminal phosphorylation mutant gene into Aspergillus oryzae via homologous recombination, thereby obtaining a CreA C-terminal phosphorylation mutant strain. Using this strain to express amylase enhances the amylase activity of Aspergillus oryzae. Experimental results confirm that mutations at the four phosphorylation sites at the CreA C-terminus (S... 414 S 415 T 416 S 419 →V 414 V 415 V 416 V 419 The mutant strain significantly increased the activity of Aspergillus oryzae amylase, with the amylase activity of the mutant strain being approximately three times that of the control strain. Attached Figure Description
[0019] Figure 1 A schematic diagram of the selected dephosphorylation mutation sites in the CreA protein;
[0020] Figure 2 This is a schematic diagram of the primer sequences and amplification fragments;
[0021] Figure 3 Electrophoresis results of the CreA C-terminal dephosphorylated fusion fragment;
[0022] Figure 4 Sanger sequencing alignment diagram of a successful phosphate site mutation;
[0023] Figure 5A comparison of the results of amylase activity determination in DPY medium using the starch-iodine colorimetric method (Shanghai Jining) with reference strain and mutant strain using the amylase (AMS) test kit;
[0024] Figure 6 This is a graph showing the amylase activity detection of CD liquid culture mutant strain 4M. Detailed Implementation
[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.
[0026] Example 1
[0027] A method to improve amylase activity expression, such as Figure 1 and Figure 2 As shown, the C-terminal phosphorylation sites of the CreA protein in Aspergillus oryzae strain RIB40 were predicted using NetPhos 3.1 and the UniProt website. The analysis results indicated that multiple potential phosphorylation sites exist in the C-terminal sequence (amino acids 410-420) of the CreA protein, among which serine 414 (S414), serine 415 (S415), threonine 416 (T416), and serine 419 (S419) were predicted as high-probability phosphorylation modification sites. Therefore, these four sites were mutated to non-phosphorylated valine (V), i.e., S... 414 S 415 T 416 S 419 Mutate to V 414 V 415 V 416 V 419 In this manner, a site-directed mutation was performed on the C-terminal phosphorylation site of CreA, and the mutation was introduced into Aspergillus oryzae using homologous recombination to obtain a CreA dephosphorylated mutant strain. This strain was then used to express amylase, thereby enhancing amylase activity. Primers designed based on the above mutations for subsequent homologous recombination are shown in Table 1.
[0028] The specific steps are as follows:
[0029] 1) Using Super Pfx MasterMix (CWBIO, China), four fragments constituting the CreA C-terminal dephosphorylation mutant expression cassette were amplified (creA-N, gene sequence as shown in SEQ ID NO.1; creA-C, gene sequence as shown in SEQ ID NO.2; pyrG, gene sequence as shown in SEQ ID NO.3; creA-ter, gene sequence as shown in SEQ ID NO.4). Figure 2 As shown, the primers and amplification targets for each fragment are as follows:
[0030] Fragment creA-N (5' homologous arm): Amplified using primers SmaI-CreA-F and CreA(creA-N)-R.
[0031] Fragment creA-C (core fragment of the CreA gene containing the mutation site): Amplification was performed using primers CreA(creA-C)-F and CreA(creA-C)-R. This primer pair contained a base variation design that mutated S414, S415, T416, and S419 to valine (V).
[0032] Fragment pyrG (nutrient selection marker gene): Amplified using primers pyrG-F and pyrG-R.
[0033] Fragment creA-ter (3' homologous arm): Amplified using primers pyrG-CreA-F and CreA-SmaI-R.
[0034] The main conditions for amplification of each fragment are as follows:
[0035] PCR system: 25 μL of 2×Super Pfx MasterMix enzyme; 2 μL of upstream and downstream primers (see Table 1 for specific primer sequences); 1 μL of A. oryzae RIB40 genomic DNA (donated by Noda Institute of Sinica, Japan); add ddH2O to 50 μL.
[0036] Table 1 Specific primer sequences
[0037]
[0038] PCR conditions: 98.0℃ for 3 min; 98.0℃ for 10 s, 55.0℃ for 30 s, 72.0℃ for 1 min, 35 cycles; 72.0℃ for 10 min; store at 16.0℃.
[0039] 2) Purify the DNA in the electrophoresis gel and recover the fragments, following these steps:
[0040] DNA was extracted using the Takara MiniBest Agarose Gel DNA Etraction kit (TaKaRa, Japan). The main process is as follows:
[0041] Preparation: Set the dry heat sterilizer to 37°C. Chop the gel and add it to a 1.5 mL microcentrifuge tube. Add 3 times the volume of Buffer GM and mix well. Incubate at 37°C for 10 min (vortex occasionally to speed up dissolution). Place the spin column in a collection tube and pipette the solution into the spin column. Centrifuge at 12000 rpm for 1 min. After centrifugation, remove the liquid at the bottom. Add 700 μL of Buffer WB to the column. Centrifuge at 12000 rpm for 30 s. After centrifugation, remove the liquid at the bottom and repeat the step. Centrifuge at 12000 rpm for 1 min. Place the spin column in a new 1.5 mL tube. Add 30 μL of water / Elution Buffer and let stand at room temperature for 1 min. Centrifuge at 12000 rpm for 1 min.
[0042] 4) Fuse gene fragments into a cassette that defines the C-terminal dephosphorylation expression of CreA.
[0043] After obtaining a linearized plasmid by digesting plasmid PUC19 with SmaI enzyme, the linearized plasmid and the above four fragments were fused using Seamless CloningMix to obtain the CreA C-terminal dephosphorylation expression cassette. The cassette was introduced into E. coli and cultured overnight, and colony PCR and electrophoresis were performed to verify that the CreA C-terminal dephosphorylation expression cassette was successfully constructed.
[0044] The PUC19 linearization system consisted of: 1 μL of SmaI enzyme; 2 μL of 10×T buffer; 2 μL of 0.1% BS; 5 μL of PUC19 plasmid; 2 μL of ddH2O; and incubation at 30℃ for 30 min.
[0045] The Seamless Cloning system consisted of: 5 μL of 2×Seamless Cloning Mix enzyme; 0.5 μL of linearized PUC19; 1 μL of creA-N; 1 μL of creA-C; 0.5 μL of pyrG; 1 μL of creA-ter; and ddH2O added to a final volume of 10 μL.
[0046] The E. coli transformation procedure is as follows: Take 100 μL of competent cells thawed on ice, add the target DNA (plasmid or ligation product), mix gently, and let stand on ice for 5 min; heat shock in a 42°C water bath for 45-60 s, quickly transfer to an ice bath, and let stand for 2 min; add 700 μL of antibiotic-free sterile liquid culture medium (SOB or LB) to a centrifuge tube, take an appropriate volume of resuscitation solution and spread it evenly on the culture medium containing Amp antibiotic, or spread it directly without adding culture medium, and incubate in an inverted incubator at 37°C overnight.
[0047] The colony PCR system consisted of: 15 μL of 2×Super Pfx MasterMix enzyme; 1 μL of SmaI-CreA-F and CreA-SmaI-R (see Table 1 for specific primer sequences); a small amount of positive E. coli cultured overnight was used as a template by pipetting a pipette tip; and ddH2O was added to a final volume of 30 μL.
[0048] Colony PCR conditions: 98.0℃ for 3 min; 98.0℃ for 10 s, 55.0℃ for 30 s, 72.0℃ for 5 min, 35 cycles; 72.0℃ for 10 min; store at 16.0℃.
[0049] 5) Confirmation by electrophoresis, results as follows: Figure 3 As shown in the figure, the CreA C-terminated dephosphorylated fusion fragment is approximately 4.6 kb. The presence of a 4.6 kb fragment in the electrophoresis image indicates successful fusion. This fragment was purified into DNA in the gel using the Takara MiniBest Agarose Gel DNA Extraction Kit (TaKaRa, Japan) for transformation.
[0050] 6) Transformation
[0051] Preparation: Solution 0 (50mM Maleate buffer, pH 5.5); Solution 2 (1.2M Sorbitol, 50mM CaCl2, 35mM NaCl, 10mM Tris-HCl, pH 7.5); Solution 3 (60% PEG4000, 50mM CaCl2, 10mM Tris-HCl, pH 7.5); CD supernatant medium (CD medium + 1.2M Sorbitol + 0.8% Agar);
[0052] Pre-culture: Aspergillus oryzae strain E-F1 (∆ku70::ptrA, ∆AF, ∆pyrG, donated by Noda Industrial Science and Technology Research Institute of Japan) was cultured in DPY liquid medium. The wild strain of this fungus is A. oryzae RIB40. The culture was carried out at 30℃, 150rpm, for 18-24h.
[0053] Transformation experiment: Solution 1 (0.1% Yatalase, 0.6M (NH4)2SO4, add 10mL Solution 0), sterilize using a 0.45μm filter and transfer to a new 50mL sterile tube; filter the cultured Aspergillus oryzae using a funnel, rinse with water to remove the culture medium (the cells are white), squeeze with a spatula to remove the water; place the cells into Solution 0+1 prepared in step 1 using a spatula, seal the tube with a seal, and incubate at 30℃, 50rpm for 3h (protoplastization); prepare ice; filter (use disposable), collect the liquid; add 10mL Solution 2, mix thoroughly; remove the tube and centrifuge at 4℃, 2000rpm for 8min (adjust the ascent rate to 3'), collect the precipitate; add 5mL Solution 2, mix well, centrifuge at 4℃, 2000rpm for 8min, discard the supernatant; add Solution 2 (1-5×10) depending on the amount of precipitate. 7 Add 1 mL), dispense 200 μL x 4 portions; add 10 μL of plasmid (not exceeding 15 μL) to each portion, mix well with a disposable pipette; place on ice for 30 min, during which time melt the CD selection medium in a microwave oven, pour it into a 50 mL tubing, and incubate at 45 °C; after 30 min, add Solution 3 in three portions: 250 μL, 250 μL, and 850 μL, mix well. Incubate at room temperature for 20 min; add 5 mL of Solution 2, mix well, centrifuge at 4 °C at 2000 rpm for 8 min, discard the supernatant; add 500 μL of Solution 2, mix well, add 10 mL of CD upper layer medium, mix well, and pour into plates; incubate in CD medium at 30 °C in the dark for 3-5 days.
[0054] 7) Perform at least two separation and purification processes using selective medium (CD), and then extract the genome, as follows:
[0055] Preparation: Add Nucleic acid lysis solution (600 μL). Treat with liquid nitrogen, grind in a mortar and pestle, and place in a 1.5 mL tube; incubate at 65°C for 15 min using a dry incubator, then cool to hand temperature; add 3 μL RNase A and incubate at 37°C for 30-60 min to hydrolyze RNA; add 200 μL protein precipitation solution and mix by inverting for 20 s to remove proteins; centrifuge at 12000 rpm for 3 min and transfer the supernatant to a new 1.5 mL tube; treat with 600 μL PCI (wearing gloves), inverting and mixing to further remove proteins and other impurities (Phenol:chloroform:Isoamyl Alcohol 25:24:1); centrifuge at 12000 rpm for 3 min and collect the supernatant; add 600 μL isopropanol to precipitate the gene; centrifuge at 12000 rpm for 5 min, remove the supernatant and collect the precipitate; wash with 600 μL 70% ethanol at 12000 rpm for 5 min. Remove the supernatant and collect the precipitate; centrifuge at 12000 rpm for 1 min to remove the supernatant, air dry for 10 min; add 100 μL of TE solution.
[0056] 8) Validation of CreA dephosphorylation mutant strains
[0057] The results were obtained by Sanger sequencing alignment. Figure 4 As shown, the phosphate site was successfully mutated, indicating that the dephosphorylated mutant strain was successfully constructed.
[0058] 9) Comparative growth experiment of the constructed 4M mutant strain
[0059] The 4M strain and the control strain were grown on CD agar medium with glucose, sucrose and starch as the sole carbon sources, respectively. No significant growth delay was found, indicating that the mutation of the C-terminal phosphorylation site of CreA in the 4M strain did not lead to significant growth differences and would not affect subsequent production practices.
[0060] Example 2: Amylase Activity Assay
[0061] Culture medium: 45 mL DPY liquid medium per conidium; Conidium density: 102 5 ; 3 repetitions.
[0062] Enzyme activity was measured on days 2, 4, and 6.
[0063] The amylase activity in DPY medium was determined using the starch-iodine colorimetric method of the Amylase (AMS) test kit (Shanghai Jining).
[0064] See results Figure 5 Con: The control strain was obtained by directly introducing the pyrG selection marker gene into *A. oryzae* E-F1 (∆ku70::ptrA,∆AF, ∆pyrG); 4M: CreA dephosphorylation mutant strain. Mutations were made at the four phosphorylation sites at the C-terminus of CreA (S...). 414 S 415 T 416 S 419 →V 414 V 415 V 416 V 419 The mutant strain 4M significantly improved the activity of Aspergillus oryzae amylase. On day 6 of culture in CD liquid medium with starch replacing glucose as the carbon source, the amylase activity of the mutant strain 4M was about 3 times that of the control strain.
[0065] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.
Claims
1. A method for enhancing the expression of Aspergillus oryzae amylase activity, characterized in that, By performing site-directed mutagenesis on CreA, a transcriptional repressor in Aspergillus oryzae, a CreA dephosphorylation mutant strain was obtained, and then the mutant strain was used to produce amylase through fermentation.
2. The method according to claim 1, characterized in that, The site-directed mutation is a dephosphorylation mutation targeting the phosphorylation modification site at the C-terminus of the CreA protein.
3. The method according to claim 2, characterized in that, The C-terminal phosphorylation modification site includes at least one of serine at position 414, serine at position 415, threonine at position 416, and serine at position 419.
4. The method according to claim 3, characterized in that, The site-directed mutagenesis involves simultaneously mutating S414, S415, T416, and S419 to valine.
5. An expression cassette for constructing the CreA dephosphorylation mutant strain according to any one of claims 1-4, characterized in that, The expression cassette comprises the following components connected in sequence: the 5' homologous arm fragment creA-N of the Aspergillus oryzae CreA gene, the CreA gene fragment creA-C containing the site-directed mutation site as described in claim 4, the nutrient selection marker gene pyrG, and the 3' homologous arm fragment creA-ter of the CreA gene, wherein the nucleotide sequences of the creA-N, creA-C, pyrG, and creA-ter fragments are as shown in SEQ ID NO. 1 to 4, respectively.
6. A CreA dephosphorylated mutant strain of Aspergillus oryzae, characterized in that, The strain was obtained by introducing the expression cassette of claim 5 into an Aspergillus oryzae strain and replacing the wild-type creA gene in its genome through homologous recombination.
7. The mutant strain according to claim 6, characterized in that, The mutant strain exhibited significantly increased amylase activity compared to the reference strain introduced with an empty vector, under conditions where starch was the primary carbon source.
8. The mutant strain according to claim 7, characterized in that, The mutant strain had three times the amylase activity of the control strain.
9. A method for producing amylase, characterized in that, include: Cultivate the Aspergillus oryzae CreA dephosphorylated mutant strain according to any one of claims 6-8, and harvest amylase from its fermentation culture.
10. An amylase, characterized in that, It is prepared by the method described in claim 9.