Penicillium oxalicum engineering bacteria and a construction method thereof

Abstract

The application discloses a penicillium oxalicum engineering bacterium and a construction method thereof. Penicillium oxalicum The engineering bacterium is named penicillium oxalicum (QL-ACxrC-ACreA) and has a preservation number of CCTCC NO: M 2026896. The penicillium oxalicum engineering bacterium is obtained by knocking out transcriptional inhibitor CxrC and transcriptional factor CreA of carbon catabolite repression of filamentous fungi, so that when starch is used as the only carbon source for fermentation, the amylase activity and extracellular protein content generated by the penicillium oxalicum engineering bacterium are significantly improved; and the knocking out of the two factors synergistically improves the starting efficiency of the promoter Pamy15A.

Description

Technical Field

[0001] This invention relates to an engineered Penicillium oxalate strain and its construction technology, specifically to an engineered Penicillium oxalate strain and its construction method. Background Technology

[0002] Penicillium oxalate ( Penicillium oxalicum *Trichoderma reesei* is a type of fungus that produces lignocellulosic enzymes. Compared to traditional industrial filamentous fungi such as *Trichoderma reesei* and *Aspergillus niger*, it possesses unique advantages such as low secretion background and high-efficiency protein secretion under non-induced conditions. The cellulase it produces has been listed in the food additive catalog. The strain exhibits high biosafety and can be widely adapted to industrial applications in food processing, feed industry, biopharmaceuticals, and biorefining. It is an excellent chassis strain for constructing high-efficiency, low-background fungal expression hosts, possessing significant research value and broad industrialization prospects.

[0003] Currently, the modification and optimization of Penicillium oxalate expression systems has become a research hotspot in the field of microbial engineering. Existing technologies mainly focus on conventional modification methods such as fermentation carbon source regulation, promoter and signal peptide modification, and single-component transcription factor editing. Furthermore, most studies only explore the function of a single transcriptional regulatory factor. For example, CN115725421A reported a genetically engineered Penicillium oxalate strain, GXUR001, classified as Penicillium oxalate TE4-10ΔCxrC::AmyR. Penicillium oxalate GXUR001 was obtained by modifying the Penicillium oxalate mutant strain A2-13 through four rounds of EMS chemical mutagenesis and two rounds of Co60-γ-ray mutagenesis to obtain the mutant strain TE4-10. Then, the transcriptional repressor gene PoxCxrC and the transcriptional activator gene PoxAmyR were simultaneously knocked out in strain TE4-10, and its fermentation broth exhibited amylase activity. CN113388531B reports a Penicillium strain that enhances amylase activity. The Penicillium strain is named *Penicillium oxalate* Δ13AO AmyRΔCreA. By knocking out the CreA gene, the expression of the expression element amylase Amy15A promoter Pamy15A is enhanced.

[0004] In fact, CreA and CxrC belong to different transcriptional regulatory pathways in fungi, with complementary functions and independent regulatory levels: CreA mainly mediates the global carbon metabolism repression effect, inhibiting the expression of degradation enzyme genes in response to external carbon source signals; CxrC is a specific transcriptional repressor of Penicillium oxalate, targeting and negatively regulating the synthesis of key functional proteins such as cellulase and amylase. Knocking out only a single repressor cannot completely remove the limitations of the multiple transcriptional repression network of Penicillium oxalate, making it difficult to fully utilize the advantages of the strain's natural low-contamination protein secretion, and failing to further reduce extracellular background secretion and effectively improve the expression efficiency and purity of target proteins. This results in existing engineered Penicillium oxalate strains still having technical shortcomings such as high background secretion, insufficient release of expression potential, and insufficient industrial adaptability, making it difficult to meet the current high-end industrial demand in the biomanufacturing field for high-expression, low-background, high-purity, and high-stability filamentous fungal expression systems.

[0005] Therefore, constructing a Penicillium oxalate engineered bacterium with both CxrC and CreA genes knocked out, and exploring the synergistic regulatory effect of dual-target knockout, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] The first technical problem to be solved by this invention is to provide an engineered Penicillium oxalate strain that significantly increases the yield of amylase produced by the synergistic effect of CxrC and CreA double gene knockout.

[0007] The second technical problem to be solved by the present invention is to provide a method for constructing the engineered Penicillium oxalate strain.

[0008] The technical solution adopted by this invention to solve its first technical problem is an engineered Penicillium oxalate strain, named Penicillium oxalate ( Penicillium oxalicum The accession number of the oxalic acid penicillium is CCTCC NO: M 2026896.

[0009] The technical solution adopted by the present invention to solve its second technical problem is a method for constructing the engineered Penicillium oxalate strain, comprising the following steps:

[0010] S1. Starting with Penicillium oxalate OamyR-QL, the CxrC gene was knocked out to obtain Penicillium oxalate QL-ΔCxrC.

[0011] S2. Using Penicillium oxalate QL-ΔCxrC as the starting strain, the CreA gene is knocked out to obtain Penicillium oxalate QL-ΔCxrC-ΔCreA.

[0012] Preferably, the knockout of the CxrC gene specifically includes the following steps:

[0013] S1a. Using pyrG-S3 plasmid as a template, the pyrG sequence was amplified using primer pair SIX-PYRG-F / SIX-PYRG-R, and the CxrC coding region was replaced by the pyrG sequence of Aspergillus nidulans.

[0014] S1b: Using the Penicillium oxalicum 114-2 genome as a template, a promoter region of about 3kb upstream of the Penicillium oxalicum CxrC coding region and a terminator region of about 3kb were amplified as exchange homologous arms.

[0015] S1c. The three sequences obtained in the above steps are fused using the Double-joint PCR method to obtain the CxrC knockout cassette.

[0016] S1d, the CxrC knockout cassette was transformed into the OamyR-QL strain to obtain the transformant QL-ΔCxrC; it was selected and verified by PCR to obtain Penicillium oxalate QL-ΔCxrc.

[0017] Preferably, in step S1a, the sequence of the primer pair is as follows:

[0018] SIX-PYRG-F: GGAAGGATACAGTCGCTAGC (SEQ ID No. 1);

[0019] SIX-PYRG-R: CGTTCACACGTGAAGC (SEQ ID No. 2).

[0020] Preferably, in step S1b, the primer pair for amplification is as follows:

[0021] Upstream arm primer pair:

[0022] cxrc-F:GTGAAGCTTCGAGAGACCCG (SEQ ID No.3)

[0023] cxrc-pyrG-R:

[0024] GAGTTGCTAGCGACTGTATCCTTCCGTCGGCGGTGTAAAGGAAAT (SEQ ID No. 4);

[0025] Downstream arm primer pair:

[0026] cxrc-pyrG-F:

[0027] ACTGCGGCCGCTTCACGTGTGAACGCCCCTCCTCTTCCACTTGAC (SEQ ID No. 5)

[0028] cxrc-R: AGCCGTCTGTGTGCTTGTT (SEQ ID No. 6).

[0029] Preferably, in step S1d, the primer pair used for PCR verification is as follows:

[0030] cxrc-NF: GGATAAAGTGGAGGAAAAGGTCGAG (SEQ ID No. 7);

[0031] cxrc-NR: CTCCAAGCATATCATACACCACCCG (SEQ ID No. 8).

[0032] Preferably, the knockout of the CreA gene specifically includes the following steps:

[0033] S2a. Using pyrG-S3 plasmid as a template, the pyrG sequence was amplified using primer pair creA-PYRG-F / creA-PYRG-R, and the creA coding region was replaced by the pyrG sequence of Aspergillus nidulans.

[0034] S2b: Using the Penicillium oxalicum 114-2 genome as a template, the promoter region of about 660 bp upstream of the Penicillium oxalicum creA coding region and the terminator region of about 800 bp were amplified as exchange homologous arms.

[0035] S2c. The three sequences obtained above are fused using the Double-joint PCR method to obtain the creA knockout cassette.

[0036] S2d, the CreA knockout cassette was transformed into the QL-ΔCxrC strain to obtain the transformant QL-ΔCxrC-ΔCreA; it was then selected and verified by PCR.

[0037] Preferably, in step S2a, the sequence of the primer pair is as follows:

[0038] creA-PYRG-F:TTCGTCAGGATCTGCCTCGCGGAAGGTATACAGTCGCTAGCAAC (SEQ IDNo.9);

[0039] creA-PYRG-R: GCGTAGTTGGAGAAGGAGTGCGTTCACACGTGAAGCGG (SEQ ID No. 10).

[0040] Preferably, in step S2b, the primer pair for amplification is as follows:

[0041] creA-up-F:CATTCCTCGTCCTCCCCTCT (SEQ ID No. 11);

[0042] creA-up-R: GCGAGGCAGATCCTGACGAA (SEQ ID No. 12);

[0043] creA-down-F:CACTCCTTCTCCAACTACGC (SEQ ID No. 13);

[0044] creA-down-R: GTAGCGCGCATGGAAAGCAA (SEQ ID No. 14).

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] This invention obtains *Penicillium oxalate* by knocking out the transcriptional repressor CxrC and the transcriptional factor CreA, which represses carbon catabolism in filamentous fungi. Penicillium oxalicum The addition of QL-ΔCxrc-ΔCreA significantly increased the activity of amylase and the content of extracellular protein when starch was used as the sole carbon source for fermentation; the knockout of the two factors synergistically improved the initiation efficiency of the Pamy15A promoter.

[0047] Information on the preservation of biological materials

[0048] Penicillium oxalate ( Penicillium oxalicum QL-ΔCxrc-ΔCreA was deposited on May 8, 2026 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China; accession number: CCTCC NO: M2026896. Attached Figure Description

[0049] Figure 1 Electrophoresis diagram validating the constructed sequence of the cxrc gene knockout cassette;

[0050] Wherein, A represents the upstream arm fragment, downstream arm fragment, and pyrG gene sequence of the CxrC gene; M: 5kb marker; lane 1: upstream arm; lane 2: downstream arm; lanes 3 and 4: selection marker pyrG; B represents the complete CxrC knockout cassette fragment, lane 1: CxrC knockout cassette.

[0051] Figure 2 Validation electrophoresis image for PCR verification of QL-ΔCxrC transformants;

[0052] In this context, A represents positive verification; M: 5 kb marker; lane 1: original strain OamyR-QL positive control; lanes 2-6: transformant positive verification. B represents negative verification of transformants; M: 5 kb marker; lane 1: original strain OamyR-QL negative control; lanes 2-6: transformant negative verification.

[0053] Figure 3 The image shows the SDS-PAGE results of extracellular secreted proteins from strains OamyR-QL, QL-ΔCxrC, QL-ΔCreA, and QL-ΔCxrC-ΔCreA.

[0054] Lane 1: OamyR-QL; Lane 2: QL-ΔCreA; Lane 3: QL-ΔCxrC; Lane 4: QL-ΔCxrC-ΔCreA.

[0055] Figure 4 Line graph showing the results of extracellular amylase activity assays for strains OamyR-QL, QL-ΔCxrC, QL-ΔCreA, and QL-ΔCxrc-ΔCreA. Detailed Implementation

[0056] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise stated, all reagents and instruments used in this invention are commercially available products.

[0058] plasmids and strains

[0059] Penicillium oxalate 114-2 (original strain): Penicillium oxalate CGMCC5032.

[0060] Penicillium oxalate Δ13A-OamyR: Starting with Penicillium oxalate 114-2, the amylase Amy13A was knocked out, and amyR was overexpressed to obtain strain Δ13A-OamyR.

[0061] OamyR-QL: Starting with Δ13A-OamyR, the G protein signaling pathway was continuously activated through point mutation using hph as a selection marker.

[0062] Penicillium oxalate QL-ΔCreA (ZL202010171977.5): A strain that knocks out the transcription factor CreA, starting with Penicillium oxalate OamyR-QL.

[0063] Penicillium oxalate QL-ΔCxrC strain: A strain that uses Penicillium oxalate OamyR-QL as the starting strain and knocks out the transcriptional repressor cxrC.

[0064] Penicillium oxalate QL-ΔCxrC-ΔCreA strain: A strain that uses Penicillium oxalate QL-ΔCxrC as the starting strain and knocks out the transcription factor CreA, which inhibits carbon catabolism.

[0065] Culture media and reagents used in the construction of engineered strains

[0066] 1. Upper solid conversion medium (120 mL): 10.92 g sorbitol, 1.2 g glucose, 2.4 mL Vogel's salt solution (50×), 1.2 g agarose, and deionized water to a final volume.

[0067] 2. Lower solid conversion medium (50 mL): 9.1 g sorbitol, 0.5 g agarose, and deionized water to a final volume.

[0068] 3. 100 mL liquid glucose medium: 1% glucose, 2% Vogel's salt solution (50×).

[0069] 4. Glucose phenotype medium: Add 2% agar powder to the liquid medium.

[0070] 5. Starch liquid culture medium: 2% Vogel's salt solution (50×), 1.5% soluble starch (dissolve by heating first and then bring to volume).

[0071] 6. Starch phenotype culture medium: Add 2% agar powder to the liquid culture medium.

[0072] 7. PDA phenotypic culture medium: 4.61 g PDA powder dissolved in 100 mL deionized water.

[0073] 8. Bran culture medium (600 mL): Weigh 60 g of bran into a ceramic jar, boil it for 30 min, filter it through 4-8 layers of gauze, and bring the filtrate to a final volume of 600 mL. First, add 0.6 g of uracil. After the uracil dissolves, slowly add 12 g of agar powder while heating at a low temperature.

[0074] 9. Vogel's salt solution (200 mL, 50×): 25 g trisodium citrate dihydrate, 50 g anhydrous potassium dihydrogen phosphate, 20 g ammonium nitrate, 2 g magnesium sulfate heptahydrate, 1 g anhydrous calcium chloride (dissolved in a small amount of water beforehand), 0.2 mL trace element solution, dilute to 200 mL with deionized water, place in an oven for 8 h to dissolve completely, and store in a refrigerator at 4 ℃.

[0075] 10. Protoplast transformation solvent

[0076] (1) Physiological saline (200 mL): 1.8 g NaCl, 100 μL Tween-80, and deionized water to a final volume of 200 mL.

[0077] (2) S1 solution (300 mL): 4.08 g anhydrous potassium dihydrogen phosphate, 65.58 g sorbitol, deionized water to a final volume of 300 mL, pH adjusted to 5.6 (using NaOH solution).

[0078] (3) S2 solution (300 mL): 54.66 g sorbitol, 1.665 g anhydrous calcium chloride, deionized water to a final volume of 300 mL, pH adjusted to 7.5 (using 1 M Tris-HCl).

[0079] (4) T1 solution (50 mL): 12.5 g PEG6000, 0.2775 g anhydrous calcium chloride, deionized water to 50 mL, pH adjusted to 7.5 (using 1 M Tris-HCl).

[0080] (5) 1 M Tris-HCl (200 mL): 24.22 g Tris, diluted to volume with deionized water. Adjust the pH to 7.5 using hydrochloric acid.

[0081] 11.1% starch substrate (500 mL) solution: Weigh 5 g of starch into 200 mL of water, heat to boiling, and make up to volume. Store at 4℃.

[0082] 12. Genomic extraction buffer (200 mL): 1.68 g EDTA, 4.28 g Tris, 2.92 g NaCl, 4 g SDS, bring to a final volume of 200 mL.

[0083] 13. SDS-PAGE staining solution (1000 mL): 100 mL glacial acetic acid, 1 g Coomassie brilliant blue, 250 mL isopropanol, bring to a final volume.

[0084] 14. SDS-PAGE decolorization solution (2000 mL): 100 mL anhydrous ethanol, 200 mL glacial acetic acid, bring to volume.

[0085] 15. PrimeSTAR® Max DNA Polymerase: Takara Bio Inc.; BCA Protein Assay Kit: Nanjing Novizan Biotechnology Co., Ltd.; DNA Marker (8K, 5K, 2K), Rapid High-Fidelity DNA Polymerase: TransGen Biotech Co., Ltd.; Protein Maeker (10-180 kDA), Column-Based Plasmid DNA Extraction Kit: Shanghai Sangon Biotech Co., Ltd.; Agarose Gel Recovery Kit: Omega (USA); PAGE Gel Rapid Preparation Kit: Yaxin Biotech Co., Ltd.

[0086] Example 1 Construction of engineered Penicillium oxalate strain

[0087] A method for constructing an engineered Penicillium oxalate strain includes the following steps:

[0088] S1. Starting with Penicillium oxalate OamyR-QL, the CxrC gene was knocked out to obtain Penicillium oxalate QL-ΔCxrC.

[0089] S1a. Using pyrG-S3 plasmid as a template, the pyrG sequence was amplified using primer pair SIX-PYRG-F / SIX-PYRG-R, and the CxrC coding region was replaced by the pyrG sequence of Aspergillus nidulans.

[0090] Bacterial plasmids were extracted using a bacterial plasmid extraction kit (Sangon Biotech). Primers for pyrG sequence amplification were designed to amplify the pyrG sequence, resulting in a pyrG sequence with six cleavage sites at both ends. The sequences of the pyrG sequence amplification primers are as follows:

[0091] SIX-PYRG-F: GGAAGGATACAGTCGCTAGC (SEQ ID No. 1);

[0092] SIX-PYRG-R: CGTTCACACGTGAAGC (SEQ ID No. 2).

[0093] PCR amplification system: This experiment used a 20 μl PCR reaction system, with 7.8 μl ddH2O, 0.6 μl upstream primer, 0.6 μl downstream primer, 1 μl template, and 10 μl enzyme.

[0094] PCR amplification program: 94 ℃ for 5 min; 94 ℃ for 30 s, 58 ℃ for 30 s, 72 ℃ (1 kb / min, set the time according to the fragment size) for 28~30 cycles; 72 ℃ for 10 min.

[0095] S1b. Using the Penicillium oxalate 114-2 genome as a template, a promoter region approximately 3 kb upstream of the Penicillium oxalate CxrC coding region and a terminator region approximately 3 kb upstream were amplified as exchanged homologous arms; the primer pairs for amplification are as follows:

[0096] Upstream arm primer pair:

[0097] cxrc-F:GTGAAGCTTCGAGAGACCCG (SEQ ID No. 3);

[0098] cxrc-pyrG-R:

[0099] GAGTTGCTAGCGACTGTATCCTTCCGTCGGCGGTGTAAAGGAAAT (SEQ ID No. 4);

[0100] Downstream arm primer pair:

[0101] cxrc-pyrG-F:

[0102] ACTGCGGCCGCTTCACGTGTGAACGCCCCTCCTCTTCCACTTGAC (SEQ ID No. 5);

[0103] cxrc-R: AGCCGTCTGTGTGCTTGTT (SEQ ID No. 6).

[0104] PCR amplification system: This experiment used a 20 μl PCR reaction system, ddH2O: 7.8 μl, upstream primer: 0.6 μl, downstream primer: 0.6 μl, template: 1 μl, enzyme: 10 μl.

[0105] PCR amplification program: 94 ℃ for 5 min; 94 ℃ for 30 s, 58 ℃ for 30 s, 72 ℃ (1 kb / min, set the time according to the fragment size) for 28~30 cycles; 72 ℃ for 10 min.

[0106] S1c. The three sequences obtained in the above steps are fused using the Double-joint PCR method to obtain the CxrC knockout cassette.

[0107] Fusion PCR reaction program: 94 ℃ for 5 min; 94 ℃ for 30 s, 58 ℃ for 8 min, 72 ℃ (1 kb / min, time set according to fragment size) for 12 cycles; 72 ℃ for 10 min.

[0108] S1d, the CxrC knockout box was transformed into OamyR-QL strain to obtain transformant QL-ΔCxrC; selected and verified by PCR to obtain Penicillium oxalate QL-ΔCxrc;

[0109] The primer pairs used for the PCR verification are as follows:

[0110] cxrc-NF: GGATAAAGTGGAGGAAAAGGTCGAG (SEQ ID No. 7);

[0111] cxrc-NR: CTCCAAGCATATCATACACCACCCG (SEQ ID No. 8).

[0112] PCR amplification system: This experiment used a 20 μl PCR reaction system, ddH2O: 7.8 μl, upstream primer: 0.6 μl, downstream primer: 0.6 μl, template: 1 μl, enzyme: 10 μl.

[0113] PCR amplification program: 94 ℃ for 5 min; 94 ℃ for 30 s, 58 ℃ for 30 s, 72 ℃ (1 kb / min, set the time according to the fragment size) for 28~30 cycles; 72 ℃ for 10 min.

[0114] S2. Using Penicillium oxalate QL-ΔCxrC as the starting strain, the CreA gene was knocked out to obtain Penicillium oxalate QL-ΔCxrC-ΔCreA;

[0115] The specific steps are as follows:

[0116] S2a. Using pyrG-S3 plasmid as a template, the pyrG sequence was amplified using primer pair creA-PYRG-F / creA-PYRG-R, and the creA coding region was replaced by the pyrG sequence of Aspergillus nidulans.

[0117] creA-PYRG-F:TTCGTCAGGATCTGCCTCGCGGAAGGTATACAGTCGCTAGCAAC (SEQ IDNo.9);

[0118] creA-PYRG-R: GCGTAGTTGGAGAAGGAGTGCGTTCACACGTGAAGCGG (SEQ ID No. 10);

[0119] PCR amplification system: This experiment used a 20 μl PCR reaction system, ddH2O: 7.8 μl, upstream primer: 0.6 μl, downstream primer: 0.6 μl, template: 1 μl, enzyme: 10 μl.

[0120] PCR amplification program: 94 ℃ for 5 min; 94 ℃ for 30 s, 58 ℃ for 30 s, 72 ℃ (1 kb / min, set the time according to the fragment size) for 28~30 cycles; 72 ℃ for 10 min.

[0121] S2b: Using the Penicillium oxalicum 114-2 genome as a template, the promoter region of about 660 bp upstream of the Penicillium oxalicum creA coding region and the terminator region of about 800 bp were amplified as exchange homologous arms.

[0122] creA-up-F:CATTCCTCGTCCTCCCCTCT (SEQ ID No. 11);

[0123] creA-up-R: GCGAGGCAGATCCTGACGAA (SEQ ID No. 12);

[0124] creA-down-F:CACTCCTTCTCCAACTACGC (SEQ ID No. 13);

[0125] creA-down-R: GTAGCGCGCATGGAAAGCAA (SEQ ID No. 14).

[0126] PCR amplification system: This experiment used a 20 μl PCR reaction system, ddH2O: 7.8 μl, upstream primer: 0.6 μl, downstream primer: 0.6 μl, template: 1 μl, enzyme: 10 μl.

[0127] PCR amplification program: 94 ℃ for 5 min; 94 ℃ for 30 s, 58 ℃ for 30 s, 72 ℃ (1 kb / min, set the time according to the fragment size) for 28~30 cycles; 72 ℃ for 10 min.

[0128] S2c. The three sequences obtained above are fused using the Double-joint PCR method to obtain the creA knockout cassette.

[0129] Fusion PCR reaction program: 94 ℃ for 5 min; 94 ℃ for 30 s, 58 ℃ for 8 min, 72 ℃ (1 kb / min, time set according to fragment size) for 12 cycles; 72 ℃ for 10 min.

[0130] S2d, the creA knockout cassette was transformed into the QL-ΔCxrC strain to obtain the transformant QL-ΔCxrC-ΔCreA; it was then selected and verified by PCR.

[0131] The primer pairs used for the PCR verification are as follows:

[0132] pyrG-yz-F: TGCCCGAACAGAAAGAGG (SEQ ID No. 15);

[0133] creA-R: AAAGTGTCAGCAGGTCTCGG (SEQ ID No. 16).

[0134] PCR amplification system: This experiment used a 20 μl PCR reaction system, ddH2O: 7.8 μl, upstream primer: 0.6 μl, downstream primer: 0.6 μl, template: 1 μl, enzyme: 10 μl.

[0135] PCR amplification program: 94 ℃ for 5 min; 94 ℃ for 30 s, 58 ℃ for 30 s, 72 ℃ (1 kb / min, set the time according to the fragment size) for 28~30 cycles; 72 ℃ for 10 min.

[0136] Penicillium oxalate protoplast transformation

[0137] 1. Inoculate the spores onto bran slant agar and incubate at 30 ℃ for 2-4 days.

[0138] 2. Wash the fresh spores with sterile physiological saline and transfer them to a 5 mL centrifuge tube for later use.

[0139] 3. Spread a layer of cellophane on the prepared bran plate, add about 200 μL of spore suspension to each plate, spread it evenly, making sure there are no air bubbles, seal it with disposable sealing film, and incubate it in a 30 ℃ incubator for 16 h to observe the mycelial growth.

[0140] 4. In a clean bench, add 0.14 g of lysin and 0.02 g of lysis enzyme to a sterile 50 mL centrifuge tube, then add 30 mL of S1 solution. Gently shake to dissolve, preparing the lysis buffer for later use. Add 10 mL of S1 solution to a sterilized agar plate. Using sterilized tweezers, peel off the cellophane and place it on the plate. Add 2-3 mL of lysis buffer for each layer of cellophane and remove any air bubbles between the layers. Place the plate inside a disposable glove and incubate at 30 °C for lysis for 2 hours.

[0141] 5. Add 10 mL of S1 solution to a new sterile plate, rinse the lysed hyphae, and filter the lysate and rinse solution together into a 50 mL centrifuge tube using a sterile funnel containing 4 layers of filter paper.

[0142] 6. Centrifuge at 2000 rpm for 12 min, discard the supernatant, add 10 mL of S2 solution, and gently resuspend the protoplasts by pipetting. (To ensure the protoplasts are in good condition, subsequent operations must be performed on ice).

[0143] 7. Centrifuge at 2000 rpm for 12 min, discard the supernatant, and add 0.4-0.6 mL of S2 solution. Gently pipette to resuspend the protoplasts.

[0144] 8. Take 20 μL of protoplast preparation slides for microscopic examination to observe the protoplast preparation. If there are about 20-25 round and well-formed protoplasts in each field of view, the subsequent transformation operation can be carried out.

[0145] 9. Add 100 μL of protoplasts to each 50 mL centrifuge tube, followed by 10 μL of DNA fragments at a concentration of at least 200. Mix thoroughly with the pipette tip, and finally add 25 μL of L1 solution. Incubate on ice for 20 min. Pour off the lower culture medium at this point for later use.

[0146] 10. Add 1 mL of T1 and let stand at room temperature for 5 min.

[0147] 11. Add 2 mL of S2 and gently mix by blowing.

[0148] 13. Pour 8-10 mL of the upper culture medium into each 50 mL centrifuge tube, then pour the transformation system onto the lower culture medium. Seal with sealing film and incubate at 30 ℃ for 4-6 days.

[0149] Single colonies of transformants from (13) were picked and cultured in glucose medium at 30 °C and 200 rpm for 24 h. The genomes of the transformants were then extracted.

[0150] Penicillium oxalate genome extraction

[0151] 1. Inoculate the spore solution into 20 mL of glucose liquid culture medium and culture it in a shaker at 200 rpm and 30 ℃ for 16 h to grow mycelium.

[0152] 2. Centrifuge 1 mL of fermentation broth at 13500 rpm for 8 min, discard the supernatant, add 150 μg of quartz sand and 0.5 mL of extraction buffer, and grind in a cryogenic grinder for 4 min (to destroy the cell wall of Penicillium oxalate and release the internal genomic DNA).

[0153] 3. Incubate in a 65 ℃ water bath for 10 min, add 0.2 mL of 7.5 mol / L ammonium acetate solution, and incubate in an ice bath for 8 min.

[0154] 4. Centrifuge at 12000 rpm for 10 min, collect the supernatant, add 0.5 times the volume of ice-cold isopropanol, mix by inversion, and place in a -20 ℃ refrigerator for 10 min.

[0155] 5. Centrifuge at 12500 rpm for 10 min, discard the supernatant, and wash the precipitate twice with 200 μL of 70% ethanol at 12000 rpm for 2 min.

[0156] 6. After drying in the oven, dissolve in 20 μL of double-distilled water.

[0157] Using the genome as a template, PCR amplification was performed with verification primers to verify whether the transformed fragment was integrated into the genome of the transformant.

[0158] Example 2: SDS-PAGE Validation and Comparative Analysis

[0159] Culture fermentation broth

[0160] The PCR-verified transformants QL-ΔCxrC and QL-ΔCxrC-ΔCreA, along with the starting strain OamyR-QL, were inoculated onto fresh wheat bran slant and cultured for 2-4 days. The spores were washed off with physiological saline and transferred to glucose liquid medium, and cultured at 30 ℃ and 200 rpm for 24 h. 0.5 g of mycelium was then transferred to starch medium for further fermentation. Three replicates were set up for each strain, and fermentation was carried out at 30 ℃ and 200 rpm with shaking for 5 days.

[0161] SDS-PAGE analysis of engineered strains

[0162] During fermentation, samples were taken from the fermentation broth after 72 h, and the supernatant was processed and preserved for SDS-PAGE analysis. The results showed that the expression levels of extracellular proteins in the engineered strains QL-ΔCxrC and QL-ΔCreA were significantly higher than those in the starting strain OamyR-QL, and the expression levels of extracellular proteins in the engineered strain QL-ΔCxrC-ΔCreA were significantly higher than those in strains QL-ΔCxrC and QL-ΔCreA. Figure 3 Amylase activity assays showed that during a 120-hour culture period, the amylase activity of QL-ΔCreA was 152%-203% of that of the starting strain OamyR-QL; QL-ΔCxrC was 167%-220% of that of the starting strain OamyR-QL; while during the culture period, the engineered strain QL-ΔCxrC-ΔCreA was 404%-612% of that of the starting strain OamyR-QL. Therefore, the simultaneous removal of cxrC and creA had a synergistic effect on the yield of amylase Amy15A. Figure 4 ).

[0163] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. An engineered strain of Penicillium oxalate, named Penicillium oxalate (… Penicillium oxalicum QL-ΔCxrC-ΔCreA, with accession number CCTCC NO: M 2026896.

Images