Genetic engineering strain for fermenting immunosuppressant mycophenolic acid

Abstract

The invention provides a genetic engineering strain for fermenting immunosuppressant mycophenolic acid as well as a preparation method and application of the genetic engineering strain, and the method is characterized in that a promoter PgpdA of 3-phosphoglycerol dehydrogenase is used, the amino acid sequence of the promoter PgpdA is SEQ ID NO: 1, and an indigenous promoter of peroxisome biogenic protein PbPex337 of a penicillium strain is replaced. According to the genetically engineered bacterium for high yield of mycophenolic acid, the yield of mycophenolic acid is effectively increased by replacing a promoter of the peroxisome biogenic protein PbPex337 gene, and the genetically engineered bacterium has positive industrial application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fermentation of biological products, and particularly relates to a genetically engineered strain for fermenting immunosuppressant mycophenolic acid. BACKGROUND

[0002] Mycophenolic acid (MPA) is a natural antibiotic derived from fungal secondary metabolites, which has antiviral, antifungal, antibacterial, antitumor, antipsoriasis and immunosuppressive activities. Studies have shown that the proliferation of T and B lymphocytes mainly depends on the de novo synthesis pathway of guanine, and mycophenolic acid has physiological activity of inhibiting inosine monophosphate dehydrogenase (IMPDH), thereby inhibiting the conversion of inosine nucleotide (IMP) to xanthine nucleotide (XMP), and ultimately inhibiting the de novo synthesis of guanine and the proliferation of lymphocytes. Therefore, mycophenolic acid has shown great application value in clinical anti-immune rejection and treatment of autoimmune diseases.

[0003] Since the discovery of mycophenolic acid, it has attracted the attention of many researchers. According to the structure of mycophenolic acid, researchers have developed multiple chemical synthesis paths. Structurally, mycophenolic acid can be regarded as a six-substituted benzene ring. Birch first realized the chemical total synthesis of mycophenolic acid in 1969. Birch used a linear synthesis method to first synthesize the basic skeleton phthalate, then introduce 3-bromopropylene at C-6, and then extend the side chain through a Claisen rearrangement and multiple chemical reactions to finally synthesize mycophenolic acid. The chemical synthesis process has many defects such as long steps, harsh reaction conditions, difficult separation, and low yield, which makes it difficult to realize industrial large-scale application. Therefore, the main way of industrial production of mycophenolic acid is through the biological synthesis strategy-fungal submerged fermentation. At present, Penicillium brevicompactum ( Penicillium brevicompactum ) and Penicillium roqueforti ( Penicillium roqueforti ) are the main strains for industrial production of mycophenolic acid. However, the current industrialization of mycophenolic acid fungal fermentation is still relatively low, and the production cost is high. The strain used for fermentation is a high-yield Penicillium brevicompactum induced by ultraviolet mutagenesis, but this mutagenic strategy has limited potential for industrial production of mycophenolic acid by Penicillium. Therefore, it is an inevitable requirement to realize the industrial production of mycophenolic acid to optimize the mycophenolic acid synthesis pathway of Penicillium through genetic means, enhance the synthesis of mycophenolic acid of Penicillium, reduce the production of mycophenolic acid byproducts of Penicillium, and develop high-yield strains. SUMMARY

[0004] The purpose of the present application is to provide a genetically engineered strain for fermenting immunosuppressant mycophenolic acid and a preparation method and application thereof, which can be used for efficient fermentation synthesis and preparation of mycophenolic acid.

[0005] The application first provides a method for constructing a high-yield mycophenolic acid penicillium strain, wherein a 3-phosphoglycerate dehydrogenase promoter PgpdA with a nucleotide sequence of SEQ ID NO:1 is used to replace a native promoter of a peroxisome biogenesis protein PbPex337 of the penicillium strain; The application further provides a genetically engineered strain for high-yield immunosuppressant mycophenolic acid, which is constructed by the method. Further, the 3-phosphoglycerate dehydrogenase promoter PgpdA further comprises a derivative promoter formed by replacing or deleting one or more nucleotides based on the nucleic acid with the nucleotide sequence of SEQ ID NO:1. As a specific embodiment, the peroxisome biogenesis protein PbPex337 has a nucleotide sequence of SEQ ID NO:2.

[0006] Further, the 3-phosphoglycerate dehydrogenase promoter PgpdA used to replace the native promoter of the peroxisome biogenesis protein PbPex337 of the strain further comprises the 3-phosphoglycerate dehydrogenase promoter PgpdA used to replace the promoter of any enzyme involved in peroxisome biogenesis proliferation in the strain.

[0007] The method is to use a homologous recombination method to replace the native promoter of the peroxisome biogenesis protein with the 3-phosphoglycerate dehydrogenase promoter PgpdA with the nucleotide sequence of SEQ ID NO:1. The recombinant expression vector used for homologous recombination comprises the 3-phosphoglycerate dehydrogenase promoter PgpdA with the nucleotide sequence of SEQ ID NO:1, the homologous arms with the nucleotide sequence of SEQ ID NO:3 and the sequence of SEQ ID NO:4, and a resistance screening marker. As a specific embodiment, the screening marker is a resistance screening marker hygromycin gene (hygB) hyg B (SEQ ID NO:5).

[0008] Further, the genetically engineered strain is Penicillium brevicompactum.

[0009] The application further provides a method for fermenting mycophenolic acid, which uses the genetically engineered strain constructed by the application to ferment mycophenolic acid.

[0010] The genetically engineered strain for high-yield mycophenolic acid of the application effectively increases the yield of mycophenolic acid by replacing the promoter of the peroxisome biogenesis protein PbPex337 (SEQ ID NO:2) gene, and has positive industrial application value. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 : Recombinant plasmid pET28a- 337HL-hyg B-PgpdA-337HR schematic diagram; Figure 2 : Split-marker technology to construct engineering strain schematic diagram; Figure 3 : PCR verification of nucleic acid picture of transformant; Figure 4 : Engineering strain and original strain fermentation yield verification data graph; Figure 5 : Engineering strain and original strain fermentation growth curve data graph. DETAILED DESCRIPTION

[0012] The present application will be described below through specific implementation cases. Unless specifically stated, the technical means used in the present application are methods known to those skilled in the art. In addition, the implementation scheme should be understood as illustrative, not limiting the scope of the present application, and the essence and scope of the present application are only limited by the claims.

[0013] The present application provides a genetically engineered strain for high-yield mycophenolic acid, the main feature of which is that the engineering strain uses Penicillium brevicompactum as a chassis cell, and uses the promoter PgpdA (SEQ ID NO: 1) derived from 3-phosphoglycerate dehydrogenase of Aspergillus nidulans to replace the original promoter of the peroxisome biogenesis protein PbPex337 (SEQ ID NO: 2) gene to start the expression of the gene.

[0014] Among them, the promoter PgpdA is derived from the nucleotide sequence 2156bp upstream of the ATG of 3-phosphoglycerate dehydrogenase of Aspergillus nidulans as the promoter sequence.

[0015] Among them, the peroxisome biogenesis protein PbPex337 (SEQ ID NO: 2) gene and the two homologous arms (337HL: SEQ ID NO: 3, 337HR: SEQ ID NO: 4) required for integration are derived from the genome of the host cell Penicillium brevicompactum.

[0016] Among them, the resistance screening marker hygromycin gene (hygB) (SEQ ID NO: 5) is derived from the pET28a- plasmid preserved in the laboratory. hyg B hyg B plasmid.

[0017] Example 1: Extraction of Penicillium brevicompactum genome Genome extraction 1. Scrape Penicillium brevicompactum spores and inoculate into 50 mL PDB medium, 200 rpm, 28°C, shake culture for 48h.

[0018] ​2, Collect bacteria, 4°C, 12000 rpm, centrifugal 10 min, discard supernatant.

[0019] 3, Add 500 μL extraction buffer (Tris: 24.22 g / L, NaCl: 14.61 g / L, EDTA: 9.3 g / L, SDS: 2 g / L, pH 8.5) and a appropriate amount of quartz sand, vortex for 1 min. Place in 65°C water bath for 10 min.

[0020] 4, Add 200 μL 7.5 M ammonium acetate solution, mix gently, and place for 8 min.

[0021] 5, 12000 rpm, 4°C centrifugal 10 min, transfer the supernatant to a new centrifuge tube, add 0.6 times the volume of pre-cooled isopropanol, mix well, and stand at -20°C for 10 min.

[0022] 6, 4°C, 12000 rpm, centrifugal 10 min, discard supernatant.

[0023] 7, Add 500 μL 75% ethanol, mix well, and centrifugal 7,500 rpm for 5 min.

[0024] 8, Discard the supernatant, stand at room temperature for 10 min, and evaporate the remaining ethanol.

[0025] 10, Add 30 μL sterile water to dissolve.

[0026] Example 2: Preparation of Penicillium brevicompactum protoplasts In this example, the preparation of Penicillium brevicompactum protoplasts is described in detail.

[0027] 1) Bacterial culture Uniformly spread Penicillium brevicompactum spores on potato dextrose agar (PDA: potato extract 4.0 g / L, glucose 20.0 g / L, agar powder 15 g / L, pH 5.1±0.2) plates and incubate at 28°C for 5-7 days. Collect the spores from the PDA plates into 100 mL potato dextrose broth (PDB: potato extract 4.0 g, glucose 20.0 g, pH 5.6±0.2) and continue to culture for 2-3 days.

[0028] 2) Protoplast preparation 1. Take 120 mg lysing enzyme, 120 mg lysozyme, 180 mg cellulase, 120 mg lywallzyme and dissolve in 12 mL 0.8 M NaCl solution, shake gently until completely dissolved. Filter sterilize with 0.22 um filter into a sterile 50 mL conical flask.

[0029] 2. Collect mycelium with a nylon cloth funnel, wash twice with 20 mL 0.8 M NaCl, pick the mycelium into a conical flask containing the complex enzyme solution, and gently break the mycelium with a sterile gun head. Enzymatic hydrolysis at 30°C, 70 rpm for 2-3 h, and take samples every 1 h for microscopic observation of the enzymatic hydrolysis.

[0030] 3. After the end of enzymatic hydrolysis, filter the protoplasts with a nylon cloth funnel, and add 30 mL 0.8 M NaCl to rinse the filter funnel.

[0031] 4. Centrifuge the collected protoplasts at 2500 x g, 4°C for 5 min, remove the supernatant, and resuspend the protoplasts with 5 mL ice-cold solution I (0.8 M NaCl, 10 mM CaCl2, 50 mM Tris-HCl, pH 7.5).

[0032] 5. Centrifuge at 2500 x g, 4°C for 5 min, remove the supernatant, and resuspend the protoplasts with an appropriate amount (1-2 mL) of ice-cold solution I.

[0033] Protoplast quality (microscopic observation) control: Calculate the protoplast concentration by a hemocytometer, and the concentration should be higher than 3 x 10 7 / mL. c = n / f x 4 x 10 6 , c = number / mL, n = protoplast count, f = number of small squares counted.

[0034] Example 3: Obtaining the target fragment for Split Marker recombination 1) Construction of recombinant plasmid pET28a- 337HL-hyg B-PgpdA-337HR Obtain the two homologous arm gene sequences (337HL: SEQ ID NO: 3, 337HR: SEQ ID NO: 4) required for homologous recombination: According to the short dense penicillium genome sequence, design and synthesize the required primers: P1: 5'-ggtggtggtggtggtTTCGCCTTGTCCAGCTC-3' ​P2: 5'-ccagcccctgggttgctcgagGCTCTTCAAGAGCGTCG-3' P3: 5'-ATGGTTGCCGACGCCCTCGT-3' P4: 5'-tcgagctccgtcgacACCGGGTCTCCAGCGTAC-3' Screening marker acquisition: obtained from the laboratory preserved hygromycin resistance vector hyg B Sequence ( hyg B : SEQ ID NO: 5), the required primers were designed and synthesized: P5: 5'-ctcgagcaacccaggggctg-3' P6: 5'-gcggccgccgacgttaactg-3' Strong promoter selection: according to the gene sequence of PgpdA from Aspergillus nidulans published in Genebank, primers were designed and synthesized: P7: 5'-aacgtcggcggccgcGAATTCCCTTGTATCTCTAC-3' P8: 5'-gcgtcggcaaccatACTAGTGTCTGCTCAAGCGGGGTA-3' Select pET28a vector as the basic backbone, select restriction enzymes Xho I and Hind III to double digest pET28a vector, and linearized vector was obtained by gel recovery.

[0035] Using the short Penicillium genome as a template, PCR was performed with P1 / P2 and P3 / P4 primers to obtain two homologous arms 337HR and 337HL. The homologous arms of the restriction enzyme digestion sites of the plasmid pET28a vector were introduced at both ends of the primers P1 and P4.

[0036] Using the laboratory preserved plasmid as a template, PCR was performed with P5 / P6 primers to obtain hyg B Sequence fragments.

[0037] Using the Aspergillus nidulans genome as a template, PCR was performed with P7 / P8 primers to obtain the gene sequence fragment of PgpdA.

[0038] The reaction system of cloning PCR was: 2 × Phanta Max Master Mix 25 μL, ddH2O 20 μL, template 1 μL (50 ng), and upstream and downstream primers 2 μL each.

[0039] PCR reaction conditions: 95°C pre-denaturation 3 min; 35 cycles of 95°C denaturation 20 s, 55°C annealing 20 s, 72°C extension according to 1 Kb / 30 s rate setting; and final 72°C incubation 10 min. After the PCR amplification reaction, 1% agarose gel electrophoresis was used to detect the PCR products.

[0040] All PCR products and linearized vector pET28a were recovered by lipid sugar gel.

[0041] The PCR fragments 337HR, hyg B , PgpdA, 337HL were subjected to homologous recombination with the linearized pET28a vector: 1 μL of each of the four fragments cloned above, 5 μL of homologous recombination enzyme 2 x ClonExpress Mix, 1 μL of ddH2O, 50°C, 15 min.

[0042] 2) The recombination product was transformed as follows: DH5α chemically competent cells were taken out of the -80°C freezer, and placed on ice for thawing 10 μL of the recombination product was pipetted into the DH5α competent cells, and left on ice for 30 min.

[0043] After 42°C water bath heat shock for 45 s, immediately place on ice for 2-3 min.

[0044] Add 900 μL of LB liquid medium (without adding antibiotics), and incubate at 37°C for 1 h (rotation speed 200-250 rpm ).

[0045] 5,000 rpm centrifugation for 3 min, discard 900 μL supernatant. Resuspend the bacterial cells with 100 μL of medium, and spread on LB solid plates containing kanamycin resistance (working concentration 50 μg / mL).

[0046] Invert and incubate in a 37°C incubator for 12-16 h.

[0047] After overnight, pick the transformants for PCR and sequencing verification, and obtain the recombinant plasmid pET28a- 337HL-hyg B-PgpdA- 337HR (refer to Figure 1 ).

[0048] Example 4: PEG-mediated protoplast transformation 1) Transformation fragment acquisition The constructed recombinant plasmid pET28a- 337HL-hygB-PgpdA-337HR was used as a template, and the following two pairs of primers were used to obtain transformation fragments by PCR, P1: 5'-ggtggtggtggtggtTTCGCCTTGTCCAGCTC-3' P9: 5'-gtcgagaagtttctgatcg-3' P10: 5'- tggggcgtcggtttccac -3' P4: 5'-tcgagctccgtcgacACCGGGTCTCCAGCGTAC-3' PCR reaction conditions: 95°C pre-denaturation 5 min; 35 cycles of 94°C denaturation for 20 s, 55°C annealing for 20 s, 72°C extension for 1 Kb / 30 s; final 72°C incubation for 10 min, and 4°C preservation after reaction.

[0049] After termination of the PCR reaction, 1% agarose gel electrophoresis was performed to detect the transformed fragments, which were purified by an agarose gel recovery kit.

[0050] 2) Transformation 1. The transformation operation was performed on ice. The volume of DNA in each transformation system was not more than 10 μL, and the total amount of DNA was not less than 10 μg.

[0051] 2. 150 μL of protoplasts were taken and 10 μL of DNA fragments were added. After gentle mixing, the mixture was placed on ice for 30 min.

[0052] 3. 1 mL of room temperature solution II (60% PEG 6000, 0.8 M NaCl, 50 mM CaCl2, 50 mM Tris-HCl, pH 7.5) was added, and the mixture was gently mixed and placed at room temperature for 30 min.

[0053] 4. 7 mL of pre-warmed 47°C PDAS soft agar (PDB + 1 M sorbitol + 0.75% agar) was added, and the mixture was gently mixed and spread on a PDAS (PDB + 1 M sorbitol + 1.5% agar) plate. After solidification, the plate was incubated at 28°C.

[0054] 5. After 5-10 days of incubation, the transformants were visible and were picked and transferred to PDA medium containing the corresponding antibiotic for subculture to obtain stable traits.

[0055] Example 5: Obtaining of recombinant genetically engineered bacteria After the transformants grew into mature spores, they were cultured in liquid PDB medium, and the genome of the above-mentioned transformants with stable traits was extracted and verified by PCR.

[0056] Primer required for PCR reaction: P11: 5’-CATTCTCAGAAATGGTGGC-3’ P9: 5’-gtcgagaagtttctgatcg-3’ P10: 5’- tggggcgtcggtttccac -3’ P12: 5’- TAGGTAGACACTCCCTGG -3’ PCR reaction system is as follows: 95℃ pre-denaturation 5 min; 35 cycles of 94℃ denaturation 20 s, 65℃ annealing 20 s, 72℃, extension time set according to 1Kb / 30 s rate. Finally 72℃ for 10 min, after the reaction 4℃ preservation.

[0057] P1 fragment is obtained by PCR with primers P11 / P9; P2 fragment is obtained by PCR with primers P10 / P12; P3 fragment is obtained by PCR with primers P11 / P12. Figure 3 ).

[0058] Investigation results: PCR products are subjected to agarose electrophoresis, and the results are shown in Figure 3 Compared with the control group, the size of the segment PCR band of the strain conforms to the expected results. The insertion of the resistance gene hyg B and the promoter PgpdA, theoretically, the PCR band size of the mutant is 7841 bp, and the PCR band size of the control group is 3142 bp, and the PCR result conforms to the expected result. At the same time, the PCR products are sequenced, and the insertion of the resistance gene hyg B and the promoter PgpdA is further determined. This shows that the promoter PgpdA of the 3-phosphoglycerate dehydrogenase of SEQ ID NO: 1 is successfully used to replace the original promoter of the peroxisome biogenesis protein PbPex337 (SEQ ID NO: 2) gene of the penicillium strain, and the obtained engineering strain is named Pb M2.

[0059] Example 6: Engineering bacteria Pb M2 shake flask fermentation for producing mycophenolic acid and detection 1. Starting strain: Pb M2 2. Shake flask seed culture: Shake flask seed culture medium: PDB medium Cultivation conditions: 250 mL triangular flask, liquid volume 100 mL, cultivation temperature 25℃, shaking bed rotation speed 200 rpm / min, cultivation time 72 hours.

[0060] 3. Fermentation culture: Fermentation medium: PDB medium Inoculation: The secondary seed liquid was inoculated into the fermentation medium at an inoculation amount of 15%.

[0061] Culture conditions: 250 mL triangular flask, liquid volume 100 mL, culture temperature 25°C, shaking speed 200 rpm / min, culture time 10 days.

[0062] 4. Mycophenolic acid yield detection: Detection sample processing: The fermentation culture was taken and the mycelium and fermentation liquid were separated by a suction filter bottle. The mycelium was collected and dried in an oven. 2 mg of chloramphenicol was added to the fermentation liquid as an internal standard, and an equal volume of ethyl acetate was added for extraction twice. 50 mL of the upper organic phase was accurately taken and the organic phase was removed by rotary evaporation. 2 mL of methanol was added to dissolve the fermentation product, and an appropriate amount was taken for high performance liquid chromatography (HPLC) detection.

[0063] High performance liquid chromatography (HPLC) detection procedure: HPCL detection selected Agilent 1260, reverse phase chromatographic column YMC-Triart C18 (250 x 4.6 mm, 5 μm), mobile phase was water (A, containing 0.1% formic acid) and acetonitrile (B phase, containing 0.1% formic acid), detection procedure was 0-3 min, 2% B; 3-23 min, 2-100% B; 23-28 min, 100% B; 28-28.5 min, 2% B; 28.5-35 min, 2% B. Agilent OpenLab was used for data analysis.

[0064] Investigation results: The engineered strain Pb M2 and the control wild strain Pb WT were monitored for mycophenolic acid yield Figure 4 (A) and growth curve Figure 5 (mycelium dry weight) for 10 days of continuous culture. Compared with the control wild strain Pb WT, the engineered strain Pb M2 reached the highest mycophenolic acid yield of 0.94 g / L at the 6th day, which was 22% higher than that of the wild strain Pb WT (mycophenolic acid yield of 0.77 g / L at the 6th day) Figure 4 (B). At the same time, the growth curves of the engineered strain Pb M2 and Pb WT were basically the same, which also indicated that the normal growth of the engineered strain Pb M2 was not affected.

[0065] The present application is not limited to the above-described embodiments, but covers all changes and modifications which come within the spirit and scope of the present application. Such changes and modifications are intended to be included within the scope of the appended claims, and it is intended to seek a fair scope of equivalents based on the appended claims.

Claims

1. A method for constructing a Penicillium strain that produces high levels of mycophenolic acids, characterized in that, The method involves using the promoter PgpdA of glycerol-3-phosphate dehydrogenase, with the amino acid sequence SEQ ID NO:1, to replace the original promoter of the peroxisome biogener protein PbPex337 of Penicillium strain.

2. The method as described in claim 1, characterized in that, The promoter PgpdA of the 3-phosphoglycerate dehydrogenase also includes a derivative promoter formed by substituting or deleting one or more nucleosides based on the nucleic acid with the nucleotide sequence SEQ ID NO:

1.

3. The method as described in claim 1, characterized in that, The peroxisome biogener protein PbPex337 has the amino acid sequence SEQ ID NO:

2.

4. The method as described in claim 1, characterized in that, The method further includes replacing the promoter of any enzyme involved in peroxisome development and proliferation in the strain with the promoter PgpdA of glycerol-3-phosphate dehydrogenase.

5. The method as described in claim 1, characterized in that, The method described involves replacing the original promoter of the peroxisome biogener with the promoter PgpdA of glycerol-3-phosphate dehydrogenase using homologous recombination.

6. The method as described in claim 5, characterized in that, The recombinant expression vector used for homologous recombination in the method comprises homologous arms with nucleotide sequences SEQ ID NO: 3 and SEQ ID NO: 4, a promoter PgpdA for glycerol phosphate dehydrogenase, and an antibiotic resistance selection marker.

7. The method as described in claim 6, characterized in that, The selection marker is the hygromycin gene, which is an anti-resistance selection marker.

8. A genetically engineered strain for high-yield production of the immunosuppressant mycophenolic acid, characterized in that, The genetically engineered strain is constructed using the method described in claim 1.

9. The genetically engineered strain as described in claim 8, characterized in that, The genetically engineered strain It is a short-dense Penicillium.

10. A method for preparing mycophenolic acid by fermentation, characterized in that, The method described herein is to use the genetically engineered strain of claim 8 to ferment and prepare mycophenolic acid.

Images