Genetically engineered bacterium of high-yield immunosuppressant mycophenolic acid as well as construction method and application of genetically engineered bacterium

Abstract

The invention provides a genetic engineering strain of high-yield immunosuppressant mycophenolic acid as well as a preparation method and application of the genetic engineering strain. The invention firstly provides a method for constructing the penicillium with the effect of high yield of mycophenolic acid. The method comprises the step of replacing an indigenous promoter of acyl-coenzyme A oxidase PbACOX323 protein participating in beta oxidation in peroxisome of a penicillium strain with a promoter PgpdA of 3-phosphoglycerol dehydrogenase with an amino acid sequence of SEQ ID NO: 1. According to the method disclosed by the invention, the genetically engineered bacterium with high yield of mycophenolic acid is constructed, the yield of mycophenolic acid is positively increased by replacing a promoter of acyl-coenzyme A oxidase PbACOX323, and the method has a good industrial application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biosynthesis of immunosuppressant mycophenolic acid, and particularly relates to a genetically engineered bacterium for high-yield production of immunosuppressant mycophenolic acid, a construction method thereof and application thereof. BACKGROUND

[0002] Mycophenolic acid (MPA for short) is a kind of polyketide produced by fungal strains such as Penicillium brevicompactum (Pb), Penicillium roqueforti (Pr) and Penicillium chrysogenum (Pc), and is one of the most widely used immunosuppressants, which is used to reduce immune rejection after organ transplantation, treat autoimmune diseases and the like. Penicillium brevicompactum The synthesis strategy of mycophenolic acid includes chemical synthesis and biosynthesis. In the field of chemical synthesis, although 12 chemical synthesis paths have been designed and developed by organic chemists, due to the unique aromatic ring six-substituted chemical structure, the main application defects are long chemical synthesis process and low yield, which are difficult to realize industrial application. Therefore, the production of mycophenolic acid through fungal liquid submerged fermentation is the main way for the industrial production of mycophenolic acid at present, which also makes the production method of mycophenolic acid and the construction and mining of high-yield strains attract high attention in the industry.

[0003] The biosynthesis based on fungal liquid submerged fermentation is the main method for the production of mycophenolic acid at present, and the strains currently applied to the production of mycophenolic acid in industry are mainly Penicillium brevicompactum (Pb) and Penicillium roqueforti (Pr), but the efficiency of the fermentation for preparing mycophenolic acid is not ideal.

[0004] The biosynthesis based on fungal liquid submerged fermentation is the main method for the production of mycophenolic acid at present, and the strains currently applied to the production of mycophenolic acid in industry are mainly Penicillium brevicompactum (Pb) and Penicillium roqueforti (Pr), but the efficiency of the fermentation for preparing mycophenolic acid is not ideal. Penicillium brevicompactum SUMMARY Penicillium roqueforti The present application provides a genetically engineered strain for high-yield production of immunosuppressant mycophenolic acid and a preparation method and application thereof, which can be used for efficient fermentation for preparing mycophenolic acid. The present application provides a genetically engineered strain for high-yield production of immunosuppressant mycophenolic acid and a preparation method and application thereof, which can be used for efficient fermentation for preparing mycophenolic acid. The present application provides a genetically engineered strain for high-yield production of immunosuppressant mycophenolic acid and a preparation method and application thereof, which can be used for efficient fermentation for preparing mycophenolic acid.

[0005] The present application provides a genetically engineered strain for high-yield production of immunosuppressant mycophenolic acid and a preparation method and application thereof, which can be used for efficient fermentation for preparing mycophenolic acid. The present application provides a genetically engineered strain for high-yield production of immunosuppressant mycophenolic acid and a preparation method and application thereof, which can be used for efficient fermentation for preparing mycophenolic acid.

[0006] The present application provides a genetically engineered strain for high-yield production of immunosuppressant mycophenolic acid and a preparation method and application thereof, which can be used for efficient fermentation for preparing mycophenolic acid. The present application provides a genetically engineered strain for high-yield production of immunosuppressant mycophenolic acid and a preparation method and application thereof, which can be used for efficient fermentation for preparing mycophenolic acid. The present application provides a genetically engineered strain for high-yield production of immunosuppressant mycophenolic acid and a preparation method and application thereof, which can be used for efficient fermentation for preparing mycophenolic acid. As a specific embodiment, the acyl-CoA oxidase in the peroxisome, PbACOX323, has an amino acid sequence of SEQ ID NO: 2. Further, the method also uses the promoter PgpdA of glycerol-3-phosphate dehydrogenase to replace the promoter of other proteins in the peroxisome of the Penicillium strain, except for the acyl-CoA oxidase, which is involved in beta oxidation.

[0007] As a specific embodiment, the promoter is replaced by homologous recombination.

[0008] More specifically, the recombination expression vector used for homologous recombination comprises the nucleotide sequence of SEQ ID NO: 3, the homologous arms of SEQ ID NO: 4, the promoter PgpdA of glycerol-3-phosphate dehydrogenase with the nucleotide sequence of SEQ ID NO: 1, and a selection marker. As a specific embodiment, the selection marker is the resistance selection marker hygromycin gene (hygB). hyg B (SEQ ID NO: 5).

[0009] Further, the genetically engineered strain is Penicillium brevicompactum.

[0010] The present application also provides a method for preparing mycophenolic acid by fermentation, which uses the genetically engineered strain constructed by the present application to prepare mycophenolic acid by fermentation.

[0011] The method of the present application constructs a genetically engineered strain of high-yield mycophenolic acid by replacing the promoter of acyl-CoA oxidase PbACOX323, thereby actively increasing the yield of mycophenolic acid, and has good industrial application value. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 : Recombinant plasmid pET28a- HL-hyg B-PgpdA-HR schematic diagram; Figure 2 : Split-marker technology for constructing an engineered strain schematic diagram; Figure 3 : Picture of PCR verification of the transformed nucleic acid; Figure 4 : Fermentation yield verification data graph of the engineered strain and the original strain; Figure 5 : Fermentation growth curve data graph of the engineered strain and the original strain. DETAILED DESCRIPTION

[0013] The application is described below through specific implementation cases. Unless specifically stated, the technical means used in the application are methods known to those skilled in the art. In addition, the implementation solutions should be understood as illustrative rather than limiting the scope of the application, and the essence and scope of the application are limited only by the claims.

[0014] The application provides a genetically engineered strain for high-yield mycophenolic acid, which is mainly characterized in that the engineered strain takes Penicillium brevi-compactum as a chassis cell, and the expression of acyl-CoA oxidase PbACOX323 gene is started by a promoter PgpdA derived from 3-phosphoglycerate dehydrogenase of Aspergillus nidulans instead of an original promoter.

[0015] Among them, the acyl-CoA oxidase PbACOX323 gene and the two homologous arms (HR, HL) required for integration are derived from the host cell Pb 864 genome.

[0016] Among them, the resistance screening marker hygromycin gene (hygB) is from the pET28a- hyg B ) plasmid preserved in the laboratory. hyg B

[0017] Example 1: Construction of recombinant plasmid pET28a- HL-hygB-PgpdA-HR According to the gene sequence of PgpdA from Aspergillus nidulans published by Genebank, the primers are designed and synthesized: P1: 5'-aacgtcggcggccgcGAATTCCCTTGTATCTCTAC-3', P2: 5'-cgttatgaaccccgggACTAGTGTCTGCTCAAGCGGGGTA-3'; According to the short compactum genome sequence obtained by sequencing in the previous laboratory, the homologous arm gene sequence (HL: SEQ ID NO: 3, HR: SEQ ID NO: 4) required for homologous recombination is obtained, and the primers are designed and synthesized, wherein the sequence information of the primers is as follows: P3: 5'-ggtggtggtggtggtATGTCCCCAGACATACAT-3', P4: 5'-cctgggttgctcgagGTAAAGGTGACGTTTCCCGT-3'; P5: 5'-CCCGGGGTTCA TAACTG-3' P6: 5'-TCAATTGACGAGAAGCGG-3' According to the hygromycin resistance vector contained in the laboratory hyg B sequence (​hyg B : SEQ ID NO.5 ), the primers were designed and synthesized: P7: 5'- ctcgagcaacccaggggctg-3', P8: 5'- gcggccgccgacgttaactg-3'.

[0018] The homologous arms of the plasmid pET28a vector were introduced at both ends of the primers P3 and P6, and the pET28a vector was cut by Xho I and Hind III as the enzyme cutting site, and the recombinant vector was obtained by homologous recombination.

[0019] The reaction system of the cloning PCR was as follows: 2 × Phanta Max Master Mix 25 μL, ddH2O 20 μL, template 1 μL, and upstream and downstream primers 2 μL each. The PCR reaction conditions were as follows: 95℃ pre-denaturation for 3 min; 35 cycles of 95℃ denaturation for 20 s, 55℃ annealing for 20 s, and 72℃ extension for 1 Kb / 30 s; finally, 72℃ incubation for 10 min. After the reaction, 1% agarose gel electrophoresis detection was performed.

[0020] Then, the PCR fragments HR, hyg B , PgpdA, and HL were subjected to homologous recombination with the linearized pET28a vector by the following method: 1 μL of each of the four fragments cloned above, 5 μL of homologous recombination enzyme 2 × ClonExpress Mix, and 1 μL of ddH2O were mixed, and the mixture was incubated at 50℃ for 15 min.

[0021] Then, the recombinant product was transformed by the following method: The DH5α chemically competent cells used for cloning were thawed on ice 10 μL of the recombinant product was added to 100 μL of the competent cells, and the mixture was incubated on ice for 30 min.

[0022] After 42℃ water bath heat shock for 45 s, the mixture was immediately placed on ice for cooling for 2-3 min.

[0023] 900 μL of LB liquid medium (without adding antibiotics) was added, and the mixture was incubated at 37℃ for 1 h (rotation speed 200-250 rpm).

[0024] The corresponding resistant LB solid medium plate was preheated in a 37℃ incubator.

[0025] Centrifugation was performed at 5,000 rpm for 5 min, and 900 μL of supernatant was discarded. The bacterial cells were resuspended with the remaining medium, and a sterile spreader was used to evenly spread the cells on the plate containing kanamycin resistance.

[0026] 37°C incubator for 12-16 h.

[0027] After overnight, pick transformants for PCR and sequencing verification, obtain recombinant plasmid pET28a- HL-hygB-PgpdA-HR Figure 1

[0028] Example 2: Preparation of Penicillium brevicompactum protoplasts The preparation of Penicillium brevicompactum protoplasts can be prepared by conventional methods, the specific steps of this embodiment are described as follows: 1) Bacterial culture Penicillium brevicompactum spores were evenly coated on potato dextrose solid medium (PDA: potato extract 4.0 g / L, glucose 20.0 g / L, agar powder 15 g / L, pH 5.1±0.2) plates, and incubated at 25°C for 5-7 days. Spores were collected 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 incubated for another 2-3 days.

[0029] 2) Protoplast preparation 1. Add 10 mg / mL lysing enzyme, 10 mg / mL lysozyme, 15 mg / mL cellulase, and 10 mg / mL lywallzyme to 12 mL 0.8 M NaCl, shake gently, filter sterilize with a 0.22 um filter into a sterile 50 mL flask.

[0030] 2. Filter the mycelium with a nylon cloth funnel, wash with 0.8 M NaCl, and then pick it into a flask containing the enzyme solution, and disperse it with a sterile syringe. Enzyme hydrolysis at 30°C, 70 rpm for 2-3 h, and take samples for microscopic observation at regular intervals.

[0031] 3. After enzyme hydrolysis, the protoplast suspension was absorbed into a 50 mL centrifuge tube, and an equal volume of 0.8 M NaCl was added, and mixed gently. Ice bath for 5 min to cool the protoplasts.

[0032] 4. Centrifuge at 2500 xg, 4°C for 5 min, remove the supernatant, and resuspend the protoplasts with 10 mL ice-cold solution I (0.8 M NaCl, 10 mM CaCl2, 50 mM Tris-HCl, pH 7.5).

[0033] ​​5. 2500 xg, 4°C for 5 min, remove supernatant, resuspend protoplasts in ice-cold solution I (1-2 mL).

[0034] Protoplast quality control (microscopic observation): count the protoplast concentration by using a hemocytometer. The concentration should be higher than 3 x 105 / mL. 7 6 c = n / f x 4 x 105, c = number / mL, n = number of protoplasts counted, f = number of small squares counted.

[0035] Example 3: PEG-mediated protoplast transformation 1) Transformation fragment preparation Using the constructed recombinant plasmid pET28a- as template, the following two pairs of primers were used to obtain the transformation fragments by PCR, HL-hygB-PgpdA-HR P3: 5'-ggtggtggtggtggtATGTCCCCAGACATACAT-3', P9: 5'-gtcgagaagtttctgatcg-3', P10: 5'-tggggcgtcggtttccac-3', P6: 5'-TCAATTGACGAGAAGCGG-3'. PCR reaction conditions: 95°C pre-denaturation for 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 then 4°C storage.

[0036] After the PCR reaction was terminated, 1% agarose gel electrophoresis was performed to detect the transformation fragments, which were purified by a gel recovery kit.

[0037] 2) Transformation

[0038] 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. 2. Take 120 μL of protoplasts and add 10 μL of DNA fragments. Mix gently and place on ice for 2 min.

[0039] 3. Add 1 mL of room temperature solution II (60% PEG 6000, 0.8 M NaCl, 50 mM CaCl2, 50 mM Tris-HCl, pH 7.5), mix gently, and incubate at room temperature for 30 min.

[0040] 3. Add 1 mL of room temperature solution II (60% PEG 6000, 0.8 M NaCl, 50 mM CaCl2, 50 mM Tris-HCl, pH 7.5), mix gently, and incubate at room temperature for 30 min.​

[0041] 4. Add 7 mL pre-warmed 47°C PDAS soft agar (PDB + 1 M sorbitol + 0.75% agar), mix gently, and spread on PDAS (PDB + 1 M sorbitol + 1.5% agar) plate. After solidification, place at 28°C for culture.

[0042] 5. After 5-10 days of culture, the transformants grow out, and the transformants are picked to PDA medium plates containing the corresponding antibiotics for subculture to stable traits.

[0043] 6. After the transformants grow out and mature spores are formed, the transformants are cultured in liquid PDB medium, the genome of the above stable transformants is extracted, and PCR verification is performed to obtain correct transformants.

[0044] Example 4: Obtain recombinant genetically engineered bacteria 1. Inoculate an appropriate amount of Penicillium brevicompactum spores into 600 μL PDB medium, 200 rpm, 30°C for 24 hours.

[0045] 2. Centrifuge at 12000 rpm, 4°C for 10 min, discard the supernatant, and collect the bacterial cells.

[0046] 3. Add an appropriate amount of quartz sand and 500 μL extraction buffer, vortex for 1 min, and then water bath at 65°C for 10 min.

[0047] 4. Add 200 μL 7.5 M ammonium acetate solution, mix well, and ice bath for 8 min.

[0048] 5. Centrifuge at 12000 rpm, 4°C for 10 min.

[0049] 6. Transfer the supernatant to a new 1.5 mL centrifuge tube, add 0.5-0.6 times the volume of ice-cold isopropanol, mix well, and place at -20°C for 10 min.

[0050] 7. Centrifuge at 12000 rpm, 4°C for 10 min, and discard the supernatant.

[0051] 8. Add 500 μL 75% ethanol, mix well, and centrifuge at 7,500 rpm for 5 min.

[0052] 9. Discard the supernatant and try to absorb the residual liquid, and place at room temperature for 10 min.

[0053] 10. Add 30 μL sterile water to dissolve.

[0054] The PCR reaction system is as follows: 95℃ pre-denaturation 5 min; 35 cycles of 94℃ denaturation 20 s, 65℃ annealing 20 s, 72℃ extension according to the setting of 1 Kb / 30 s rate. Final 72℃ incubation for 10 min, and 4℃ preservation after reaction.

[0055] The PCR products were subjected to agarose electrophoresis, and the results are shown in Figure 3 Compared with the control group, the PCR band sizes of the two mutant strains (mutant 1 and mutant 2) were obviously larger than that of the wild type. The insertion of the resistance gene hyg B and the promoter PgpdA theoretically resulted in a PCR band size of 7226 bp for the mutant and a PCR band size of 3000 bp for the control group, and the PCR results were consistent with the expected results. Meanwhile, the PCR products were sequenced to further confirm the insertion of the resistance gene hyg B and the promoter PgpdA. This indicates that the endogenous promoter of the acyl-coenzyme A oxidase PbACOX323 protein involved in β oxidation in the peroxisome of the Penicillium strain is successfully replaced by the promoter PgpdA of the 3-phosphoglycerate dehydrogenase of SEQ ID NO: 1, and the obtained engineering strain is named Pb M1.

[0056] Example 5: Engineering strain Pb M1 shake flask fermentation for producing mycophenolic acid and detection 1. Starting strain: Pb M1 2. Shake flask seed culture: Shake flask seed culture medium: PDB medium Culture conditions: 250 mL flask, liquid volume 100 mL, culture temperature 25℃, shaking speed 200 rpm / min, culture time 72 hours.

[0057] 3. Fermentation culture: Fermentation medium: PDB medium Inoculation: according to the inoculation amount of 15%, the secondary seed liquid was inoculated into the fermentation medium.

[0058] Culture conditions: 250 mL flask, liquid volume 100 mL, culture temperature 25℃, shaking speed 200 rpm / min, culture time 10 days.

[0059] 4. Mycophenolic acid yield detection: Detection sample processing: Take the fermentation culture, separate the bacteria and the fermentation broth with a suction filter bottle, collect the bacteria and dry them in an oven. Add 2 mg of chloramphenicol to the fermentation broth as an internal standard, add an equal volume of ethyl acetate, and extract twice. Accurately pipette 50 mL of the upper organic phase, and remove the organic phase by rotary evaporation. Add 2 mL of methanol to dissolve the fermentation product, and take an appropriate amount for high-performance liquid chromatography (HPLC) detection.

[0060] High-performance liquid chromatography (HPLC) detection procedure: HPCL detection uses Agilent 1260, reverse-phase chromatographic column YMC-Triart C18 (250 x 4.6 mm, 5 μm), mobile phase is water (A, containing 0.1% formic acid) and acetonitrile (B phase, containing 0.1% formic acid), detection procedure is 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 is used for data analysis.

[0061] The engineered strain Pb M1 and the control wild-type strain Pb WT was continuously cultured for 10 days, and the mycophenolic acid yield Figure 4 A) and the growth curve Figure 5 , the dry weight of the bacteria, were monitored. Compared with the control wild-type strain Pb WT, the engineered strain Pb M1 reached the highest mycophenolic acid yield of 0.85 g / L at day 6, which was 10.3% higher than that of the wild-type strain Pb WT (mycophenolic acid yield of 0.77 g / L at day 6) Figure 4 B). At the same time, the growth curves of the engineered strain Pb M1 and Pb WT were basically the same, which also indicated that the normal growth of the engineered strain Pb M1 was not affected.

[0062] The present application is not limited to the above-described embodiments, but covers all changes and modifications within the spirit and scope of the present application. These changes and modifications should not be considered to be outside the spirit and scope of the present application, and all modifications such as those apparent to those skilled in the art should be included within the scope of the appended claims.

Claims

1. A method for constructing a Penicillium with high mycophenolic acid production capacity, characterized in that, The method involves using the promoter PgpdA of 3-phosphoglycerate dehydrogenase with the amino acid sequence SEQ ID NO:1 to replace the original promoter of the acyl-CoA oxidase PbACOX323 protein involved in β-oxidation in the peroxisomal of Penicillium strains.

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 the substitution or deletion of one or more nucleosides on the nucleic acid with the nucleotide sequence SEQ ID NO:

1.

3. The method as described in claim 1, characterized in that, The amino acid sequence of the acyl-CoA oxidase PbACOX323 is SEQ ID NO:

2.

4. The method as described in claim 1, characterized in that, The method also uses the promoter PgpdA of glycerol 3-phosphate dehydrogenase to replace the promoters of other proteases involved in β-oxidation in the peroxisome of Penicillium strains, except for acyl-CoA oxidase.

5. The method as described in claim 1, characterized in that, The replacement of the original promoter of the acyl-CoA oxidase PbACOX323 protein involved in β-oxidation in the peroxisomal of Penicillium strains with the promoter PgpdA of 3-phosphoglycerate dehydrogenase with the amino acid sequence SEQ ID NO:1 is achieved by homologous recombination.

6. The method as described in claim 5, characterized in that, The homologous recombination expression vector used in the above homologous recombination includes homologous arms with nucleotide sequences SEQ ID NO: 3 and SEQ ID NO: 4, a promoter PgpdA of 3-glycerol phosphate dehydrogenase with nucleotide sequence SEQ ID NO: 1, and a 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 is Penicillium breviculae.

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 as a host strain to ferment and prepare mycophenolic acid.

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