Penicillium citrinum strain for producing pravastatin as well as construction method and application of penicillium citrinum strain

Abstract

The invention relates to a Penicillium citrinum strain for producing pravastatin and a construction method of the Penicillium citrinum strain. The penicillium citrinum strain for producing the pravastatin through one-step fermentation is constructed by performing metabolic engineering transformation and heterologous expression of a P450 enzyme catalysis module on the penicillium citrinum, and in order to relieve insufficient reducing power supply and overexpression of NADPH related genes, reinforce intracellular reducing power supply and further improve the yield of the pravastatin. As a mevastatin industrial production strain, the penicillium citrinum is mature in fermentation process, higher in yield, single in product component, few in impurity, clear in information and beneficial to industrial application, so that a cell factory for producing pravastatin through one-step fermentation, which is constructed by taking the penicillium citrinum as a chassis cell, has an important application value.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and more particularly relates to a pravastatin-producing Penicillium citrinum strain and a construction method and application thereof. Background Art

[0002] Cardiovascular disease is a major disease that threatens human health. For many years, it has been ranked first among the causes of death from diseases, far higher than tumors and other diseases. Dyslipidemia is a common cause of cardiovascular disease. Controlling blood lipid levels can effectively prevent secondary cardiovascular diseases. Lipid-lowering drugs are currently the main means of treating hyperlipidemia, so they have great market prospects. Statins are a class of HMG-CoA reductase inhibitors that can inhibit the synthesis of cholesterol in the body to lower blood lipid levels, accounting for about 80% of the entire lipid-lowering drug market.

[0003] Pravastatin is one of the most commonly used statin lipid-lowering drugs. Its good water solubility, special pharmacological characteristics, and low side effects make it always competitive, with annual global sales of about US$1 billion. In industry, the production of pravastatin requires two fermentations. The first is the fermentation of Penicillium citrinum to produce mevastatin. After separation, purification and appropriate pretreatment, mevastatin is fermented by Streptomyces carbophilus for the second time, and pravastatin is catalyzed by P450 enzymes. Since the substrate mevastatin is toxic to cells, it needs to be slowly fed during the secondary fermentation process; in addition, the transmembrane transport efficiency between the substrate and the product pravastatin is low, and the conversion rate is only 70%. This makes the entire production process of pravastatin complicated, inefficient, and costly, especially the second step of fermentation by Streptomyces carbophilus, which accounts for a large part of the total fermentation cost.

[0004] DSM of the Netherlands used Penicillium chrysogenum as the chassis cell, heterologously integrated the Penicillium citrinum mevastatin biosynthetic gene cluster to achieve mevastatin synthesis, and then heterologously reconstructed the P450 enzyme catalytic module to achieve the direct synthesis of pravastatin.

[0005] However, the chrysogenycin-producing Penicillium strains are rich in β-lactamase, which can hydrolyze the 2-methylbutyric acid side chains of mevastatin and pravastatin to produce impurities without side chains. Even after knocking out the main β-lactamase gene, a certain amount of unknown impurities still remain in the fermentation product, which is not conducive to the subsequent separation and purification of the product, resulting in high production costs and failure to achieve industrialization.

[0006] The Teva Pharmaceutical Factory in Hungary disclosed a method for constructing a strain with the ability to hydroxylate mevastatin (publication number CN 101076595A), in which cytochrome P450 and ferredoxin were transferred into Streptomyces lividans to obtain an engineered strain capable of producing pravastatin using mevastatin as a substrate. However, it still failed to construct a strain capable of synthesizing pravastatin in one step.

[0007] Therefore, the present invention uses the mevastatin industrial strain with clear background and few by-products as the chassis, introduces heterologous mevastatin P450 enzyme, strengthens the reducing power supply of the bacteria, and constructs a one-step efficient synthesis of pravastatin strain.

[0008] Microbial Deposit

[0009] The starting strain Penicillium citrinum MEFC10-1 used in the present invention is deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.40531 and a deposit date of March 23, 2023. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355. Summary of the invention

[0010] During our research, we found that after expressing cytochrome P450 and the reducing chaperone protein RhFRed in the Penicillium citrinum strain, the Penicillium citrinum strain could produce pravastatin, and further, after overexpressing some NADPH synthesis-related genes, the production of pravastatin could be increased.

[0011] Based on the above findings, the present invention provides a method for constructing a pravastatin-producing Penicillium citrinum strain, comprising the step of introducing a cytochrome P450 gene expression cassette into the Penicillium citrinum strain.

[0012] In a specific embodiment, the amino acid sequence of cytochrome P450 in the cytochrome P450 gene expression cassette is as shown in SEQ ID NO:1.

[0013] In a specific embodiment, the method further comprises the step of introducing a reduced chaperone gene expression cassette into the Penicillium citrinum strain.

[0014] In a specific embodiment, the reduction chaperone protein is RhFRed. Preferably, the amino acid sequence of RhFRed is shown in SEQ ID NO:3.

[0015] Although the embodiments of the present invention limit specific cytochrome P450 gene sequences and reduced chaperone protein RhFRed gene sequences, in fact, as long as active cytochrome P450 and chaperone protein RhFRed can be expressed in cells, the production of pravastatin can be achieved, but the yield is different. The cytochrome P450 gene sequence and reduced chaperone protein RhFRed gene sequence in the preferred embodiments of the present invention should not be used to limit the protection scope of the present invention. All cytochrome P450 and reduced chaperone protein RhFRed that already exist or will exist in the field should be covered within the protection scope of the present invention.

[0016] In a specific embodiment, the method further comprises the step of increasing the NADPH synthesis capacity in the Penicillium citrinum strain.

[0017] In a specific embodiment, the NADPH synthesis ability of the Penicillium citrinum strain is improved by introducing a glucose-6-phosphate dehydrogenase gene expression cassette and / or a glyceraldehyde-3-phosphate dehydrogenase gene expression cassette into the Penicillium citrinum strain.

[0018] In a specific embodiment, the amino acid sequence of glucose-6-phosphate dehydrogenase in the glucose-6-phosphate dehydrogenase gene expression cassette is as shown in SEQ ID NO: 7 or 8, and the amino acid sequence of glyceraldehyde-3-phosphate dehydrogenase in the glyceraldehyde-3-phosphate dehydrogenase gene expression cassette is as shown in SEQ ID NO: 9.

[0019] The present invention also provides a pravastatin-producing Penicillium citrinum strain constructed by the above method.

[0020] The present invention also provides the use of the Penicillium citrinum strain in producing pravastatin or mevastatin.

[0021] The present invention constructs a one-step fermentation pravastatin-producing Penicillium citrinum strain by metabolic engineering transformation of Penicillium citrinum and heterologously expressing a P450 enzyme catalytic module, and in order to relieve the insufficient supply of reducing power, overexpresses NADPH-related genes, strengthens the intracellular reducing power supply, and further improves the yield of pravastatin. As an industrial production strain of mevastatin, Penicillium citrinum has a mature fermentation process, a higher yield, a single product component, few impurities and clear information, which is conducive to industrial application. Therefore, a one-step fermentation pravastatin cell factory constructed with Penicillium citrinum as a chassis cell has important application value.

[0022] It should be noted that the above gene expression cassette can directly combine the target gene with the selected promoter into a gene expression cassette during the construction of the expression cassette, or the coding sequence can be inserted downstream of the in situ promoter in the genome so that the coding sequence and the in situ promoter constitute a gene expression cassette.

[0023] In order to illustrate the effect of the present invention, we used our own preserved Penicillium citrinum MEFC10-1 as the starting strain, but the scope of the present invention should not be limited to this strain, and all strains in the genus Penicillium that have the mevastatin synthesis pathway should be covered within the scope of protection of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 PCR verification of the mutant strain MEFC10-1-Pra. Lanes 1-8: Successful insertion of the gene encoding P450 Prava -RhFRed gene transformant; Lane 9: positive control; WT: wild-type MEFC10-1.

[0025] Figure 2 The fermentation analysis of mutant strain MEFC10-1-Pra01 / 02 and wild-type MEFC10-1. A: Liquid phase spectrum of mutant strain MEFC10-1-Pra01 / 02 and wild-type MEFC10-1; B: Statistical graph of statin production in fermentation broth of mutant strain MEFC10-1-Pra01 / 02 and wild-type MEFC10-1.

[0026] Figure 3 This is the time curve of reducing power level of mutant strains MEFC10-1-Pra01 / 02 and MEFC10-1 during fermentation.

[0027] Figure 4 This is the PCR verification of the mutant strain MEFC10-1-Pra02-gene. A: verification of overexpression me1 / 2 transformants; B: verification of overexpression pgd transformants; C: verification of overexpression gapd1 transformants; D: verification of overexpression g6pd2 transformants; E: verification of overexpression g6pd3 transformants; WT: wild-type MEFC10-1-Pra02.

[0028] Figure 5 Results of statin production analysis in the fermentation broth of mutant strains MEFC10-1-Pra02-gene and MEFC10-1-Pra02. A: statistic of pravastatin production in gapd1 overexpression strain; B: statistic of pravastatin production in g6pd2 overexpression strain; C: statistic of pravastatin production in g6pd3 overexpression strain. * represents P < 0.05, ** represents P < 0.01. DETAILED DESCRIPTION

[0029] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0030] Example 1 Mutant strain MEFC10-1-Pra

[0031] 1. Construction of the mutant strain MEFC10-1-Pra

[0032] 1) Starting strain

[0033] In this embodiment, the starting strain used is Penicillium fungus, Penicillium citrinum MEFC10-1, which is deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.40531 and a deposit date of March 23, 2023. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355.

[0034] 2) Construction of overexpression fragments of cytochrome P450 and reduced chaperone protein RhFRed

[0035] The cytochrome P450 used in the present invention is a mutant of P450CYP105AS1 derived from Amycolatopsis orientalis DSM40040 (the amino acid sequence is shown in SEQ ID NO: 1, and the nucleic acid sequence is shown in SEQ ID NO: 2), and the reduced chaperone protein RhFRed used is derived from Rhodococcus sp. (the amino acid sequence is shown in SEQ ID NO: 3, and the nucleic acid sequence is shown in SEQ ID NO: 4).

[0036] The above target genes were connected to the downstream of promoters PgpdAt and TtrpC respectively to obtain the expression fragment PgpdAt-P450 of cytochrome P450 and reduced chaperone protein RhFRed. Prava -RhFred-TtrpC.

[0037] 3) Protoplast transformation

[0038] Using Penicillium citrinum MEFC10-1 as the starting bacteria, a small amount of hyphae was first taken from the PDA plate and the nucleic acid was extracted using a nucleic acid extraction instrument. After crushing, take 1mL of seed liquid and inoculate it into 50mL of seed culture medium, and culture it in a 250mL Erlenmeyer flask at 220rpm and 25℃. After 36h, filter and collect the mycelium through a 500-mesh sterile filter cloth. Wash the mycelium 3 times with 0.6M MgCl2. Weigh 1g of mycelium, add 10mL of enzymatic solution, and treat at 30℃ and 130rpm for 2-3h until a large number of protoplasts are observed. The enzymatic solution consists of: 1% lytic enzyme, 0.5% snail enzyme, 0.6M MgCl2, and sterilizes by filtration through a 0.22μm sterile filter. Filter the above protoplast reaction solution through a sterile filter cloth, and collect the protoplasts by centrifugation at 3800rpm and 4℃. Wash once with precooled STC, and resuspend the protoplasts in precooled STC to obtain a protoplast suspension.

[0039] Mix PgpdAt-P450 in a molar ratio of 5:1 Prava -RhFred-TtrpC and resistance fragment (hph or neo) fragment, take 10 μL and add it to 200 μL of the above protoplast suspension, then add 10 μL of pre-cooled PSTC, mix gently, and place on ice for 25 minutes. Then, add 1 mL of PSTC, mix well and place at room temperature for 20 minutes. Finally, mix with 20 mL of top agar and pour on 6 regeneration screening medium plates PDAS-H / PDAS-HG, culture at 25 ° C for 10 days to obtain transformants. Pick transformants with hygromycin / geneticin resistance from the transformation screening plate for single spore separation and purification, and extract the transformant genome after single spore separation for PCR verification.

[0040] After two protoplast transformations, Figure 1 As shown in Figure 2, these transformants were able to amplify a band of about 3.7 kb, while MEFC10-1 could not amplify a band, indicating that they were all positive transformants, indicating that the gene encoding P450 Prava -RhFRed gene expression fragment has been successfully inserted into the Penicillium citrinum MEFC10-1 strain. Positive mutants MEFC10-1-Pra01 and MEFC10-1-Pra02 were selected for the next experiment.

[0041] 2. Fermentation verification of mutant strain MEFC10-1-Pra

[0042] The positive mutant strain MEFC10-1-Pra01 / 02 and the control strain Penicillium citrinum MEFC10-1 were inoculated on the screening medium PDA-H / PDA-HG and PDA plate, respectively, and cultured at 25°C for 5 days. A small amount of hyphae was picked and extracted using a nucleic acid extraction instrument. The mycelium was crushed and inoculated into 20 mL of seed culture medium, and cultured at 25°C and 220 rpm for 48 h. 2 mL of the above cultured seed solution was transferred into 20 mL of fermentation medium, and cultured on a shaker at 25°C and 220 rpm for 9 days, with 3 parallels for each strain. 1 mL of fermentation broth was added with 5 mL of methanol (containing 0.02 M NaOH), and the mixture was extracted by oscillation for 2 h, allowed to stand, and the supernatant was filtered through a 0.22 μm filter, and the sample was analyzed by HPLC.

[0043] HPLC analysis method: liquid chromatography column is Agile Element Eclipse Plus C18, 5μm, 4.6×150mm; mobile phase A: 0.05% formic acid acetonitrile solution, mobile phase B: 0.05% formic acid aqueous solution; flow rate is 1mL / min, UV detection wavelength is 340nm, 30℃, total elution time is 20min. Gradient elution conditions: 0-5min, mobile phase A accounts for the volume of mobile phase linearly increased from 10% to 30%, 5-15min, mobile phase A accounts for the volume of mobile phase linearly increased from 30% to 100%, 15-17min, mobile phase A accounts for 100% of the volume of mobile phase, 17.1-20min, mobile phase B accounts for 90% of the volume of mobile phase.

[0044] The results are as follows Figure 2 As shown, the fermentation products of mutant strain MEFC10-1-Pra01 / 02 and wild-type strain had an obvious difference peak of pravastatin at 9.032 min, indicating that the integrated P450 Prava -RhFred gene, Penicillium citrinum has the ability to synthesize pravastatin, and its yield is 2.91±0.1 g / L. The conversion rate of this reaction is defined as the percentage of pravastatin yield to total statins, and its conversion rate is about 62%.

[0045] Example 2 NADPH reducing power rebalancing

[0046] 1) NADPH / NADP in mutant strains + Reduced content

[0047] Detection of NADPH / NADP in cells during fermentation + The results are as follows Figure 3 As shown, NADPH / NADP in mutant MEFC10-1-Pra01 / 02 + The content is significantly lower than that of the wild type, and it is speculated that NADPH / NADP + The decrease in content may affect the synthesis of pravastatin.

[0048] 2) Construction of recombinant plasmids for overexpression of NADPH-related genes

[0049] We selected 6 NADPs in our study + The related genes that are dependent on the gene include: 2 genes me1 / 2 encoding malic enzyme (the amino acid sequences are shown in SEQ ID NOs: 5 and 6, respectively), 2 genes g6pd2 / 3 encoding 6-phosphate glucose dehydrogenase (the amino acid sequences are shown in SEQ ID NOs: 7 and 8, respectively), 1 gene pgd encoding gluconate 6-phosphate dehydrogenase (the amino acid sequence is shown in SEQ ID NO: 9), and 1 gene gapd1 encoding glyceraldehyde-3-phosphate dehydrogenase (the amino acid sequence is shown in SEQ ID NO: 10), and are synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0050] The PgpdA fragment was connected to the above genes respectively into the pEASY-Blue vector to obtain 6 recombinant plasmids with NADPH-related gene overexpression elements, collectively referred to as pEASY-Blue-gene.

[0051] 3) Protoplast transformation

[0052] Using the mutant strain MEFC10-1-Pra02 as the starting bacteria, a small amount of hyphae was first taken from the PDA-HG plate and the nucleic acid was extracted using a nucleic acid extraction instrument. Crush, take 1mL of seed liquid and inoculate it into 50mL of seed culture medium, and culture it in a 250mL triangular flask at 220rpm and 25℃. After 36h, filter and collect the mycelium through a 500-mesh sterile filter cloth. Wash the mycelium 3 times with 0.6M MgCl2. Weigh 1g of mycelium, add 10mL of enzymatic solution, and treat at 30℃ and 130rpm for 2-3h until a large number of protoplasts are observed. The enzymatic solution consists of: 1% lytic enzyme, 0.5% snail enzyme, 0.6M MgCl2, and sterilize by filtration through a 0.22μm sterile filter. Filter the above protoplast reaction solution through a sterile filter cloth, and collect the protoplasts by centrifugation at 3800rpm and 4℃. Wash once with precooled STC, and resuspend the protoplasts in precooled STC to obtain a protoplast suspension.

[0053] Mix PgpdA-gene-Tpgk (gene represents me1 / 2, pgd, gapd1, g6pd2 / 3 respectively) and resistance gene (nat) fragments in a molar ratio of 5:1, take 10uL and add it to 200μL of the above protoplast suspension, then add 10μL of pre-cooled PSTC, mix gently, and place on ice for 25 minutes. Then, add 1mL PSTC, mix well and place at room temperature for 20 minutes. Finally, mix with 20mL of top agar and pour on 6 regeneration screening medium plates PDAS-HGN, culture at 25℃ for 10 days to obtain transformants. Pick the transformants with spore resistance from the transformation screening plate for single spore isolation and purification, extract the transformant genome after single spore isolation, and perform PCR verification with primers PgpdA-F and Tpgk-R. Figure 4 As shown, these transformants were able to amplify a gene with a size of approximately 3.5 kb ( Figure 4 A), 3.6kb( Figure 4 B) 3.1kb( Figure 4 C), 2.7kb( Figure 4 D) 3.3kb( Figure 4 E), a 3.3 kb band, while no band could be amplified from MEFC10-1-Pra02, indicating that they were all positive transformants, indicating that the me1 / 2, pgd, gapd1, and g6pd2 / 3 genes had been successfully inserted into the MEFC10-1-Pra02 strain, and the mutant strains MEFC10-1-Pra02-me1 / 2, MEFC10-1-Pra02-pgd, MEFC10-1-Pra02-gapd1, and MEFC10-1-Pra02-g6pd2 / 3 were obtained.

[0054] 4) Fermentation verification of mutant strain MEFC10-1-Pra02-gene

[0055] The positive mutant strain MEFC10-1-Pra02-gene (gene represents me1 / 2, pgd, gapd1, g6pd2 / 3, respectively) and the control strain MEFC10-1-Pra02 were inoculated on PDA-HGN and PDA-HG solid plates, cultured and fermented for product detection. The culture and detection methods are as described above.

[0056] The results showed that after overexpression of me1 / 2 genes, the pravastatin production of mutant strains was slightly reduced; overexpression of pgd had no significant effect on the pravastatin production of MEFC10-1-Pra02; while overexpression of gapd1 and g6pd2 / 3 significantly increased the pravastatin production of MEFC10-1-Pra02, among which the conversion rate of the mutant strain overexpressing g6pd2 could reach up to about 70%, and the pravastatin production was 5.62±0.34g / L ( Figure 5 ).

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing a pravastatin-producing Penicillium citrinum strain, characterized in that: The method comprises the steps of introducing a cytochrome P450 gene expression cassette into a Penicillium citrinum strain.

2. The method according to claim 1, characterized in that The amino acid sequence of cytochrome P450 in the cytochrome P450 gene expression cassette is shown in SEQ ID NO:

1.

3. The method according to claim 1, characterized in that The method also includes the step of introducing a gene expression cassette for the reduced chaperone protein of cytochrome P450 into the Penicillium citrinum strain.

4. The method according to claim 3, characterized in that The reducing chaperone protein is RhFRed.

5. The method according to claim 4, characterized in that The amino acid sequence of RhFRed is shown in SEQ ID NO:

3.

6. The method according to any one of claims 1 to 5, characterized in that The method also includes the step of increasing the NADPH synthesis capacity of the Penicillium citrinum strain.

7. The method according to claim 6, characterized in that The NADPH synthesis ability of the Penicillium citrinum strain is improved by introducing a 6-phosphate glucose dehydrogenase gene expression cassette and / or a glyceraldehyde-3-phosphate dehydrogenase gene expression cassette into the Penicillium citrinum strain.

8. The method according to claim 7, characterized in that The amino acid sequence of glucose-6-phosphate dehydrogenase in the glucose-6-phosphate dehydrogenase gene expression cassette is shown in SEQ ID NO: 7 or 8, and the amino acid sequence of glyceraldehyde-3-phosphate dehydrogenase in the glyceraldehyde-3-phosphate dehydrogenase gene expression cassette is shown in SEQ ID NO:

9.

9. A Penicillium citrinum strain producing pravastatin, characterized in that: The method is constructed by any one of claims 1 to 8.

10. Use of the Penicillium citrinum strain according to claim 9 in the production of pravastatin or mevastatin.

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