A method for catalytic production of 5-fluorouracil and its application

Abstract

The application discloses a method for catalytically producing 5-fluorouracil and application, and belongs to the technical field of biology. The method uses 5-fluoroorotic acid as a substrate and orotidine-5'-phosphate decarboxylase as a catalyst to efficiently catalyze the synthesis of 5-fluorouracil. The catalytic efficiency is further improved by molecular modification of orotidine-5'-phosphate decarboxylase, the efficient and green production of 5-fluorouracil is realized, and the application of 5-fluorouracil in the fields of medicine and the like is promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for catalytic production of 5-fluorouracil and application, belonging to the field of biotechnology. BACKGROUND

[0002] 5-fluorouracil is one of the important antitumor drugs, and is also a key intermediate for synthesizing fluorinated pyrimidine antitumor drugs. The synthesis method of 5-fluorouracil is chemical synthesis method, including: direct synthesis method, condensation cyclization method, and other methods (such as fluorinated ethyl acetate and ethyl formate condensation method). Direct fluorination method is to use uracil as raw material, react with fluorine gas diluted by nitrogen, and obtain 5-fluorouracil after treatment. The process is relatively simple, and the yield is high. However, due to the high price of fluorine gas, the source of fluorine gas is restricted, at the same time, fluorine gas is highly toxic, and the equipment requirement is high, so it is less used in industrial production. Other methods mainly use derivatives of nitrogen heterocyclic compounds as starting materials, which are difficult to obtain and high in cost, and are not suitable for industrialization. With the maturity, safety and reliability of electrolytic fluorine process, fluorine gas is no longer a bottleneck for direct synthesis of 5-fluorouracil. Based on this, CN105693628A provides a one-step synthesis method for producing 5-fluorouracil. However, the current mainstream industrialization route is condensation cyclization method, which uses fluorinated methyl acetate as starting material, and reacts with ethyl formate under the catalysis of sodium methoxide to obtain methyl fluorinated propionic acid enol sodium salt. This intermediate is cyclized with methyl isourea to synthesize 2-methoxy-5-fluorouracil, and then subjected to acid hydrolysis, recrystallization and other processes to obtain 5-fluorouracil. However, this method has long route and low efficiency, and it is urgent to find a simple and efficient method for producing 5-fluorouracil. SUMMARY

[0003] [Technical problem]

[0004] The technical problem to be solved by the present application is that the method and process steps for catalytic production of 5-fluorouracil by chemical method in the prior art are complex and have low synthesis efficiency.

[0005] [Technical solution]

[0006] In view of the above problems, the present application uses recombinant expression to highly express orotidine-5'-phosphate decarboxylase gene pyrF in Escherichia coli. The expressed orotidine-5'-phosphate decarboxylase is used to catalyze the synthesis of 5-fluorouracil with 5-fluoroorotic acid as substrate, realizing the efficient and green production of 5-fluorouracil and promoting its application in the fields of medicine and the like.

[0007] The present application provides an orotidine-5'-phosphate decarboxylase mutant, which is based on the amino acid sequence shown in SEQ ID NO. 1 and has any one of the following mutations:

[0008] (1) the 21st leucine is mutated into histidine, to obtain mutant L21H;

[0009] (2) the 72nd leucine is mutated into histidine, to obtain mutant L72H;

[0010] (3) the 45th valine is mutated into alanine, to obtain mutant V45A.

[0011] The application also provides a gene encoding the mutant.

[0012] In one embodiment, the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

[0013] The application also provides an expression vector carrying the gene.

[0014] The application also provides a microbial cell expressing the mutant, or carrying the expression vector.

[0015] The application also provides a recombinant E. coli, taking E. coli BL21(DE3) as a host, and taking pETDuet-1 as an expression vector, to express the mutant.

[0016] The application also provides a method for producing 5-fluorouracil, taking the mutant, or the microbial cell, or the recombinant E. coli as a catalyst, and taking 5-fluoroorotic acid as a substrate to perform a reaction.

[0017] In one embodiment, the reaction system further contains phosphate, and the phosphate includes sodium phosphate and potassium phosphate.

[0018] In one embodiment, the reaction system contains, in terms of final concentration, 5-fluoroorotic acid 0.01-1.0 g / L, phosphate 50-300 mM, and orotidine-5'-phosphate decarboxylase 1.0-100 mg / mL.

[0019] In one embodiment, the reaction is performed at pH 5.5-8.5 and 20-40℃ for at least 3.5 h.

[0020] The application also provides application of the mutant, or the microbial cell, or the recombinant E. coli in production of 5-fluorouracil or a product containing 5-fluorouracil.

[0021] Advantages

[0022] The application realizes efficient catalytic production of 5-fluorouracil by using orotic acid riboside-5'-phosphate decarboxylase as a catalyst and 5-fluoroorotic acid as a raw material. After replacing the valine at the 45th position of orotic acid riboside-5'-phosphate decarboxylase with alanine, the expression enzyme activity of orotic acid riboside-5'-phosphate decarboxylase is increased to 1.6 times of the wild enzyme, and the efficiency of catalytic production of 5-fluorouracil is increased by 1.4 times. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 SDS-PAGE of overexpression of orotic acid riboside-5'-phosphate decarboxylase;

[0024] Figure 2 The tertiary structure of orotic acid riboside-5'-phosphate decarboxylase;

[0025] Figure 3 HPLC of orotic acid riboside-5'-phosphate decarboxylase catalytic production of 5-fluorouracil. DETAILED DESCRIPTION

[0026] The application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application rather than limit the scope of the application.

[0027] HPLC detection conditions of 5-fluorouracil: liquid chromatograph SHIMADZU 10A, chromatographic column: INERTSIL ODS-SP 5μm 4.6*250mm, mobile phase: buffer preparation: 0.1mol / L potassium dihydrogen phosphate aqueous solution: 0.01mol / L tetrabutylammonium hydroxide: methanol = 95: 95: 10, then adjust pH to 4.5 with phosphoric acid, wavelength: 262nm, flow rate: 1.0mL / min.

[0028] Definition of orotic acid riboside-5'-phosphate decarboxylase enzyme activity unit: under catalytic conditions, the enzyme volume required to catalyze the generation of 1μmol 5-fluorouracil per minute is an enzyme activity unit (U / mL).

[0029] Recombinant strains constructed in the following examples:

[0030] Strains involved in the specific embodiments of table 1

[0031]

[0032] Table 2 primers

[0033]

[0034] The reagents involved in the following examples are conventional reagents in the art and can be commonly purchased.

[0035] Construction and in vitro cultivation of recombinant enzyme expression strain of Example 1

[0036] 1. Construction of E. coli pETDuet-1-pyrF strain

[0037] The gene sequence pyrF of orotidine-5'-phosphate decarboxylase was obtained by PCR amplification using E. coli BL21 genome as a template and pyrF-FW and pyrF-RS as primers, as shown in SEQ ID NO. 2. The obtained pyrF gene and plasmid pETDuet-1 were respectively subjected to enzyme cutting at BamHI and Hind III sites and then linked to construct a recombinant plasmid pETDuet-1-pyrF. The recombinant plasmid pETDuet-1-pyrF was transformed into E. coli BL21 host to construct a recombinant bacterium E. coli pETDuet-1-pyrF.

[0038] The PCR amplification conditions were as follows: pre-denaturation at 98℃ for 5 min, denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ (the extension speed of the enzyme was 1 kb / min, and the specific time was set according to the length of the amplified fragment), 30 cycles were set, and then extension at 72℃ for 10 min, and finally 16℃ was kept. The PCR amplification system was as follows: 5×PS buffer 20 μL, dNTP 10 μL, upstream / downstream primer (10 μmol·L -1 ) 2 μL, template 1 μL, enzyme 1 μL, and water 66 μL.

[0039] 2. Expression of recombinant enzyme orotidine-5'-phosphate decarboxylase

[0040] The above constructed recombinant bacterium E. coli pETDuet-1-pyrF was inoculated into LB liquid medium and cultured at 37℃ and 200 rpm for 8 h to prepare a seed liquid. The above seed liquid was inoculated into TB liquid medium at an inoculation amount of 1% (v / v), and when the OD 600 = 0.6, 1.0 mM of inducer IPTG was added, and the fermentation was induced at 28℃ for 24 h to prepare a fermentation liquid.

[0041] The fermentation liquid was centrifuged to obtain bacterial cells, and the bacterial cells were broken by ultrasonic crushing method. After centrifugation of the broken bacterial cells, the supernatant of the broken cells was subjected to agarose-gel electrophoresis analysis, and it could be known that the results showed that orotidine-5'-phosphate decarboxylase pyrF (26.35 kDa) was successfully expressed in the fermentation liquid of the recombinant bacterium E. coli pETDuet-1-pyrF, and the enzyme activity was 9.8 U / mL. Figure 2

[0042] ​Example 2 Recombinant enzyme orotidine-5'-phosphate decarboxylase catalyzing production of 5-fluorouracil

[0043] The enzyme solution orotidine-5'-phosphate decarboxylase pyrF produced by fermentation in Example 1 was used to catalyze production of 5-fluorouracil from 5-fluoroorotic acid. The reaction system was 100 mL, containing 0.1 g / L 5-fluoroorotic acid, 200 mM sodium phosphate or potassium phosphate (pH = 7.0), 10 mg / mL (protein concentration) orotidine-5'-phosphate decarboxylase pyrF, the reaction pH was 7.0, the reaction temperature was 30°C, and the catalysis time was 5 h. After catalysis, most of the 5-fluoroorotic acid was catalytically completely converted into product 5-fluorouracil (5.465 min peak is 5-fluorouracil), and the conversion rate reached 96%. The results proved that the technical route of catalyzing production of 5-fluorouracil from 5-fluoroorotic acid was feasible and efficient. Conversion rate = (initial 5-fluoroorotic acid content - residual 5-fluoroorotic acid content) / initial 5-fluoroorotic acid content x 100%. Figure 3 ,5.465 min peak is 5-fluorouracil), and the conversion rate reached 96%. The results proved that the technical route of catalyzing production of 5-fluorouracil from 5-fluoroorotic acid was feasible and efficient. Conversion rate = (initial 5-fluoroorotic acid content - residual 5-fluoroorotic acid content) / initial 5-fluoroorotic acid content x 100%.

[0044] Adjusting the concentrations of various substances in the reaction system to 5-fluoroorotic acid 0.01-1.0 g / L, phosphate 50-300 mM, orotidine-5'-phosphate decarboxylase 1.0-100 mg / mL, reaction pH 5.5-8.5, reaction temperature 20-40°C, all can achieve efficient synthesis of 5-fluorouracil.

[0045] Example 3 Molecular modification to improve the enzyme activity of orotidine-5'-phosphate decarboxylase

[0046] 1. Homology modeling of tertiary structure

[0047] The orotidine-5'-phosphate decarboxylase (amino acid sequence is shown as SEQ ID NO. 1) was subjected to homology modeling using SWISS-MODEL software to obtain the tertiary structure of the enzyme. Figure 2 Through analysis of the tertiary structure, the active amino acid residue sites of orotidine-5'-phosphate decarboxylase were determined to be: 20, 22, 44, 46, 71, 73, 130, 131, 189, 221, 222.

[0048] 2. Molecular modification of orotidine-5'-phosphate decarboxylase

[0049] Using molecular modification, the amino acid residues Leu21 and Leu72 were replaced with histidine His to construct enzyme mutants L21H and L72H, and the amino acid residue Val45 was replaced with alanine Ala to construct enzyme mutant V45A.

[0050] The primer sequences used for constructing the mutants are shown in Table 2. The primer L21H-FW and L21H-RS were used as the mutation primers, and the recombinant plasmid pETDuet-1-pyrF was used as the template for PCR amplification. The PCR product was obtained, and the template plasmid was eliminated by DpnI digestion. The obtained product was transformed into the competent cells of E. coli BL21, and the recombinant plasmid pETDuet-1-L21H containing the mutant gene L21H of the enzyme and the recombinant bacteria E. coli pETDuet-1-L21H were obtained by screening and sequencing verification. Similarly, the primers V45A-FW and V45A-RS were used to construct the recombinant plasmid pETDuet-1-V45A containing the mutant gene V45A of the enzyme and the recombinant bacteria E. coli pETDuet-1-V45A; the primers L72H-FW and L72H-RS were used to construct the recombinant plasmid pETDuet-1-L72H containing the mutant gene L71H of the enzyme and the recombinant bacteria E. coli pETDuet-1-L72H. The information of the recombinant bacteria is shown in Table 1.

[0051] The PCR amplification conditions were as follows: pre-denaturation at 98℃ for 5 min; denaturation at 95℃ for 30 s; annealing at 55℃ for 30 s; extension at 72℃ (the extension speed of the enzyme was 1 kb / min, and the specific time was set according to the length of the amplified fragment); 30 cycles were set; re-extension at 72℃ for 10 min; and finally, 16℃ was kept. The PCR amplification system was as follows: 5×PS buffer 20 μL, dNTP 10 μL, upstream / downstream primer (10 μmol·L -1 ) 2 μL, template 1 μL, enzyme 1 μL, and water 66 μL.

[0052] The recombinant bacteria E. coli pETDuet-1-L21H, E. coli pETDuet-1-V45A, and E. coli pETDuet-1-L72H obtained by the above construction were inoculated into TB culture medium, and the induction fermentation was performed by referring to the method in Example 1. The induction conditions were as follows: the fermentation culture was performed at 37℃ until OD 600 = 0.6, 1.0 mM of the inducer IPTG was added, and the induction fermentation was performed at 28℃ for 24 h. The enzyme activity of the orotidine-5'-phosphate decarboxylase mutants L21H, V45A, and L72H was 6.2, 15.6, and 5.1 U / mL, respectively. The enzyme activity of the orotidine-5'-phosphate decarboxylase mutant V45A was increased to 1.6 times of the wild enzyme. The enzyme activity of the orotidine-5'-phosphate decarboxylase mutants L21H and L72H was reduced compared with the wild enzyme. According to the method in Example 2, when the orotidine-5'-phosphate decarboxylase mutant V45A was used to catalyze 5-fluoroorotic acid to produce 5-fluorouracil, the catalytic time for achieving a conversion rate of 96% was shortened from 5 h of the wild enzyme to 3.5 h.

[0053] While the application has been described by way of example and in terms of the preferred embodiment, it is to be understood that certain modifications can be made to the disclosed apparatus without departing from the scope of the application, and the scope of the application should be determined not by the embodiment but by the appended claims.

Claims

1. A process for the production of 5-fluorouracil, characterized in that, The reaction is carried out with 5-fluoroorotidine as a substrate and with a mutant of orotic acid nucleoside-5'-phosphate decarboxylase or a microbial cell expressing the mutant of orotic acid nucleoside-5'-phosphate decarboxylase as a catalyst; The mutant of orotic acid nucleoside-5'-phosphate decarboxylase is a mutant in which a valine at position 45 is replaced by alanine based on the amino acid sequence shown in SEQ ID NO.

1. The reaction system further contains a phosphate, and the phosphate includes sodium phosphate and potassium phosphate.

2. The method of claim 1, wherein, The microbial cell is recombinant Escherichia coli.

3. The method of claim 2, wherein, The reaction system contains, in terms of final concentrations, 5-fluoroorotidine 0.01-1.0 g / L, a phosphate 50-300 mM, and orotic acid nucleoside-5'-phosphate decarboxylase 1.0-100 mg / mL.

4. The method of any one of claims 1 to 3, wherein, The reaction is carried out at pH 5.5-8.5 and 20-40°C for at least 3.5 h.

5. Use of an orotate phosphoribosyltransferase mutant or a microbial cell expressing an orotate phosphoribosyltransferase mutant for the production of 5-fluorouracil or a product containing 5-fluorouracil, characterized in that, The mutant of orotic acid nucleoside-5'-phosphate decarboxylase is a mutant in which a valine at position 45 is replaced by alanine based on the amino acid sequence shown in SEQ ID NO.

1.

6. The use according to claim 5, characterized in that, The microbial cell is recombinant Escherichia coli.

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