Pseudomonas hydrolytic dehalogenase encoding gene, genetically engineered bacteria and application thereof

Abstract

The present application belongs to the field of bioengineering technology, and particularly relates to a coding gene of Pseudomonas hydrolysis dehalogenase, a recombinant expression vector for expressing the Pseudomonas hydrolysis dehalogenase, a genetically engineered bacterium for expressing the Pseudomonas hydrolysis dehalogenase, a fermentation culture method of the genetically engineered bacterium, and a method for preparing a favipiravir intermediate by using the genetically engineered bacterium for expressing the Pseudomonas hydrolysis dehalogenase. The Pseudomonas hydrolysis dehalogenase gene sequence is shown as SEQ ID NO. 1. Compared with the prior art, the present application first uses the Pseudomonas hydrolysis dehalogenase to catalytically prepare a favipiravir intermediate, and enables the Pseudomonas hydrolysis dehalogenase gene to be efficiently expressed in an engineered bacterium. Under preferred conditions, the enzyme activity can reach 214.5 U / L, the catalytic preparation of the favipiravir intermediate is efficient, the substrate conversion rate can reach 97.2%, and the present application has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to a gene encoding a Pseudomonas hydrolytic dehalogenase, a recombinant expression vector expressing the Pseudomonas hydrolytic dehalogenase, a genetically engineered bacterium expressing the Pseudomonas hydrolytic dehalogenase, a fermentation culture method for the genetically engineered bacterium, and a method for preparing favipiravir intermediates using the genetically engineered bacterium expressing the Pseudomonas hydrolytic dehalogenase. Background Technology

[0002] Favipiravir (chemical name: 6-fluoro-3-hydroxypyrazine-2-carboxamide) is an RNA-dependent RNA polymerase inhibitor. It is converted to T-705-furanose ribosyl 5'-triphosphate by the host enzyme and may act as a nucleotide analog that selectively inhibits viral RNA-dependent RNA polymerase or induce lethal mutagenesis upon incorporation into viral RNA without cytotoxicity to mammalian cells. It is primarily used to treat novel or recurrent influenza in adults.

[0003] The existing technology uses the following route to synthesize pipivir: In existing literature reports on the synthesis of intermediate I (6-fluoro-3-hydroxy-2-cyanopyrazine cyclohexanediamine salt) from intermediate II (3,6-difluoro-2-cyanopyrazine), Chinese patent CN101809003A discloses the organic amine salt of 6-fluoro-3-hydroxy-2-cyanopyrazine and its preparation method. However, the preparation process of the amine salt of intermediate I has the following drawbacks: First, many organic solvents such as N,N-dimethylformamide, toluene, and acetone are used in the preparation of the amine salt of intermediate I; second, the addition of 2-3 equivalents of potassium acetate aqueous solution and 2 equivalents of triethylamine to participate in the reaction results in a large amount of salt in the reaction waste, increasing the difficulty of environmental treatment; third, the organic amine is difficult to completely remove in the post-treatment of the amine salt, resulting in solvent and impurity residues, which increases the drug production cost and the difficulty of drug quality control.

[0004] Compared to the numerous organic solvents and excessive organic bases used in chemical synthesis, bio-enzyme catalysis can be carried out in a green and safe manner in aqueous solution, and does not use a large amount of organic bases during the enzyme catalysis process.

[0005] Currently, there are no reports, either domestically or internationally, on the preparation of 6-fluoro-3-hydroxy-2-cyanopyrazine by hydrolysis and defluorination of the substrate 3,6-difluoro-2-cyanopyrazine using a bioenzymatic method. Summary of the Invention

[0006] Given the lack of existing technologies for preparing 6-fluoro-3-hydroxy-2-cyanopyrazine from the substrate 3,6-difluoro-2-cyanopyrazine via hydrolysis and defluorination using a bioenzymatic method, this invention provides a gene encoding a Pseudomonas hydrolytic dehalogenase, a recombinant expression vector expressing the Pseudomonas hydrolytic dehalogenase, a genetically engineered bacterium expressing the Pseudomonas hydrolytic dehalogenase, a fermentation culture method for the genetically engineered bacterium, and a method for preparing favipiravir intermediates using the genetically engineered bacterium expressing the Pseudomonas hydrolytic dehalogenase.

[0007] This invention constructs a genetically engineered bacterium that efficiently expresses Pseudomonas hydrolytic dehalogenase for the preparation of favipiravir intermediates, thereby reducing the cost of biocatalysts and improving the economics of the favipiravir preparation process. This genetically engineered bacterium exhibits strong hydrolytic dehalogenation production capacity, low enzyme production cost, and high activity.

[0008] The objective of this invention can be achieved through the following technical solutions: The first aspect of this invention provides a gene encoding a Pseudomonas hydrolytic dehalogenase. Based on the original Pseudomonas hydrolytic dehalogenase gene sequence shown in SEQ ID NO.2, the expression level of the enzyme in *E. coli* was optimized using the codon optimization tool ExpOptimizer. The structure and binding free energy of the 5' mRNA in the translation initiation region (TIR) ​​were predicted and calculated using RNA Folding Form software to identify gene sequences more conducive to enzyme protein activity expression. Combining the results of the two optimization tools yielded the nucleotide sequence shown in SEQ ID NO.1, which is the codon-optimized enzyme gene sequence. The amino acid sequence of the corresponding Pseudomonas hydrolytic dehalogenase remained unchanged after codon optimization.

[0009] A second aspect of the present invention provides a recombinant expression vector for expressing the Pseudomonas hydrolytic dehalogenase, the recombinant expression vector containing the Pseudomonas hydrolytic dehalogenase gene with the nucleotide sequence shown in SEQ ID NO.1.

[0010] In one embodiment of the present invention, the recombinant expression vector refers to a recombinant expression vector constructed by linking the coding gene of Pseudomonas hydrolytic dehalogenase to the pET29a(+) plasmid, that is, the recombinant expression vector refers to the Pseudomonas hydrolytic dehalogenase recombinant plasmid.

[0011] In one embodiment of the present invention, the gene encoding the hydrolytic dehalogenase of Pseudomonas aeruginosa is linked to the restriction enzyme site of the pET29a(+) plasmid.

[0012] In one embodiment of the present invention, the recombinant plasmid map of Pseudomonas hydrolytic dehalogenase is as follows: Figure 2 As shown.

[0013] A third aspect of the present invention provides a genetically engineered bacterium expressing a hydrolytic dehalogenase from Pseudomonas, wherein the genetically engineered bacterium is a host microorganism carrying the recombinant expression vector.

[0014] In one embodiment of the present invention, the genetically engineered bacterium is a recombinant genetically engineered bacterium constructed by transforming the recombinant expression vector into Escherichia coli BL21(DE3).

[0015] The fourth aspect of the present invention provides a biocatalyst for preparing favipiravir intermediate products, wherein the biocatalyst is a wet cell obtained by inducing culture of genetically engineered bacteria or an enzyme solution obtained by breaking down the wet cell.

[0016] The fifth aspect of the present invention provides a fermentation culture method for the genetically engineered bacteria, wherein the genetically engineered bacteria are cultured in LB medium to obtain a seed culture, and then the seed culture is inoculated into a fermentation medium for further culture to obtain the genetically engineered bacteria.

[0017] In one embodiment of the present invention, the fermentation medium is formulated as follows: 30 g / L yeast extract, 10 g / L glycerol, 12.54 g / L K2HPO4 and 2.32 g / L KH2PO4, with a pH of 6.0 to 8.0.

[0018] Preferably, the fermentation medium is formulated as follows: 30g yeast extract, 10g glycerol, 12.54g K2HPO4 and 2.32g KH2PO4, with an initial pH of 7.0.

[0019] Preferably, the fermentation medium contains 50 μg / mL of kanamycin.

[0020] In one embodiment of the present invention, the fermentation culture conditions of the genetically engineered bacteria are as follows: After culturing the genetically engineered bacteria in LB solid medium for 12 hours, a single colony was picked and inoculated into LB seed medium, and cultured at 37°C and 200 rpm for 12 hours to obtain seed culture solution. The seed culture was inoculated into the fermentation medium at an inoculum rate of 2% (v / v), and incubated at 37°C until the OD of the fermentation broth reached a certain level. 600 When the concentration is 2.0, zinc chloride with a final concentration of 0.25 mM and IPTG with a final concentration of 0.1~0.6 mM are added to the culture medium. The induction culture temperature is 18~30℃. After culturing for 20 h, the bacterial cells are harvested.

[0021] Preferably, the culture conditions are as follows: IPTG is added to a final concentration of 0.4 mM, the induction culture temperature is 25°C, and the bacterial cells are harvested after 20 h of culture.

[0022] The sixth aspect of the present invention provides a method for preparing favipiravir intermediates, wherein the halocyanopyrazine compound is dehalogenated and hydrolyzed in an aqueous system using the Pseudomonas hydrolytic dehalogenase or wet cells obtained based on genetically engineered bacteria, or bacterial cells obtained after treating wet cells as a biocatalyst, to produce favipiravir intermediate products.

[0023] The reaction formula for the formation of 6-fluoro-3-hydroxy-2-cyanopyrazine from 3,6-difluoro-2-cyanopyrazine catalyzed by Pseudomonas hydrolytic dehalogenase is as follows: Figure 1 As shown.

[0024] The general structural formulas of the halocyanopyrazine compound (II) and the favipiravir intermediate product (I) are C5HN3(X)2 and C5H2ON3X, respectively, wherein the substituent X is selected from -F or -Cl. The structural formulas are shown below: .

[0025] Specifically, the favipiravir intermediate product (I) is 6-fluoro-3-hydroxy-2-cyanopyrazine.

[0026] In the prior art, the Pseudomonas hydrolytic dehalogenase involved in this invention is mainly used for the hydrolytic dechlorination of chlorothalonil; no one has studied the use of this enzyme for the hydrolytic defluorination to prepare favipiravir intermediates. This invention provides a method for dehalogenating and hydrolyzing halocyanopyrazine compounds into favipiravir intermediate products in an aqueous system using the aforementioned Pseudomonas hydrolytic dehalogenase, wet bacterial cells obtained from genetically engineered bacteria, or bacterial cells obtained after treating wet bacterial cells as a biocatalyst.

[0027] Furthermore, the substrates of defluorination hydrolases reported in existing technical literature are all fluorinated carboxylic acid structures. There are no reports in the prior art on the use of the Pseudomonas hydrolytic dehalogenase of this application to defluorinate the substrate 3,6-difluoro-2-cyanopyrazine to prepare 6-fluoro-3-hydroxy-2-cyanopyrazine.

[0028] The method for preparing favipiravir intermediates provided by this invention can improve the catalytic efficiency of engineered Pseudomonas hydrolytic dehalogenase.

[0029] In one embodiment of the present invention, the entire reaction system is 10 mL. The reaction conditions are as follows: 10 g dcw / L of Pseudomonas hydrolytic dehalogenase genetically engineered bacterial cells were suspended in 9 mL of PBS buffer (0.3 M) with a pH of 6.0-8.0. 1 mL of 3,6-difluoro-2-cyanopyrazine methanol solution with a final concentration of 30 mM was added. The reaction was carried out at 25-40℃ and 1000 rpm for 8 h. The reaction was terminated by adding the same volume of acetonitrile solution as the reaction system.

[0030] Preferably, the reaction conditions are as follows: 10g dcw / L of Pseudomonas hydrolytic dehalogenase genetically engineered bacterial cells were suspended in 9 mL of 0.3 M PBS buffer (pH 7.0) and incubated at 30 °C for 8 min. Then, 1 mL of a methanol solution of 3,6-difluoro-2-cyanopyrazine (final concentration 30 mM) was added, and the reaction was carried out at 30 °C and 1000 rpm for 8 h. The reaction was terminated by adding an equal volume of acetonitrile solution. The conversion rate of the product 6-fluoro-3-hydroxy-2-cyanopyrazine reached 99.1%.

[0031] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to utilize Pseudomonas hydrolytic dehalogenase to achieve the green preparation of 6-fluoro-3-hydroxy-2-cyanopyrazine, providing technical support for establishing a technical platform for the preparation of dehalogenated drug intermediates and other organic compounds by biocatalysis based on Pseudomonas hydrolytic dehalogenase, and promoting the application of hydrolytic dehalogenase in the synthesis of pharmaceutical intermediates.

[0032] 2. This invention, starting from the composition of the culture medium and the culture conditions, finds a culture method that can efficiently express the hydrolytic dehalogenase protein of *Pseudomonas*. Using the optimized fermentation medium and culture conditions, the cellular enzyme activity of the *Pseudomonas* hydrolytic dehalogenase can reach 214.5 U / L. This has important guiding significance for the development of fermentation processes for the production of this enzyme and the synthesis of favipiravir drug intermediates.

[0033] 3. Through the technical solution described in this invention, the catalytic efficiency of Pseudomonas hydrolytic dehalogenase engineered bacteria is improved, and the conversion rate of 0.1M substrate 3,6-difluoro-2-cyanopyrazine reaches 97.2%, and the conversion rate of 0.1M substrate 3,6-dichloro-2-cyanopyrazine reaches 93.4%. Attached Figure Description

[0034] Figure 1 The reaction formula for the formation of 6-fluoro-3-hydroxy-2-cyanopyrazine from 3,6-difluoro-2-cyanopyrazine catalyzed by hydrolytic dehalogenase of Pseudomonas; Figure 2 A map of recombinant plasmids containing hydrolytic dehalogenase from Pseudomonas aeruginosa. Figure 3 This is a polyacrylamide gel electrophoresis image of the protein expression of the hydrolytic dehalogenase gene of recombinant Escherichia coli in Example 1. Where M is the standard molecular weight of protein, 1 represents cell lysis supernatant, and 2 represents the precipitate after cell lysis. Detailed Implementation

[0035] Unless otherwise specified, the following examples employ conventional methods used by those skilled in the art or are performed in accordance with the product manual.

[0036] Genetically engineered strains in the following examples E.coli BL21(DE3), E.coli DH5α was purchased commercially and synthesized in its entirety by Shanghai Jierui Biotechnology Co., Ltd., using codon-optimized chd genes as shown in SEQ ID NO.1. Cd 1), and the original encoding chd gene as shown in SEQ ID NO.2 ( Cd 2), and then ligated them separately to plasmid pET29a(+), to construct recombinant plasmids pET29a(+)- Cd 1 and pET29a(+)- Cd 2. Plasmid extraction was performed using commercially available plasmid extraction and transformation kits, following the instructions provided.

[0037] Enzyme activity assay method: 300 μL of cell suspension (final concentration 2 g / L after adding stop solution) was preheated at 42 °C, followed by the addition of 195 μL of PBS buffer (pH 7.0) and 5 μL of substrate acetonitrile solution (final concentration 1.67 mM). The reaction was incubated at 42 °C for 10 min. Immediately after the reaction was complete, 1 mL of acetonitrile solution was added to terminate the reaction. After vortexing, the mixture was centrifuged at 12000 rpm for 2 min. The supernatant was aspirated using a sterile syringe, filtered through a microporous membrane, and then analyzed by HPLC.

[0038] Detection method (HPLC): The chromatographic column was a reversed-phase C18 column (Diamonsil plus, 4.6 mm × 250 mm × 5 μm); the mobile phase was water:acetonitrile = 2:1; the detection wavelength was 254 nm; the column temperature was 40 ℃; the flow rate was 0.8 mL / min; and the injection volume was 10 μL. The peak times of the substrate 3,6-difluoro-2-cyanopyrazine and the product 6-fluoro-3-hydroxy-2-cyanopyrazine were 11.2 min and 6.3 min, respectively. The peak times of the substrate 3,6-dichloro-2-cyanopyrazine and the product 6-chloro-3-hydroxy-2-cyanopyrazine were 19.6 min and 8.8 min, respectively.

[0039] In the embodiments of the present invention, the cell enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol of 6-fluoro-3-hydroxy-2-cyanopyrazine product per unit time under the conditions of pH 7.0 and temperature 42°C, which is defined as one unit U.

[0040] All reagents used in the embodiments of this invention are of analytical grade or HPLC grade.

[0041] The conversion rate of the product 6-fluoro-3-hydroxy-2-cyanopyrazine was calculated as follows: Conversion rate α = (3.18 × 10) -7×(A-A0))×B / C×100% Where A represents the peak area integrated by external standard method; A0 represents the peak area of ​​spontaneous defluorination and hydrolysis of the substrate; B represents the dilution factor of the overall reaction system; C: concentration of substrate 3,6-difluoro-2-cyanopyrazine (mM). The relationship is y = (3.18 × 10⁻⁶ m² / m²). -7 )x is the external standard curve of 6-fluoro-3-hydroxy-2-cyanopyrazine.

[0042] The conversion rate of the product 6-chloro-3-hydroxy-2-cyanopyrazine was calculated as follows: Conversion rate α = (1.72 × 10) -6 ×(A-A0))×B / C×100% Where A represents the peak area integrated by external standard method; A0 represents the peak area of ​​spontaneous dechlorination and hydrolysis of the substrate; B represents the dilution factor of the overall reaction system; C: concentration of substrate 3,6-dichloro-2-cyanopyrazine (mM). The relationship is y = (1.72 × 10⁻⁶ m² / m²). -6 )x represents the external standard curve of 6-chloro-3-hydroxy-2-cyanopyrazine.

[0043] The preparation method of crude enzyme solution in this embodiment of the invention is as follows: the bacterial cells obtained by centrifugation after fermentation are washed with 50mM PBS (pH 7.0) buffer, and the above operation is repeated twice. Then, the cells are resuspended with 300mM PBS (pH 7.0) buffer, and the cell concentration is controlled at 11g / L. The cells are disrupted twice at 4°C (pressure 1200 bar) using a low-temperature ultra-high pressure cell disruptor. The cell disruption solution is centrifuged at 12000rpm for 30min at 4°C. The supernatant obtained is decanted to obtain crude enzyme solution, which is used for biocatalytic reaction.

[0044] Example 1 Construction of recombinant Pseudomonas genetically engineered bacteria Includes Cd Recombinant plasmid pET29a(+)- of gene 1 Cd 1. The gene was synthesized from the whole genome, then transformed into E. coli DH5α. Positive clones were selected, and a large number of recombinant plasmids pET29a(+)- were obtained by plasmid extraction. Cd 1 ， It contains a codon-optimized Pseudomonas hydrolytic dehalogenase gene, the sequence of which is SEQ ID NO.1.

[0045] Includes Cd Recombinant plasmid pET29a(+)- of the two genes Cd 2. The gene was synthesized from the whole genome, then transformed into E. coli DH5α. Positive clones were selected, and a large number of recombinant plasmids pET29a(+)- were obtained by plasmid extraction. Cd 2 ， It contains the original Pseudomonas hydrolytic dehalogenase gene, the sequence of which is SEQ ID NO.2.

[0046] Take 100 μL E.coli BL21(DE3) competent cells were placed in pre-chilled sterile EP tubes and thawed in an ice bath to ensure uniform cell suspension. 10 μL of recombinant plasmid pET29a(+)- was added. Cd 1. Gently mix and incubate on ice for 30 minutes, while simultaneously preheating the thermostat. Transfer the EP tubes to a 42°C dry thermostat for 90 seconds of heat shock, then quickly transfer them to an ice bath to cool the cells for 15 minutes. Add 900 μL of antibiotic-free LB liquid medium and incubate at 37°C with shaking at 250 rpm for 1 hour. Then, spread 400 μL of the bacterial culture onto an LB agar plate containing antibiotics. Once there is no liquid flow on the gel surface, incubate upside down at 37°C for 12 hours. Single colonies of recombinant *E. coli* will appear, yielding the genetically engineered bacteria. E.coli BL21(DE3) / pET29a(+)- Cd 1 is used for subsequent induction of expression. 。 The genetically engineered bacteria E.coli BL21(DE3) / pET29a(+)- Cd 1 is carrying the recombinant plasmid pET29a(+)- Cd 1. Escherichia coli BL21(DE3).

[0047] Genetically engineered bacteria containing the original Pseudomonas hydrolytic dehalogenase gene sequence were constructed using the same plasmid transformation method as described above. E.coli BL21(DE3) / pET29a(+)- Cd 2 is used for subsequent induction of expression. 。 The genetically engineered bacteria E.coli BL21(DE3) / pET29a(+)- Cd 2 is for carrying the recombinant plasmid pET29a(+)- Cd 2. Escherichia coli BL21(DE3).

[0048] Example 2 The recombinant Pseudomonas hydrolytic dehalogenase-producing fermentation medium of the present invention was used under the condition of an initial pH of 6.0. E.coli BL21(DE3) / pET29a(+)- Cd Fermentation enzyme production results of 1: Fermentation medium: 30g yeast extract, 10g glycerol, 12.54g K2HPO4 and 2.32g KH2PO4, initial pH 6.0, prepared with distilled water; Seed culture: Single colonies transformed from the plate were picked and inoculated into a shake flask containing 50 mL of seed culture medium (containing 50 μg / mL kanamycin), i.e., LB medium, and cultured at 37°C and 200 rpm for 12 h with shaking. The seed culture medium formula was: 10 g / L tryptone, 10 g / L yeast extract, and 5 g / L NaCl, with an initial pH of 7.0.

[0049] Shake flask fermentation: Inoculate at a rate of 2% (v / v) E.coli BL21(DE3) / pET29a(+)- Cd 1. Seed culture solution was inoculated into fermentation medium (containing 50 μg / mL kanamycin) and incubated at 37°C until the OD of the fermentation broth reached a certain level. 600 When the concentration is 2.0, add zinc chloride to a final concentration of 0.25 mM and IPTG to a final concentration of 0.1 mM. Induce culture at a temperature of 25°C for 20 h. Centrifuge and wash the fermentation broth at 4°C and collect the cells for cell viability testing. Using the method described in the specific implementation, the cellular enzyme activity of the hydrolytic dehalogenase of recombinant Pseudomonas aeruginosa reached 76.0 U / L.

[0050] Example 3 The recombinant Pseudomonas hydrolytic dehalogenase-producing fermentation medium of the present invention was used under the condition of an initial pH of 7.0. E.coli BL21(DE3) / pET29a(+)- Cd Fermentation enzyme production results of 1: Fermentation medium: 30g yeast extract, 10g glycerol, 12.54g K2HPO4 and 2.32g KH2PO4, initial pH 7.0, prepared with distilled water; Shake flask fermentation: Inoculate at a rate of 2% (v / v) E.coli BL21(DE3) / pET29a(+)- Cd 1. Seed culture solution was inoculated into fermentation medium (containing 50 μg / mL kanamycin) and incubated at 37°C until the OD of the fermentation broth reached a certain level. 600 When the concentration is 2.0, add zinc chloride to a final concentration of 0.25 mM and IPTG to a final concentration of 0.1 mM. Induce culture at a temperature of 25°C for 20 h. Centrifuge and wash the fermentation broth at 4°C and collect the cells for cell viability testing. Using the method described in the specific implementation, the cellular enzyme activity of the hydrolytic dehalogenase of recombinant Pseudomonas aeruginosa reached 113.1 U / L.

[0051] Example 4 The recombinant Pseudomonas hydrolytic dehalogenase-producing fermentation medium of the present invention was used under the condition of an initial pH of 8.0. E.coli BL21(DE3) / pET29a(+)- Cd Fermentation enzyme production results of 1: Fermentation medium: 30g yeast extract, 10g glycerol, 12.54g K2HPO4 and 2.32g KH2PO4, initial pH 8.0, prepared with distilled water; Shake flask fermentation: Inoculate at a rate of 2% (v / v) E.coli BL21(DE3) / pET29a(+)- Cd 1. Seed culture solution was inoculated into fermentation medium (containing 50 μg / mL kanamycin) and incubated at 37°C until the OD of the fermentation broth reached a certain level. 600 When the concentration is 2.0, add zinc chloride to a final concentration of 0.25 mM and IPTG to a final concentration of 0.1 mM. Induce culture at a temperature of 25°C for 20 h. Centrifuge and wash the fermentation broth at 4°C and collect the cells for cell viability testing. Using the method described in the specific implementation, the cellular enzyme activity of the hydrolytic dehalogenase of recombinant Pseudomonas aeruginosa reached 98.7 U / L.

[0052] Example 5 Using the hydrolytic dehalogenase fermentation medium of recombinant Pseudomonas aeruginosa from Example 3, and with the addition of IPTG to a final concentration of 0.4 mM... E.coli BL21(DE3) / pET29a(+)- Cd Fermentation enzyme production results of 1: Shake flask fermentation: Inoculate at a rate of 2% (v / v) E.coli BL21(DE3) / pET29a(+)- Cd 1. Seed culture solution was inoculated into fermentation medium (containing 50 μg / mL kanamycin) and incubated at 37°C until the OD of the fermentation broth reached a certain level. 600 When the concentration is 2.0, add zinc chloride to a final concentration of 0.25 mM and IPTG to a final concentration of 0.4 mM to the culture medium. The induction culture temperature is 25℃ and the culture time is 20 h. The fermentation broth is centrifuged and washed at 4℃, and the cells are collected for cell viability testing. Using the method described in the specific implementation, the cellular enzyme activity of the hydrolytic dehalogenase of recombinant Pseudomonas aeruginosa reached 214.5 U / L.

[0053] Example 6 Using the hydrolytic dehalogenase fermentation medium of recombinant Pseudomonas aeruginosa from Example 3, and with the addition of IPTG at a final concentration of 0.6 mM... E.coliBL21(DE3) / pET29a(+)- Cd Fermentation enzyme production results of 1: Shake flask fermentation: Inoculate at a rate of 2% (v / v) E.coli BL21(DE3) / pET29a(+)- Cd 1. Seed culture solution was inoculated into fermentation medium (containing 50 μg / mL kanamycin) and incubated at 37°C until the OD of the fermentation broth reached a certain level. 600 When the concentration is 2.0, add zinc chloride to a final concentration of 0.25 mM and IPTG to a final concentration of 0.6 mM. Induce culture at a temperature of 25°C for 20 h. Centrifuge and wash the fermentation broth at 4°C and collect the cells for cell viability testing. Using the method described in the specific implementation, the cellular enzyme activity of the hydrolytic dehalogenase of recombinant Pseudomonas aeruginosa reached 133.1 U / L.

[0054] Example 7 The recombinant Pseudomonas hydrolytic dehalogenase-producing fermentation medium from Example 3 was used, and the culture was carried out at 18°C. E.coli BL21(DE3) / pET29a(+)- Cd Fermentation enzyme production results of 1: Shake flask fermentation: Inoculate at a rate of 2% (v / v) E.coli BL21(DE3) / pET29a(+)- Cd 1. Seed culture solution was inoculated into fermentation medium (containing 50 μg / mL kanamycin) and incubated at 37°C until the OD of the fermentation broth reached a certain level. 600 When the concentration is 2.0, add zinc chloride to a final concentration of 0.25 mM and IPTG to a final concentration of 0.4 mM. Induce culture at 18°C ​​for 20 h. Centrifuge and wash the fermentation broth at 4°C and collect cells for cell viability testing. Using the method described in the specific implementation, the cellular enzyme activity of the hydrolytic dehalogenase of recombinant Pseudomonas aeruginosa reached 23.0 U / L.

[0055] Example 8 The recombinant Pseudomonas hydrolytic dehalogenase-producing fermentation medium from Example 3 was used, and the culture was carried out at 30°C. E.coli BL21(DE3) / pET29a(+)- Cd Fermentation enzyme production results of 1: Shake flask fermentation: Inoculate at a rate of 2% (v / v) E.coli BL21(DE3) / pET29a(+)- Cd 1. Seed culture solution was inoculated into fermentation medium (containing 50 μg / mL kanamycin) and incubated at 37°C until the OD of the fermentation broth reached a certain level.600 When the concentration is 2.0, add zinc chloride to a final concentration of 0.25 mM and IPTG to a final concentration of 0.4 mM. Induce culture at a temperature of 30°C for 20 h. Centrifuge and wash the fermentation broth at 4°C and collect the cells for cell viability testing. Using the method described in the specific implementation, the cellular enzyme activity of the hydrolytic dehalogenase of recombinant Pseudomonas aeruginosa reached 122.5 U / L.

[0056] Example 9 The recombinant Pseudomonas hydrolytic dehalogenase-producing fermentation medium described in Example 5 was used to obtain the culture method. E.coli BL21(DE3) / pET29a(+)- Cd 1. Pseudomonas hydrolytic dehalogenase genetically engineered cells were used to determine the conversion rate of 3,6-difluoro-2-cyanopyrazine in PBS buffer (0.3M) at pH 6.0.

[0057] The total reaction volume is 10 mL. The reaction conditions are as follows: 10 g dcw / L of Pseudomonas hydrolytic dehalogenase genetically engineered cells were suspended in 9 mL of 0.3 M PBS buffer (pH 6.0) and incubated at 25 °C for 8 min. Then, 1 mL of 3,6-difluoro-2-cyanopyrazine methanol solution (final concentration 30 mM) was added, and the reaction was carried out at 25 °C and 1000 rpm for 8 h. The reaction was terminated by adding the same volume of acetonitrile solution as the reaction system. After vortexing, the sample was centrifuged at 12000 rpm for 2 min. The supernatant was aspirated with a sterile syringe, filtered through a microporous membrane, and then analyzed by HPLC.

[0058] The conversion rate of 3,6-difluoro-2-cyanopyrazine was determined to be 48.8% using the analytical method described in the specific implementation method.

[0059] Example 10 The recombinant Pseudomonas hydrolytic dehalogenase-producing fermentation medium described in Example 5 was used to obtain the culture method. E.coli BL21(DE3) / pET29a(+)- Cd Results of the determination of 3,6-difluoro-2-cyanopyrazine conversion rate in Pseudomonas hydrolytic dehalogenase genetically engineered cells in PBS buffer (0.3 M) at pH 7.0: The total reaction volume is 10 mL. The reaction conditions are as follows: 10 g dcw / L of Pseudomonas hydrolytic dehalogenase genetically engineered bacterial cells were suspended in 9 mL of PBS buffer (pH 7.0, 0.3 M) and incubated at 25 °C for 8 min. Then, 1 mL of 3,6-difluoro-2-cyanopyrazine methanol solution (final concentration 30 mM) was added, and the reaction was carried out at 25 °C and 1000 rpm for 8 h. The reaction was terminated by adding the same volume of acetonitrile solution as the reaction system. After vortexing, the sample was centrifuged at 12000 rpm for 2 min. The supernatant was aspirated with a sterile syringe, filtered through a microporous membrane, and then analyzed by HPLC.

[0060] The conversion rate of 3,6-difluoro-2-cyanopyrazine can reach 88.6% when determined using the analytical method described in the specific implementation method.

[0061] Example 11 The recombinant Pseudomonas hydrolytic dehalogenase-producing fermentation medium described in Example 5 was used to obtain the culture method. E.coli BL21(DE3) / pET29a(+)- Cd Results of the determination of 3,6-difluoro-2-cyanopyrazine conversion rate in Pseudomonas hydrolytic dehalogenase genetically engineered cells in PBS buffer (0.3 M) at pH 8.0: The total reaction volume is 10 mL. The reaction conditions are as follows: 10 g dcw / L of Pseudomonas hydrolytic dehalogenase genetically engineered bacterial cells were suspended in 9 mL of PBS buffer (pH 8.0, 0.3 M) and incubated at 25 °C for 8 min. Then, 1 mL of 3,6-difluoro-2-cyanopyrazine methanol solution (final concentration 30 mM) was added, and the reaction was carried out at 25 °C and 1000 rpm for 8 h. The reaction was terminated by adding the same volume of acetonitrile solution as the reaction system. After vortexing, the sample was centrifuged at 12000 rpm for 2 min. The supernatant was aspirated with a sterile syringe, filtered through a microporous membrane, and then analyzed by HPLC.

[0062] The conversion rate of 3,6-difluoro-2-cyanopyrazine can reach 83.3% when determined using the analytical method described in the specific implementation method.

[0063] Example 12 The recombinant Pseudomonas hydrolytic dehalogenase-producing fermentation medium described in Example 5 was used to obtain the culture method. E.coli BL21(DE3) / pET29a(+)- Cd Results of determining the conversion rate of 3,6-difluoro-2-cyanopyrazine in Pseudomonas hydrolytic dehalogenase genetically engineered cells at a reaction temperature of 30℃: The total reaction volume is 10 mL. The reaction conditions are as follows: 10 g dcw / L of Pseudomonas hydrolytic dehalogenase genetically engineered cells were suspended in 9 mL of PBS buffer (pH 7.0, 0.3 M) and incubated at 30 °C for 8 min. Then, 1 mL of 3,6-difluoro-2-cyanopyrazine methanol solution (final concentration 30 mM) was added, and the reaction was carried out at 30 °C and 1000 rpm for 8 h. The reaction was terminated by adding the same volume of acetonitrile solution as the reaction system. After vortexing, the sample was centrifuged at 12000 rpm for 2 min. The supernatant was aspirated with a sterile syringe, filtered through a microporous membrane, and then analyzed by HPLC.

[0064] The conversion rate of 3,6-difluoro-2-cyanopyrazine can reach 99.1% when determined using the analytical method described in the specific implementation method.

[0065] Example 13 The recombinant Pseudomonas hydrolytic dehalogenase-producing fermentation medium described in Example 5 was used to obtain the culture method. E.coli BL21(DE3) / pET29a(+)- Cd Results of determining the conversion rate of 3,6-difluoro-2-cyanopyrazine in Pseudomonas hydrolytic dehalogenase genetically engineered cells at a reaction temperature of 35°C: The total reaction volume is 10 mL. The reaction conditions are as follows: 10 g dcw / L of Pseudomonas hydrolytic dehalogenase genetically engineered cells were suspended in 9 mL of 0.3 M PBS buffer (pH 7.0) and incubated at 35 °C for 8 min. Then, 1 mL of 3,6-difluoro-2-cyanopyrazine methanol solution (final concentration 30 mM) was added, and the reaction was carried out at 35 °C and 1000 rpm for 8 h. The reaction was terminated by adding the same volume of acetonitrile solution as the reaction system. After vortexing, the sample was centrifuged at 12000 rpm for 2 min. The supernatant was aspirated with a sterile syringe, filtered through a microporous membrane, and then analyzed by HPLC.

[0066] The conversion rate of 3,6-difluoro-2-cyanopyrazine can reach 78.0% when determined using the analytical method described in the specific implementation method.

[0067] Example 14 The recombinant Pseudomonas hydrolytic dehalogenase-producing fermentation medium described in Example 5 was used to obtain the culture method. E.coli BL21(DE3) / pET29a(+)- Cd Results of determining the conversion rate of 3,6-difluoro-2-cyanopyrazine in Pseudomonas hydrolytic dehalogenase genetically engineered cells at a reaction temperature of 40℃: The total reaction volume is 10 mL. The reaction conditions are as follows: 10 g dcw / L of Pseudomonas hydrolytic dehalogenase genetically engineered bacterial cells were suspended in 9 mL of PBS buffer (pH 7.0, 0.3 M) and incubated at 40 °C for 8 min. Then, 1 mL of 3,6-difluoro-2-cyanopyrazine methanol solution (final concentration 30 mM) was added, and the reaction was carried out at 40 °C and 1000 rpm for 8 h. The reaction was terminated by adding an equal volume of acetonitrile solution. After vortexing, the sample was centrifuged at 12000 rpm for 2 min. The supernatant was aspirated with a sterile syringe, filtered through a microporous membrane, and then analyzed by HPLC.

[0068] The conversion rate of 3,6-difluoro-2-cyanopyrazine can reach 74.5% when determined using the analytical method described in the specific implementation method.

[0069] Example 15 The recombinant Pseudomonas hydrolytic dehalogenase-producing fermentation medium described in Example 5 was used to obtain the culture method. E.coli BL21(DE3) / pET29a(+)- Cd 1. Using Pseudomonas hydrolytic dehalogenase genetically engineered cells and the recombinant Pseudomonas hydrolytic dehalogenase catalytic reaction conditions described in Example 12, 3,6-difluoro-2-cyanopyrazine was dehalogenated and hydrolyzed into favipiravir intermediate product on a scale-up basis.

[0070] The entire reaction system consisted of 50 mL. 10 g / L wet bacterial cells were added to 0.3 M PBS buffer (pH 7.0). After incubation for 8 min in a shaker, 5 mL of 0.1 M 3,6-difluoro-2-cyanopyrazine was added. The reaction was stopped after 8 h at 30 °C and 250 rpm. The substrate and product concentrations were determined by HPLC. The supernatant was collected by centrifugation and extracted three times with an equal volume of ethyl acetate. The ethyl acetate phases were combined, dried overnight with anhydrous sodium sulfate, filtered to remove the anhydrous sodium sulfate, and the solvent was recovered under reduced pressure. The residue was purified by column chromatography under the following conditions (ethyl acetate: petroleum ether = 3:1). The product-containing chromatographic solution was recovered under reduced pressure. The residue was dried under vacuum to obtain 0.51 g of a pale yellow oily liquid.

[0071] Using the analytical method described in the specific implementation method, the conversion rate of 0.1 M 3,6-difluoro-2-cyanopyrazine reached 97.2%, and the separation yield was 73.4%.

[0072] Example 16 The 3,6-dichloro-2-cyanopyrazine was dehydrolyzed into favipiravir intermediate product using the hydrolytic dehalogenase catalytic reaction conditions of recombinant Pseudomonas hydrolysis in Example 12.

[0073] The entire reaction system is 50 mL. Add a final concentration of 10 g / L to 0.3 M PBS buffer (pH 7.0). E.coli BL21(DE3) / pET29a(+)- Cd 1. Wet bacterial cells were incubated in a shaker for 8 min, then 5 mL of 0.1 M 3,6-dichloro-2-cyanopyrazine was added. The reaction was carried out at 30 °C and 250 rpm for 8 h, and the reaction was stopped. The substrate and product concentrations were determined by HPLC. The reaction solution was centrifuged and the supernatant was extracted three times with an equal volume of ethyl acetate. The ethyl acetate phases were combined, dried overnight with anhydrous sodium sulfate, filtered to remove anhydrous sodium sulfate, and the solvent was recovered under reduced pressure. The residue was purified by column chromatography under the chromatographic conditions (ethyl acetate: petroleum ether = 1:5). The chromatographic solution containing the product was recovered under reduced pressure. The residue was dried under vacuum to obtain 0.53 g of a pale yellow oily liquid as the final product.

[0074] Using the analytical method described in the specific implementation method, the conversion rate of 0.1 M 3,6-dichloro-2-cyanopyrazine reached 93.4%, and the separation yield was 68.2%.

[0075] Example 17 The difference from Example 15 is that in this example, recombinant Pseudomonas hydrolysis dehalogenase crude enzyme solution is used to dehalogenate 3,6-difluoro-2-cyanopyrazine into favipiravir intermediate product.

[0076] The entire reaction system consisted of 50 mL. The culture method used in Example 5, which produced recombinant Pseudomonas hydrolytic dehalogenase fermentation medium, was employed. E.coli BL21(DE3) / pET29a(+)- Cd 1. Recombinant Pseudomonas hydrolytic dehalogenase genetically engineered bacterial cells were used to prepare crude recombinant Pseudomonas hydrolytic dehalogenase solution according to the specific implementation method. 45 mL of the crude enzyme solution was incubated in a shaker at 30°C for 8 min, followed by the addition of 5 mL of 0.1 M 3,6-difluoro-2-cyanopyrazine. The reaction was carried out at 30°C and 250 rpm for 8 h, after which the reaction was stopped. The substrate and product concentrations were determined by HPLC. The reaction solution was centrifuged, and the supernatant was extracted three times with an equal volume of ethyl acetate. The ethyl acetate phases were combined, dried overnight with anhydrous sodium sulfate, filtered to remove the anhydrous sodium sulfate, and the solvent was recovered under reduced pressure. The residue was purified by column chromatography under the conditions of ethyl acetate:petroleum ether = 1:5. The chromatographic solution containing the product was recovered under reduced pressure, and the residue was dried under vacuum to obtain 0.50 g of a pale yellow oily liquid as the final product.

[0077] Using the analytical method described in the specific implementation method, the conversion rate of 0.1 M 3,6-difluoro-2-cyanopyrazine reached 96.8%, and the separation yield was 72.0%.

[0078] Comparative Example 1 Using the same culture medium components and fermentation conditions as in Example 5, E.coliBL21(DE3) / pET29a(+)- Cd Fermentation enzyme production results of 2: Shake flask fermentation: Inoculate at a rate of 2% (v / v) E.coli BL21(DE3) / pET29a(+)- Cd 2. Seed culture broth was inoculated into fermentation medium (containing 50 μg / mL kanamycin) and incubated at 37°C until the OD of the fermentation broth reached a certain level. 600 When the concentration is 2.0, add zinc chloride to a final concentration of 0.25 mM and IPTG to a final concentration of 0.4 mM to the culture medium. The induction culture temperature is 25℃ and the culture time is 20 h. The fermentation broth is centrifuged and washed at 4℃, and the cells are collected for cell viability testing. Using the methods described in the specific implementation embodiment, the cellular enzyme activity of the recombinant Pseudomonas hydrolytic dehalogenase reached 63.3 U / L. This enzyme production level is comparable to that of codon-optimized strains under the same conditions. E.coli BL21(DE3) / pET29a(+)- Cd 1 of 29.5%. The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be easily made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

[0079] The sequences involved in this invention are as follows: SEQ ID NO.1: Encoding gene for hydrolytic dehalogenase in Pseudomonas aeruginosa atgccgccgg gttgtagcgg tctgtgtagc tttgtgggcc tgaccatgcc gctgaaattt 60 tcaggtgtgc tgtgtgcatc actgctgacc attaccctga gcgttgcagc acatgccacc 120 gaattaattc tggattttaa taaagttcag atgcgtagtc agcagttagc tccaggcgtg 180 tatgcacatc tgccagctga tagcgcagaa ctgaatgcca aaggcggtgt tgcaggtacc 240 tcaggtggct taattgtggg tacccgcggc gcaatgttaa ttgaaaccat gctgaatcgt 300 cgcttatttg atcaggttca ggccctggcc aaaaaagaag ccttaggcct gccgttactg 360 tatgcagtga atacctcata tcatggtgat catagctatg gcaatatgta tctgaaagcc 420 ccgacccgtg ttattcagtc taccaaaacc cgcgattatg tggatggtca tttagccgat 480 gataaagcct ttatggttaa aaatttggc gcaggtcgcg gcgtggaaca gattaccgca 540 cgtaccggcg atattctggt tccgccggc ggtcgcgtga gcgttgatct gggcggtaaaa 600 accgttgaaa ttattgattt tggctttgca cagaccggtg gcgatctgtt tgtttgggaa 660 ccgcagagta aagtgatgtg gaccggtaat gcagttgttg cctctaaacc ggcattaccg 720 tggttactgg atggtaaatt agtggaaacc ttagccacct tacagaaagt gtatgattttt 780 ctgccgccgg atgccaccat tgttccgggt catggtgttc cgatggcacg cgaaggctta 840 cgctggcatt tagattattt agcagcagtt caggcaggcg ttaaagatgc cctggcacgc 900 aaactgtctt tagaacagac cgtgaccgaa ctgaaaatgc cggaatttcg cggctatgtg 960 ctgtttgatt gggtgcatcc ggatctgaat gttccagcag cctataagga tctggcagca 1020 cgtccgtaa 1029 SEQ ID NO. 2: Original coding gene of Pseudomonas hydrolytic dehalogenase atgccgcctg gatgttccgg gctttgtagc tttgtaggat tgacgatgcc actcaagttt 60 tcgggcgtgc tctgcgccag cctcctgacc atcacccttt cggtagccgc gcatgcaacc 120 gaactcatct tggacttcaa caaggtgcag atgcgcagcc aacaactcgc acctggggtg 180 tacgcgcatc taccggcaga ctccgccgag ctgaatgcaa aaggcggtgt agcaggcacc 240 agcggtggct tgatcgtcgg gacacgtggc gcgatgctga tcgagaccat gctcaaccgt 300 cgccttttcg atcaggtcca agcactcgcc aaaaaggagg cattggggct gccgctgctg 360 tacgcagtca acaccagtta tcatggcgat cactcgtatg ggaacatgta cctgaaggcg 420 ccgacgcgtg tcatccaaag caccaaaacg cgtgattatg tcgatggtca cctcgccgac 480 gacaaggcat tcatggtgaa gaatttcggt gccgggcgcg gtgttgagca gatcacggca 540 aggacgggcg acatcctcgt gcctccggga ggtcgcgtga gcgtcgacct tggcggcaag 600 actgttgaga tcatcgactt cgggttcgca cagaccggcg gggacctgtt cgtctgggag 660 ccgcaatcca aagttatgtg gacaggcaac gcagtggtcg ccagcaagcc agctctacct 720 tggctgctcg atggcaagct ggtagagacg ttggcgacgc tacagaaagt ctacgatttt 780 ctaccgcctg atgccaccat cgtccctgga catggtgtac ccatggcccg cgaaggcctg 840 cgctggcatc tggactacct cgcagccgtg caagcaggcg tcaaggatgc tttggctcgc 900 aaactgagcc tcgaacagac agtgaccgaa ctcaagatgc cggagttccg cggctacgta 960 ctgttcgact gggttcatcc agatctcaac gtccccgctg cttacaagga tctcgcagcc 1020 aggccttga 1029

Claims

1. A gene encoding a hydrolytic dehalogenase from Pseudomonas, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A recombinant expression vector for expressing Pseudomonas hydrolytic dehalogenase, characterized in that, The recombinant expression vector is a recombinant expression vector constructed by linking the encoding gene of the Pseudomonas hydrolytic dehalogenase as described in claim 1 to the pET29a(+) plasmid.

3. A genetically engineered bacterium expressing a Pseudomonas hydrolytic dehalogenase, characterized in that, The genetically engineered bacteria are recombinant genetically engineered bacteria constructed by transforming the recombinant expression vector of claim 2 into Escherichia coli BL21(DE3).

4. A biocatalyst for preparing favipiravir intermediate products, characterized in that, The biocatalyst is a wet bacterial cell obtained by inducing culture of the genetically engineered bacteria as described in claim 3, or an enzyme solution obtained by breaking down the wet bacterial cell.

5. A fermentation culture method for the genetically engineered bacteria as described in claim 3, characterized in that, The genetically engineered bacteria were cultured in LB medium to obtain a seed culture, which was then inoculated into a fermentation medium for further culture to obtain the genetically engineered bacteria.

6. The fermentation culture method of the genetically engineered bacteria according to claim 5, characterized in that, The fermentation medium has the following formula: 30 g / L yeast extract, 10 g / L glycerol, 12.54 g / L K2HPO4 and 2.32 g / L KH2PO4, with a pH of 6.0~8.

0.

7. The fermentation culture method of the genetically engineered bacteria according to claim 5, characterized in that, The fermentation culture conditions for the genetically engineered bacteria are as follows: After culturing the genetically engineered bacteria in LB solid medium for 12 hours, a single colony was picked and inoculated into LB seed medium, and cultured at 37°C and 200 rpm for 12 hours to obtain seed culture solution. The seed culture was inoculated into the fermentation medium at an inoculum rate of 2% v / v, and incubated at 37°C until the OD of the fermentation broth reached a certain level. 600 When the concentration is 2.0, zinc chloride with a final concentration of 0.25 mM and IPTG with a final concentration of 0.1~0.6 mM are added to the culture medium. The induction culture temperature is 25~30℃. After culturing for 20 h, the bacterial cells are harvested.

8. A method for preparing favipiravir intermediates, characterized in that, Using the biocatalyst described in claim 4, a halocyanopyrazine compound is dehalogenated and hydrolyzed in an aqueous system to produce a favipiravir intermediate. The general structural formulas of the halocyanopyrazine compound and the favipiravir intermediate are C5HN3(X)2 and C5H2ON3X, respectively, as shown in II and I: ； In Formula II, the substituents X at positions 3 and 6 are the same and are selected from -F or -Cl. In Formula I, the selection of X is the same as in Formula II.

9. The method for preparing favipiravir intermediate according to claim 8, characterized in that, The reaction conditions are as follows: react for 8 hours in phosphate buffer solution at 25~40℃ and pH 6~8. After the reaction, add the same volume of acetonitrile solution as the reaction system to terminate the reaction.

Images