A method for synthesizing fosfomycin by chemical enzymatic method

Abstract

The application discloses a method for synthesizing fosfomycin by a chemical enzyme method, and belongs to the technical field of bioengineering. The method uses chloroacetone and trialkyl phosphite as raw materials to prepare dialkyl acetonyl phosphonate, and then obtains a key substrate 2-oxo-propyl phosphonic acid or a salt compound thereof, i.e. 2-OPP, through a hydrolysis reaction; subsequently, a cascade catalytic system is constructed by using a reductase Psf3 or a functional variant or mutant thereof and a cyclase Psf4 or a functional variant or mutant thereof, so that 2-OPP is converted into fosfomycin or a salt compound thereof through 2-hydroxypropyl phosphonic acid or a salt compound thereof, i.e. 2-HPP. The method can realize the conversion of phosphonate precursors into fosfomycin through 2-OPP, has a simple route, mild reaction conditions, and good application value and industrialization prospect.

Description

Technical Field

[0001] This invention relates to a method for the chemical enzymatic synthesis of fosfomycin, belonging to the field of bioengineering technology. Background Technology

[0002] Fosfomycin is a broad-spectrum antibiotic containing phosphonic acid groups, exhibiting antibacterial activity against both Gram-positive and Gram-negative bacteria. Fosfomycin exerts its bactericidal effect by inhibiting bacterial cell wall synthesis and by affecting bacterial biofilm formation. Clinically, fosfomycin is mainly used in the forms of fosfomycin sodium, fosfomycin calcium, and fosfomycin tromethamine, and can be used to treat various infectious diseases. Therefore, developing efficient, green, and scalable methods for preparing fosfomycin is of great significance.

[0003] Currently, the main methods for preparing fosfomycin include chemical synthesis, bio-fermentation, and enzymatic synthesis. Traditional chemical synthesis has a relatively good industrial foundation, but some routes suffer from problems such as numerous reaction steps, stringent reaction conditions, difficulty in selecting control, the need for chiral resolution or chiral construction, and heavy environmental burden. Bio-fermentation and de novo microbial synthesis have the advantages of mild reaction conditions and good selectivity, but they are usually limited by factors such as precursor supply, metabolic flux distribution, product toxicity, and host tolerance; therefore, the accumulation level of the target product and process stability still need improvement.

[0004] Enzymatic and chemoenzymatic synthesis methods offer new approaches for fosfomycin preparation. Compared to purely chemical synthesis, enzyme-catalyzed reactions typically exhibit better regioselectivity and stereoselectivity. Compared to de novo microbial synthesis, enzymatic transformation using specific phosphonic acid intermediates as substrates reduces dependence on host-centric metabolism and complex natural biosynthetic pathways. However, existing enzymatic or multi-enzyme cascade systems still suffer from problems such as insufficient connection between key phosphonic acid substrate preparation and subsequent enzymatic transformation, limited catalytic efficiency of natural enzymes, low intermediate transfer efficiency in multi-enzyme cascade reactions, and complex preparation processes and high application costs for purified enzyme systems.

[0005] Therefore, existing technologies still require a method for preparing fosfomycin that can take into account substrate source, reaction steps, catalytic efficiency, and feasibility of engineering applications, in order to improve the overall efficiency of the conversion of phosphonic acid precursors to fosfomycin and simplify the preparation and application process of enzyme catalysts. Summary of the Invention

[0006] The purpose of this invention is to provide a chemical enzymatic method for synthesizing fosfomycin, in order to solve the problems of insufficient connection between phosphonic acid substrate preparation and subsequent enzymatic conversion in the existing fosfomycin preparation process, limited catalytic efficiency of natural enzymes, low transfer efficiency of intermediates in multi-enzyme cascade reactions, and complex enzyme catalyst preparation process.

[0007] The first technical solution provided by the present invention is a chemical enzymatic synthesis method for fosfomycin, the method comprising a step of chemically preparing 2-OPP and a step of enzymatically catalyzing 2-OPP to generate fosfomycin.

[0008] In one embodiment, the method includes the following steps: The first step involves reacting chloroacetone and trialkyl phosphite as raw materials to prepare acetone-based dialkyl phosphonate. The second step involves hydrolyzing the acetone-phosphonic acid dialkyl ester to prepare 2-oxopropylphosphonic acid or its salt compound, namely 2-OPP. The third step involves using 2-OPP as a substrate and converting it into 2-hydroxypropylphosphonic acid or its salt compounds, namely 2-HPP, under the catalysis of Psf3 catalytic protein. The fourth step involves converting 2-HPP into fosfomycin or its salt compounds under the catalytic action of the Psf4 catalytic protein. Among them, the Psf3 catalytic protein is the wild-type Psf3 protein or the Psf3 mutant described above.

[0009] The Psf4 catalytic protein is a wild-type Psf4 protein, a Psf4 homolog, or a Psf4 mutant.

[0010] In one embodiment, the trialkyl phosphite is a compound of formula P(OR)3, wherein R is a C1-C6 alkyl group. The C1-C6 alkyl group is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, or n-hexyl. Preferably, the trialkyl phosphite is trimethyl phosphite or triethyl phosphite.

[0011] In one embodiment, the acetone-phosphonate dialkyl ester is a compound of the formula CH3COCH2P(O)(OR)2, wherein R is a C1-C6 alkyl group. Preferably, when the trialkyl phosphite is trimethyl phosphite, chloroacetone reacts with trimethyl phosphite to generate acetone-phosphonate dimethyl ester; when the trialkyl phosphite is triethyl phosphite, chloroacetone reacts with triethyl phosphite to generate acetone-phosphonate diethyl ester.

[0012] The reaction route is as follows: Chloroacetone + Trialkyl Phosphite → Dimethyl Pyruvate → 2-OPP → 2-HPP → Fosfomycin; Preferably: Chloroacetone + Trimethyl phosphite → Dimethyl pyruvate → 2-OPP → 2-HPP → Fosfomycin; or Chloroacetone + Triethyl phosphite → Diethyl acetone phosphonate → 2-OPP → 2-HPP → Fosfomycin.

[0013] In one embodiment, the hydrolysis reaction is an acidic hydrolysis reaction, and the acid used in the acidic hydrolysis reaction includes one or more of hydrochloric acid, sulfuric acid, phosphoric acid, and trifluoroacetic acid.

[0014] In one embodiment, the reaction system includes one or more of NADH, NADPH, metal ions, and flavin cofactors. The metal ion is ferrous ion or ferric ion, preferably ferrous ion; the flavin cofactor is flavin mononucleotide or flavin adenine dinucleotide, preferably flavin mononucleotide.

[0015] In one embodiment, the nucleotide sequence of the gene encoding the Psf3 wild-type protein is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0016] In one embodiment, the nucleotide sequence of the gene encoding the Psf4 wild-type protein is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0017] In another embodiment, the amino acid sequence of the Psf4 homolog is as shown in SEQ ID NO.5.

[0018] In one embodiment, the Psf3 mutant is a protein with the amino acid sequence shown in SEQ ID NO.2 containing one or more mutations selected from the following: E180Q, K173R, N177Q, V234I, V238I, R239H, D242N. Preferably, the Psf3 mutant is Psf3. E180Q / V234I .

[0019] In one embodiment, the Psf4 mutant is a protein with the amino acid sequence shown in SEQ ID NO.2 containing one or more mutations selected from the following: R87K, Y95H, L112I, N125Q, F173Y, A186V. Preferably, the Psf4 mutant is Psf4. L112I / N125Q .

[0020] The second technical solution provided by this invention is a Psf3 mutant, wherein the Psf3 mutant contains one or more mutations selected from the following, relative to the wild-type Psf3 with the amino acid sequence shown in SEQ ID NO.2: E180Q, K173R, N177Q, V234I, V238I, R239H, D242N.

[0021] The Psf3 mutant is used to catalyze the conversion of 2-OPP to 2-HPP.

[0022] In one embodiment, the Psf3 mutant is a single-point mutant, including but not limited to: Psf3-E180Q, Psf3-K173R, Psf3-N177Q, Psf3-V234I, Psf3-V238I, Psf3-R239H, Psf3-D242N.

[0023] In another embodiment, the Psf3 mutant is a combined mutant, including but not limited to a Psf3 mutant containing any two or more mutations of E180Q, K173R, N177Q, V234I, V238I, R239H and D242N.

[0024] In a preferred embodiment, the Psf3 mutant is used to improve the conversion efficiency of 2-OPP to 2-HPP.

[0025] In one embodiment, the Psf3 catalytic protein and the Psf4 catalytic protein can form a fusion protein.

[0026] The third technical solution provided by this invention is a Psf4 mutant, wherein the Psf4 mutant contains one or more mutations selected from the following, relative to the wild-type Psf4 with the amino acid sequence shown in SEQ ID NO.4: R87K, Y95H, L112I, N125Q, F173Y, A186V.

[0027] The Psf4 mutant is used to catalyze the conversion of 2-HPP into fosfomycin or its salt compounds.

[0028] In one embodiment, the Psf4 mutant is a single-point mutant, including but not limited to: Psf4-R87K, Psf4-Y95H, Psf4-L112I, Psf4-N125Q, Psf4-F173Y, Psf4-A186V.

[0029] In another embodiment, the Psf4 mutant is a combined mutant, including but not limited to a Psf4 mutant containing any two or more mutations of R87K, Y95H, L112I, N125Q, F173Y and A186V.

[0030] In a preferred embodiment, the Psf4 mutant is used to improve the conversion efficiency of 2-HPP to fosfomycin.

[0031] The fourth technical solution provided by the present invention is a fusion protein, which includes a Psf3 unit, a linker peptide, and a Psf4 unit. The Psf3 unit is selected from Psf3 or the Psf3 mutant described in the second technical solution; the Psf4 unit is selected from Psf4, a Psf4 homolog, or the Psf4 mutant described in the third technical solution.

[0032] In one embodiment, the fusion protein has a structure of Psf3 unit-linker-Psf4 unit or Psf4 unit-linker-Psf3 unit.

[0033] In one embodiment, the linker peptide is a flexible linker peptide or a rigid linker peptide. The flexible linker peptide includes one of GSGGS, (GSGGS)2, (GSGGS)3, or (GSGGS)4; the rigid linker peptide includes one of EKAAK, (EKAAK)2, A(EKAAK)2A, A(EKAAK)3A, or A(EKAAK)4A.

[0034] In a preferred embodiment, the fusion protein includes Psf3. E180Q / V234I -GSGGS-Psf4 L112I / N125Q Psf4 L112I / N125Q -GSGGS-Psf3 E180Q / V234I The fifth technical solution provided by the present invention is a gene encoding the mutant described in the second technical solution, the mutant described in the third technical solution, or the fusion protein described in the fourth technical solution.

[0035] The sixth technical solution provided by the present invention is a recombinant vector, wherein the recombinant vector carries the gene described in the fifth technical solution.

[0036] The seventh technical solution provided by the present invention is to express the mutant described in the second technical solution, the mutant described in the third technical solution, or the fusion protein described in the fourth technical solution, or to contain the gene described in the fifth technical solution, or to transform a host cell with the recombinant vector described in the sixth technical solution.

[0037] In one embodiment, the host cell is Escherichia coli.

[0038] The eighth technical solution provided by the present invention is an enzyme composition for synthesizing fosfomycin, the enzyme composition comprising: Psf3 or its functional variants or mutants and Psf4 or its functional variants or mutants; or, the enzyme composition comprising a fusion protein formed by Psf3 units and Psf4 units.

[0039] In one embodiment, the enzyme composition may further include one or more of NADH, NADPH, buffer solution, metal ions, or flavin cofactors.

[0040] In one embodiment, the metal ion is ferrous ion or ferric ion, preferably ferrous ion; the flavin cofactor is flavin mononucleotide or flavin adenine dinucleotide, preferably flavin mononucleotide.

[0041] The ninth technical solution provided by the present invention is the application of the Psf3 mutant described in the second technical solution, the Psf4 mutant described in the second technical solution, the fusion protein described in the third technical solution, the gene described in the fourth technical solution, the recombinant vector described in the fifth technical solution, the host cell described in the sixth technical solution, or the enzyme composition described in the seventh technical solution in the preparation of fosfomycin or its salt compounds.

[0042] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention combines chemical substrate preparation with enzymatic cascade catalysis. Using chloroacetone and trialkyl phosphite as raw materials, acetone-based dialkyl phosphonate is prepared, and the key intermediate 2-OPP is obtained by hydrolysis. Fosfomycin or its salt compounds are then synthesized through Psf3 / Psf4 cascade catalysis. The route is simple and the reaction conditions are mild.

[0043] This invention uses trialkyl phosphite as a phosphonate precursor source, and can prepare corresponding acetone-based dialkyl phosphonates according to different alkyl ester raw materials; wherein, trimethyl phosphite and triethyl phosphite can be used to prepare acetone-based dimethyl phosphonate and acetone-based diethyl phosphonate respectively, providing different raw material options for the preparation of 2-OPP.

[0044] This invention obtained Psf3 and Psf4 mutants through enzyme engineering, which can improve the catalytic efficiency of the conversion of 2-OPP to fosfomycin via 2-HPP. Specifically, Psf3... E180Q / V234I The conversion ability of Psf4 to 2-OPP is 4.2 times that of wild-type Psf3. L112I / N125Q Its relative activity in the cascade reaction system is 6.3 times that of wild-type Psf4.

[0045] This invention constructed a Psf3-Psf4 fusion protein and verified that both the flexible linker peptide GSGGS and the rigid linker peptide EKAAK can be used to construct a catalytically active Psf3. E180Q / V234I -Psf4 L112I / N125Q Fusion proteins. Among them, Psf4... L112I / N125Q -GSGGS-Psf3 E180Q / V234IThe fusion protein can generate 4.3 mM fosfomycin in the in vitro cascade reaction, indicating that the type of linker peptide and the fusion direction affect the efficiency of the cascade reaction.

[0046] This invention further constructs a recombinant E. coli whole-cell catalytic system expressing the Psf3-Psf4 fusion protein. Psf3 expression... E180Q / V234I -GSGGS-Psf4 L112I / N125Q Recombinant E. coli BL21 with fusion protein ( DE3 Whole cells can catalyze the conversion of 20 mM 2-OPP to 18 mM fosfomycin with a conversion rate of about 90%, which can reduce protein purification steps and simplify catalyst preparation process, and has good application value and industrialization prospects. Attached Figure Description

[0047] Figure 1 The 1H NMR spectrum of 2-OPP obtained in Example 1; Figure 2 The mass spectrometry result of 2-OPP obtained in Example 1 is shown below. Figure 3 The diagrams show the expression and purification analysis of enzyme elements in the cascade reaction system; (a) Protein expression and purification analysis of reductase Psf3; (b) Protein expression and purification analysis of cyclase Psf4. Figure 4 HPLC chromatograms of 2-OPP standard, fosfomycin standard, and Psf3 / Psf4 cascade reaction products; Figure 5 The phosphorus NMR spectrum of fosfomycin, a product of the Psf3 / Psf4 cascade reaction; Figure 6 Mass spectrometry of fosfomycin, a product of the Psf3 / Psf4 cascade reaction; Figure 7 An expression analysis diagram of Psf3-Psf4 or Psf4-Psf3 fusion proteins constructed with different linker peptides; Figure 8 HPLC chromatograms of different fusion proteins catalyzing the formation of fosfomycin from 2-OPP; Figure 9 This is the standard curve for fosfomycin.

[0048] Figure 10 Recombinant E. coli BL21 expressing the Psf4*-GSGGS-Psf3* fusion protein ( DE3 HPLC detection diagram of whole-cell catalysis of 2-OPP to generate fosfomycin. Detailed Implementation

[0049] refer to Figures 1-10The preferred embodiments of the present invention will be described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0050] Detection method: High-performance liquid chromatography (HPLC) detection conditions: A ChromCore SAA column (4.6 × 150 mm, 5 µm) was used; the column temperature was 30°C, and the detection wavelength was 210 nm. The mobile phase was a two-phase solvent system of acetonitrile and double-distilled water, wherein the double-distilled water contained 100 mM ammonium acetate, and the pH was 5.2; the acetonitrile to water ratio was 30:70; the mobile phase flow rate was 1 mL / min; and the elution time was 20 min.

[0051] Materials used in the examples: The antibiotics used in this invention, such as ampicillin sodium, kanamycin sulfate, and streptomycin sulfate, were all sourced from Shanghai Sangon Biotech. The PCR enzymes and homologous recombinant enzymes involved in the molecular experiments in this invention were all purchased from Takara Bio Engineering (Dalian) Co., Ltd. The tryptone, yeast extract, and sodium chloride used in this invention were purchased from Shanghai Sangon Biotech. The chemical reagents, such as chloroacetone, trimethyl phosphite, and triethyl phosphite, were purchased from Sinopharm Chemical Reagent Co., Ltd., and the methanol, acetonitrile, and deuterated reagents were purchased from Shanghai Titan Technology Co., Ltd.

[0052] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0053] 2YT medium: tryptone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L.

[0054] Example 1: Preparation of 2-OPP from chloroacetone and trialkyl phosphite This embodiment provides a method for preparing 2-OPP from chloroacetone and trialkyl phosphite.

[0055] The specific process is as follows: This embodiment provides a method for preparing 2-oxopropylphosphonic acid or its salt compound, i.e., 2-OPP, from chloroacetone and a trialkyl phosphite. The trialkyl phosphite is either trimethyl phosphite or triethyl phosphite, and correspondingly, the resulting dialkyl pyruvate is either dimethyl pyruvate or diethyl pyruvate. The specific process is as follows: Under nitrogen protection, 6.40 g (38.6 mmol) of potassium iodide and 2.9 mL (36 mmol) of chloroacetone were dissolved in a mixed solvent of 20 mL acetone and 20 mL acetonitrile. Separately, 6.0 mL (35 mmol) of triethyl phosphite was dissolved in a mixed solvent of 10 mL acetone and 10 mL acetonitrile to obtain a triethyl phosphite solution. At room temperature, the triethyl phosphite solution was slowly added dropwise to the reaction system containing potassium iodide and chloroacetone over 30 min. After the addition was complete, the reaction mixture was stirred at room temperature for 14 h. After the reaction was complete, the reaction solution was filtered through a glass filter lined with diatomaceous earth, and the filtrate was collected and concentrated under reduced pressure to obtain 7.1 g of crude diethyl acetone phosphonate. The crude product was used directly for subsequent hydrolysis without further purification.

[0056] Using the same method, but replacing triethyl phosphite with trimethyl phosphite, dimethyl pyruvate can be prepared. Specifically, under nitrogen protection, potassium iodide, chloroacetone, and trimethyl phosphite are reacted in an acetone / acetonitrile mixed solvent. After the reaction, the mixture is filtered through diatomaceous earth and concentrated under reduced pressure to obtain the crude product, dimethyl pyruvate. The obtained dimethyl pyruvate is used directly in subsequent hydrolysis reactions without further purification.

[0057] Subsequently, the obtained diethyl or dimethyl pyruvate was added to an acidic hydrolysis system for hydrolysis. Specifically, diethyl or dimethyl pyruvate was added to 3 M hydrochloric acid and reacted under reflux at 100 °C for 10 h to hydrolyze the phosphonate groups. After the reaction, the solvent and excess acid were removed by vacuum concentration, and the pH was adjusted or a salt was formed to obtain approximately 6.5 g of 2-OPP or its salts. The product was used directly for subsequent enzymatic reactions without further purification.

[0058] The structure of the obtained hydrolysis products was further confirmed using 1H NMR and mass spectrometry. The 1H NMR results are shown below. Figure 1 As shown, the mass spectrometry results are as follows: Figure 2 As shown in the figure. The 1H NMR results showed that the hydrolysis product had a characteristic hydrogen signal consistent with the structure of 2-OPP; the mass spectrometry results showed that the hydrolysis product had a characteristic ion peak consistent with the molecular weight of 2-OPP. These results indicate that 2-OPP can be prepared from dialkyl pyruvate ester via hydrolysis. The obtained 2-OPP can be further used as a substrate for subsequent Psf3 / Psf4 cascade enzymatic reactions.

[0059] Example 2: Expression of Psf3 and Psf4 The Psf3 encoding gene (SEQ ID NO.1) and the Psf4 encoding gene (SEQ ID NO.3) were ligated to plasmid pET-21b(+) to construct recombinant vectors pET-21b(+)-Psf3 and pET-21b(+)-Psf4, respectively, and then transformed into Escherichia coli BL21 (…). DE3 In the expression host, recombinant expression strains of Psf3 and Psf4 were obtained.

[0060] The obtained recombinant expression strains were inoculated into 2YT liquid medium containing 100 μg / mL ampicillin and cultured. The culture conditions were 115 rpm and 37℃. When the bacterial culture OD... 600 When the concentration reaches 0.6-0.8, the culture temperature is lowered to 18℃, and isopropyl-β-D-thiogalactoside (IPTG) is added to a final concentration of 0.1 mmol / L. Induction culture continues for another 8 h. The induced bacterial culture is centrifuged (8000 rpm, 5 min, 4℃), the supernatant is discarded, and the bacterial cells are collected. The bacterial cells are resuspended in lysis buffer at a ratio of 10 mL of lysis buffer per 1 g of bacterial cells. The cells are homogenized using a high-pressure homogenizer, and then centrifuged (40000×g, 30 min). The supernatant is collected to obtain the crude enzyme solution.

[0061] The crude enzyme solution was purified by affinity chromatography using a Ni-NTA column. After loading, impurities were washed with 10-fold lysis buffer, and the target protein was eluted with elution buffer. The eluted target protein was collected and desalted using a protein concentration tube with desalting buffer (25 mmol / L Tris-HCl, 150 mmol / L NaCl, 10% glycerol). After desalting, the concentration was increased to 10 mg / mL for subsequent reactions. The purified protein was analyzed by 10% SDS-PAGE gel electrophoresis. The results are shown in the figure. Figure 3 We successfully obtained purified protein with clear target bands that matched the theoretical molecular weight.

[0062] Example 3: Psf3 / Psf4 cascade reaction with 2-OPP as substrate Using 2-OPP obtained in Example 1 as a substrate and the purified Psf3 and Psf4 proteins obtained in Example 2 as catalysts, a Psf3 / Psf4 dual-enzyme cascade reaction system was constructed to catalyze the generation of fosfomycin from 2-OPP.

[0063] Specifically, 2-OPP, purified Psf3 protein, purified Psf4 protein, NADH and / or NADPH, ferrous ions, flavin mononucleotide, and buffer were mixed to construct an enzymatic reaction system. In this reaction system, the final concentrations of 2-OPP, Psf3 protein, and Psf4 protein were 5 μM, NADH and / or NADPH were 5 mM, ferrous ions were 2 mM, and flavin mononucleotide were 1 mM. The buffer solution was 50 mM HEPES with a pH of 7.5. The reaction system was incubated at 35 °C for 3 h. After the reaction, 5 volumes of methanol were added to terminate the reaction, followed by centrifugation at 20000 × g for 5 min, and the supernatant was collected. The supernatant was used for subsequent HPLC analysis. The HPLC results are shown below. Figure 4 As shown, the product obtained by co-catalysis of Psf3 and Psf4 is consistent with the fosfomycin standard, indicating that Psf3 and Psf4 can generate fosfomycin via a cascade catalytic reaction using 2-OPP as a substrate. The NMR spectrum and mass spectrum of the fosfomycin product are shown below. Figure 5 and Figure 6 As shown.

[0064] To quantitatively analyze the generated fosfomycin, a standard curve was established using fosfomycin standards. Specifically, a linear regression was performed with different concentrations of fosfomycin standards as the x-axis and the corresponding fosfomycin peak area at a detection wavelength of 210 nm as the y-axis to obtain the fosfomycin standard curve. Figure 7 The amount of fosfomycin produced in the dual-enzyme cascade reaction system was calculated based on the standard curve. The results showed that, under the above conditions, the yield of fosfomycin in the reaction solution was 0.5 mM.

[0065] This example demonstrates that Psf3 and Psf4 can form a dual-enzyme cascade catalytic system to achieve the enzymatic synthesis of fosfomycin using 2-OPP as a substrate.

[0066] Example 4: Construction and screening of Psf3 mutants Using the recombinant plasmid pET-21b(+)-Psf3 as a template, full-plasmid PCR was performed using primers E180Q-F / E180Q-R, K173R-F / K173R-R, N177Q-F / N177Q-R, V234I-F / V234I-R, V238I-F / V238I-R, R239H-F / R239H-R, and D242N-F / D242N-R (primers are shown in Table 1) to obtain plasmids of the corresponding Psf3 mutants. Subsequently, the protein expression method described in Example 2 was used to obtain the corresponding Psf3 mutant proteins, and the ability of each Psf3 mutant to catalyze the production of 2-HPP was detected using 2-OPP as a substrate. Specifically, the final concentration of 2-OPP was 5 mM, the final concentration of Psf3 and its mutant purified protein was 5 μM, and the final concentration of NADH and / or NADPH was 5 mM. The buffer solution was 50 mM HEPES with a pH of 7.5. The above reaction system was incubated at 35 °C for 3 h. The post-reaction treatment was the same as in Example 3.

[0067] Table 1 Primer names and primer sequences

[0068] Using wild-type Psf3 as a control, the relative activity of different mutants was represented by the conversion rate of the substrate 2-OPP after the mutant reaction. The results are shown in Table 2. The conversion rate of 2-OPP by wild-type Psf3 was 20%; the conversion rate of mutant Psf3 was... E180Q Psf3 V234I Psf3 V238I and Psf3 D242N They exhibited 2-OPP conversion rates of 72%, 40%, 46%, and 50%, respectively; among them, the combined mutant Psf3... E180Q / V234I The genotype Psf3* achieved an 84% conversion rate for 2-OPP, with a relative activity 4.2 times that of wild-type Psf3. This result indicates that Psf3… E180Q / V234I It can significantly improve the conversion efficiency of 2-OPP to 2-HPP.

[0069] Table 2. Relative enzyme activities of the Psf3 reductase mutant

[0070] Example 5: Construction and screening of Psf4 mutants Using the recombinant plasmid pET-21b(+)-Psf4 as a template, full plasmid PCR was performed using primers R87K-F / R87K-R, Y95H-F / Y95H-R, L112I-F / L112I-R, N125Q-F / N125Q-R, F173Y-F / F173Y-R, and A186V-F / A186V-R (primers are shown in Table 3) to obtain the plasmid of the corresponding Psf4 mutant. Subsequently, the protein expression method described in Example 2 was used to obtain the corresponding Psf4 mutant protein. Since the intermediate 2-HPP did not exhibit a clear, stable, and quantitatively usable characteristic chromatographic peak at a detection wavelength of 210 nm, this example did not directly use 2-HPP as the HPLC quantitative detection target; instead, Psf3 was used. E180Q / V234I In-situ catalytic conversion of 2-OPP to 2-HPP was performed, and the ability of different Psf4 mutants to promote fosfomycin production in the cascade reaction system was further evaluated. Specifically, the final concentration of 2-OPP was 5 mM, and the concentration of Psf3 was... E180Q / V234I The final concentrations were 5 μM for Psf4 or its mutant purified protein, 5 mM for NADH and / or NADPH, 2 mM for ferrous ions, and 1 mM for flavin mononucleotides. The buffer solution was 50 mM HEPES with a pH of 7.5. The reaction system was incubated at 35 °C for 3 h. After the reaction was complete, 5 volumes of methanol were added to terminate the reaction, followed by centrifugation at 20000 × g for 5 min. The supernatant was collected for HPLC analysis.

[0071] Table 3 Primer names and primer sequences

[0072] Using wild-type Psf4 as a control, the relative activity of different Psf4 mutants was evaluated based on the fosfomycin yield after the reaction. The results showed that the fosfomycin yield in the cascade reaction system involving wild-type Psf4 was 0.5 mM; the mutant Psf4… N125Q and Psf4 F173Y The corresponding fosfomycin yields were 2.15 mM and 1.6 mM, respectively; among them, the combined mutant Psf4 L112I / N125Q The resulting product, Psf4*, yielded 3.15 mM of fosfomycin, with a relative activity 6.3 times that of wild-type Psf4. This result indicates that Psf4… L112I / N125Q It can significantly improve the generation efficiency of fosfomycin in cascade reaction systems.

[0073] Table 4. Relative enzyme activities of the Psf4 cyclase mutant

[0074] Example 6: Construction and application of Psf3-Psf4 fusion protein To improve the efficiency of the Psf3 and Psf4 cascade catalysis of 2-OPP to fosfomycin, a fusion protein of Psf3 and Psf4 was constructed. In this example, Psf3* represents Psf3. E180Q / V234I Psf4* represents Psf4 L112I / N125Q .

[0075] Specifically, using the Psf3* and Psf4* encoding genes as templates, Psf3*-GSGGS-Psf4*, Psf3*-EKAAK-Psf4*, Psf4*-GSGGS-Psf3*, and Psf4*-EKAAK-Psf3* fusion genes were constructed via gene synthesis or overlap extension PCR. The obtained fusion genes were cloned into the expression vector pET-21b(+) to obtain the corresponding recombinant vectors. Subsequently, these recombinant vectors were transformed into *E. coli* BL21(…). DE3 In the expression host, the corresponding recombinant expression strain of fusion protein was obtained.

[0076] The corresponding fusion protein was obtained using the protein expression and purification method described in Example 2. The purified fusion protein was analyzed by 10% SDS-PAGE gel electrophoresis, and the results are shown below. Figure 7 As shown, the target bands consistent with the theoretical molecular weights were detected in the fusion proteins Psf3*-GSGGS-Psf4*, Psf3*-EKAAK-Psf4*, Psf4*-GSGGS-Psf3*, and Psf4*-EKAAK-Psf3*, indicating that the above fusion proteins could be successfully expressed and purified in the E. coli expression system.

[0077] Furthermore, using 2-OPP as a substrate, the ability of different fusion proteins to catalyze the formation of fosfomycin was investigated. The in vitro reaction, post-reaction processing, and detection were performed using the method described in Example 3. HPLC results are as follows: Figure 8 As shown, the Psf3*-GSGGS-Psf4*, Psf3*-EKAAK-Psf4*, Psf4*-GSGGS-Psf3*, and Psf4*-EKAAK-Psf3* fusion proteins can all catalyze the production of fosfomycin from 2-OPP. Specifically, the fosfomycin yield was 3.15 mM in the free Psf3* and Psf4* combination system; 2.8 mM in the Psf3*-GSGGS-Psf4* fusion protein system; 3.0 mM in the Psf3*-EKAAK-Psf4* fusion protein system; 4.3 mM in the Psf4*-GSGGS-Psf3* fusion protein system; and 2.7 mM in the Psf4*-EKAAK-Psf3* fusion protein system. The above results indicate that both GSGGS and EKAAK linker peptides can be used to construct catalytically active Psf3*-Psf4* fusion proteins. Among them, the Psf4*-GSGGS-Psf3* fusion protein exhibited a higher fosfomycin production capacity, suggesting that fusion expression of Psf3* and Psf4* via linker peptides can promote the transfer of the cascade reaction intermediate 2-HPP between the two catalytic units, thereby improving the overall conversion efficiency of 2-OPP to fosfomycin.

[0078] Example 7: Whole-cell catalytic synthesis of fosfomycin from 2-OPP To further simplify the enzyme catalyst preparation process and reduce the impact of protein purification steps on the reaction application, this example uses recombinant E. coli BL21 expressing the Psf4*-GSGGS-Psf3* fusion protein. DE3 Using whole cells as catalysts and 2-OPP as substrates, the formation of fosfomycin was detected.

[0079] Specifically, recombinant Escherichia coli BL21 expressing the Psf4*-GSGGS-Psf3* fusion protein was constructed according to the method described in Example 6. DE3 The recombinant Escherichia coli BL21 was expressed as a strain. DE3 Inoculate into 2YT liquid medium containing 100 μg / mL ampicillin and incubate at 115 rpm and 37 ℃ until OD. 600 After the concentration reached 0.6-0.8, the culture temperature was lowered to 18 ℃, and IPTG was added to a final concentration of 0.1 mM. Induction culture was continued for another 8 h. After induction, the bacterial culture was centrifuged at 4 ℃ and 7000 rpm for 8 min, the supernatant was discarded, and recombinant *E. coli* cells expressing the Psf4*-GSGGS-Psf3* fusion protein were collected. The cells were then washed three times with 50 mM HEPES buffer, and the washed cells were resuspended in 50 mM HEPES buffer at pH 7.5 to obtain the whole-cell catalyst.

[0080] A whole-cell catalytic reaction system was constructed using 2-OPP as a substrate. The reaction system included 2-OPP, recombinant *E. coli* BL21 expressing the Psf3*-GSGGS-Psf4* fusion protein, and... DE3 Whole cells, 50 mM HEPES buffer, and ferrous ions. The final concentration of 2-OPP was 20 mM, and the amount of recombinant E. coli whole cells added was OD0.05. 600= 40, with a final ferrous ion concentration of 5 mM. The above reaction system was placed at 35 ℃ and 250 rpm for 12 h. After the reaction was completed, 5 times the volume of methanol was added to the reaction solution to terminate the reaction, followed by centrifugation at 20000 × g for 5 min, and the supernatant was collected. The obtained supernatant was used for subsequent HPLC analysis. The amount of fosfomycin generated in the reaction solution was calculated according to the fosfomycin standard curve and converted to mM according to the molecular weight of fosfomycin free acid.

[0081] HPLC detection results are as follows Figure 10 As shown, in the above whole-cell catalytic system, a product peak with the same retention time as the fosfomycin standard was detected in the reaction solution. Based on the fosfomycin standard curve, the 20 mM 2-OPP reaction was carried out by recombinant E. coli BL21 expressing the Psf4*-GSGGS-Psf3* fusion protein. DE3 After whole-cell catalysis for 12 h, 18 mM fosfomycin can be generated, with a molar conversion rate of approximately 90% for 2-OPP to fosfomycin. This result indicates that recombinant *E. coli* BL21 expressing the Psf4*-GSGGS-Psf3* fusion protein... DE3 Whole cells can be directly used as catalysts for the enzymatic conversion of 2-OPP to fosfomycin, which can reduce protein purification steps, simplify the catalyst preparation process, and improve the application convenience of whole-cell catalysis systems.

[0082] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A Psf3 mutant, characterized in that, The Psf3 mutant contains one or more mutations selected from the following, relative to the wild-type Psf3 with the amino acid sequence shown in SEQ ID NO.2: E180Q, K173R, N177Q, V234I, V238I, R239H, D242N.

2. A Psf4 mutant, characterized in that, The Psf4 mutant contains one or more mutations selected from the following, relative to the wild-type Psf4 with the amino acid sequence shown in SEQ ID NO.4: R87K, Y95H, L112I, N125Q, F173Y, A186V.

3. A fusion protein, characterized in that, The fusion protein comprises a Psf3 unit, a linker peptide, and a Psf4 unit, wherein the Psf3 unit is selected from one of the wild-type Psf3 protein and the Psf3 mutant of claim 1; the Psf4 unit is selected from one of the wild-type Psf4 protein, the Psf4 homolog, or the Psf4 mutant of claim 2; the amino acid sequences of the wild-type Psf3 protein, the wild-type Psf4 protein, and the Psf4 homolog are shown in SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.5, respectively; the linker peptide is a flexible linker peptide or a rigid linker peptide; the flexible linker peptide includes one of GSGGS, (GSGGS)2, (GSGGS)3, or (GSGGS)4; the rigid linker peptide includes one of EKAAK, (EKAAK)2, A(EKAAK)2A, A(EKAAK)3A, or A(EKAAK)4A.

4. A biomaterial, characterized in that, The biomaterial is any one of the following: (1) A gene encoding the Psf3 mutant of claim 1, the Psf4 mutant of claim 2, or the fusion protein of claim 3; (2) The gene recombination vector described in (1); (3) Expressing the Psf3 mutant of claim 1, the Psf4 mutant of claim 2, or the fusion protein of claim 3, or containing the gene of (1), or a host cell transformed with the recombinant vector of (2).

5. A chemical enzymatic synthesis method for fosfomycin, characterized in that, The method includes the following steps: The first step involves reacting chloroacetone and trialkyl phosphite as raw materials to prepare acetone-based dialkyl phosphonate. The second step involves hydrolyzing the acetone-phosphonic acid dialkyl ester to prepare 2-oxopropylphosphonic acid or its salt compound, namely 2-OPP. The third step involves using 2-OPP as a substrate and converting it into 2-hydroxypropylphosphonic acid or its salt compounds, namely 2-HPP, under the catalysis of Psf3 catalytic protein. The fourth step involves converting 2-HPP into fosfomycin or its salt compounds under the catalytic action of the Psf4 catalytic protein. Wherein, the Psf3 catalytic protein is the wild-type Psf3 protein or the Psf3 mutant as described in claim 1; The Psf4 catalytic protein is a wild-type Psf4 protein, a Psf4 homolog, or a Psf4 mutant as described in claim 2.

6. The method according to claim 5, characterized in that, The trialkyl phosphite is a compound of formula P(OR)3, wherein R is a C1-C6 alkyl group. The C1-C6 alkyl group is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, or n-hexyl; the dialkyl acetone phosphonate is a compound of formula CH3COCH2P(O)(OR)2, wherein R is a C1-C6 alkyl group.

7. The method according to claim 5, characterized in that, The reaction system in the third and fourth steps also includes one or more of NADH, NADPH, metal ions, and flavin cofactors; wherein the metal ion is ferrous ion or ferric ion; and the flavin cofactor is flavin mononucleotide or flavin adenine dinucleotide.

8. An enzyme composition for synthesizing fosfomycin, characterized in that, The enzyme composition comprises a Psf3 catalytic protein and a Psf4 catalytic protein, wherein the Psf3 catalytic protein is a wild-type Psf3 protein or a Psf3 mutant as described in claim 1, and the Psf4 catalytic protein is a wild-type Psf4 protein or a Psf4 homolog or a Psf4 mutant as described in claim 2. Alternatively, the enzyme composition may comprise the fusion protein of claim 3.

9. The enzyme composition according to claim 8, characterized in that, The enzyme composition also includes one or more of NADH, NADPH, buffer solution, metal ions, or flavin cofactors.

10. The use of the Psf3 mutant of claim 1, the Psf4 mutant of claim 2, the fusion protein of claim 3, the biomaterial of claim 4, or the enzyme composition of any one of claims 8-9 in the preparation of fosfomycin or its salt compounds.

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