Penicillin acylase gene and method for synthesizing beta-lactam antibiotics

Abstract

The invention belongs to the technical field of biology, and discloses penicillin acylase or a mutant thereof, a constructed strain and a method for synthesizing beta-lactam antibiotics by an enzymic method. The penicillin acylase obtained by the invention can catalyze beta-lactam antibiotic parent nucleus and an acyl donor to generate acylation reaction, is used for preparing beta-lactam antibiotics, and has good industrial application prospects in synthesis of cephalosporin drug intermediates catalyzed by a biological enzyme method.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a penicillin acylase production strain, penicillin acylase and mutant, and application in enzymatic synthesis of beta-lactam antibiotics. Background Art

[0002] Penicillin acylase (PGA), also known as penicillin amidase or penicillin aminohydrolase. It is mainly derived from Escherichia coli and Bacillus megaterium cells. The enzyme has been widely used in the industrial production of key intermediates and semi-synthetic β-lactam antibiotics. Under acidic and neutral conditions, the enzyme can be used to catalyze the acylation reaction of 6-aminopenicillin acid (6-APA), 7-aminocephalosporanic acid (7-ACA) or 7-aminodeacetoxycephalosporanic acid (7-ADCA) with acyl donors to prepare semi-synthetic antibiotics; under alkaline conditions, it can catalyze the hydrolysis of penicillin potassium salt, cephalosporin sodium salt, etc. into 6-APA, 7-ADCA or 7-ACA. 6-APA and 7-ADCA (7-ACA) are important mother nuclei for the preparation of semi-synthetic antibiotics, and can be used to prepare highly efficient and broad-spectrum new beta-lactam antibiotics. Therefore, penicillin acylase is an enzyme with high industrial application value.

[0003] Cephalosporins are β-lactam antibiotics with high efficacy and are commonly used to treat mild to severe infectious diseases. According to records from the healthcare database from 2004 to 2014, the number of prescriptions for cephalosporins in the United States (47.5% of total antibiotics) exceeded the number of narrow-spectrum and broad-spectrum penicillins (39.7% of total antibiotics) (Bush and Bradford. β-Lactams and β-Lactamase Inhibitors: An Overview.). Semisynthetic cephalosporins have a wide range of activities against both Gram-positive and Gram-negative bacteria. They are synthesized based on a bicyclic core composed of a six-membered dihydrothiazine ring connected to a β-lactam ring. The two carbons of the cephalosporin skeleton, C3 and C7, provide great possibilities for the introduction of variable side chains, which can significantly expand the antibacterial activity and enhance the structural stability against β-lactamases.

[0004] Cefradine is a first-generation semi-synthetic cephalosporin with similar antibacterial effects to cephalexin. This product is acid-resistant and can be taken orally, has good absorption, and has a high blood concentration. It is characterized by resistance to β-lactamase and has a rapid and reliable bactericidal effect on highly resistant Staphylococcus aureus and other bacilli resistant to broad-spectrum antibiotics. According to existing literature reports, the enzymatic catalytic synthesis activity of cefradine cannot meet the needs of industrial applications. Therefore, it is necessary to explore penicillin acylases with excellent performance, such as strains with good pH tolerance, a wide range of synthetic substrates, and higher thermal stability, to provide a better choice for industrial production. Summary of the invention

[0005] The invention discloses a penicillin acylase production strain. The penicillin acylase expression vector is constructed and the enzyme is heterologously expressed in Escherichia coli (E.coli) BL21 (DE3) to obtain a recombinant enzyme. The penicillin acylase synthesizes a beta-lactam antibiotic with 6-APA, 7-ACA and 7-ADCA as a mother nucleus.

[0006] The invention provides a penicillin acylase or a mutant thereof, which is from Klebsiella sp. WP8-S18-ESBL-06. The penicillin acylase has an amino acid sequence shown in SEQ ID NO.1; the mutant has substitution mutations at the following amino acid sites in the amino acid sequence shown in SEQ ID NO.1: S356, I466, A72, S175, R171, A72, S290, R171, A72, S290, R171, A109, S290, F172, M168, Y316, F360, M168, Y316, F360, F313 Y316, F360, F313, L542, R409, H481, F545, R409, Y537, F545, F748, F543, F545, F748, Y6 28. L546, F748, A651, L546, 544H, T770, L546, 679E, 544H, 664C, 679C, 544H, 666H, 679A.

[0007] Preferably, the mutant has substitution mutations at the following amino acid positions in the amino acid sequence shown in SEQ ID NO.1: M168F / F313G, M168G / Y316C, M168G / F360G, M168G / R409H, M168G / F545G, M168G / F748S, M168G / T770A, F313G / Y316C, F313G / F360G, F313G / R409H, F313G / F545E, F313G / F748S, F313 G / T770A, Y316C / F360G, Y316C / R409H, Y316C / F748S, Y316C / T770A, F360G / R409H, F360G / F545E, F360G / F7 48S, F360G / T770A, R409H / F545E, R409H / F748S, R409H / T770A, F545E / F748S, F545E / T770A, F748S / T770A.

[0008] The present invention provides a nucleic acid encoding the penicillin acylase or its mutant, wherein the nucleic acid is DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid is RNA, such as mRNA or hnRNA.

[0009] The present invention further provides a recombinant vector comprising the coding gene. Specifically, the starting vector is pET-28a.

[0010] The present invention provides a recombinant strain comprising the recombinant vector, specifically, Escherichia coli, more specifically, an expression vector constructed by the nucleic acid and the vector pET-28a is transformed into Escherichia coli BL21 (DE3).

[0011] The present invention further provides the use of the penicillin acylase or its mutant in the synthesis of β-lactam antibiotics, such as cephradine.

[0012] Specifically, the penicillin acylase synthesizes cephalosporin antibiotics with 6-APA, 7-ACA or 7-ADCA as the parent nucleus.

[0013] The present invention also provides a method for preparing β-lactam antibiotics, comprising: using β-lactam antibiotics 6-APA, 7-ACA, 7-ADCA as substrates, using the cells of the recombinant strain according to claim 6 or its lysate to carry out a catalytic reaction to obtain β-lactam antibiotics;

[0014] The catalytic reaction temperature is 10-40°C, specifically 15°C; the reaction is carried out in a phosphate buffer solution with a pH of 6.5 to 9.5, specifically pH 6.5 to 7.5, more specifically pH 7.0;

[0015] The reaction time is 0.5-24 hours, specifically 1 hour.

[0016] The present invention first discovered that penicillin acylase from Klebsiella sp. WP8-S18-ESBL-06 can be used to catalyze the synthesis of β-lactam antibiotics from β-lactam antibiotic nuclei 7-ADCA, 7-ACA, and 6-APA. Furthermore, the penicillin acylase strain provided by the present invention has a relatively wide substrate spectrum and has good industrial application prospects in the biocatalytic preparation of β-lactam antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The present invention is a reaction route diagram for the synthesis of cefradine.

[0018] Figure 2 It is the HPLC detection result of the synthesis of L1 cefradine in the present invention.

[0019] Figure 3 It is the pH curve of the synthesis reaction in the present invention.

[0020] Figure 4 It is the synthesis reaction temperature curve in the present invention. DETAILED DESCRIPTION

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0022] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0023] Example 1 Acylase sequence mining and bacterial cell preparation

[0024] 1. Penicillin acylase sequence mining

[0025] Through NCBI search, the acylase sequence SEQ ID NO.1 (named L1) derived from Klebsiella sp. WP8-S18-ESBL-06 was obtained.

[0026] 2. Bacterial culture and induced expression of penicillin acylase

[0027] The nucleic acid sequence containing the amino acid sequence of SEQ ID NO.1 was transformed into Escherichia coli BL21 (DE3) and the bacterial solution was evenly spread on a kanamycin-resistant LB plate. After culturing at 37°C for 14 hours, a single colony grew out, which was the transformant containing the cephalosporin C acylase nucleic acid sequence.

[0028] The above transformants were transferred to a test tube containing 5 mL of LB medium and cultured at 37°C, 220 rpm overnight; then inoculated into 100 mL of TB medium and cultured at 37°C, 220 rpm for 3 h. 600 When the value reaches 0.6-0.8, add IPTG and continue shaking culture at 18°C ​​and 220rpm for 16h; collect the bacteria by centrifugation at 4°C and 4000rpm for 10min; wash the bacteria with phosphate buffer, centrifuge, discard the supernatant, and obtain wet bacteria.

[0029] 3. Obtaining crude penicillin acylase solution

[0030] Add 10 mL of phosphate buffer to the wet bacteria to resuspend and obtain a bacterial suspension. Use ultrasonic disruption for 15 minutes, centrifuge for 60 minutes, and take the supernatant to obtain a crude enzyme solution.

[0031] 4. Penicillin acylase catalyzes the condensation of 7-ADCA and DHME to produce cefuroxime

[0032] Take 100 μL of supernatant, add 7-ADCA and DHME at a final concentration of 50 mM in a 1 mL system. Reaction conditions: 37°C 1000 rpm, 30 min, take 100 μL of sample, add methanol to terminate and dilute the reaction system. Analyze 7-ADCA conversion product cephradine by HPLC. The conversion rate of 7-ADCA to cephradine was 20%.

[0033] Detection conditions: chromatographic column Agilent ZORBAX SB-C18 (4.6*150mm 4um); mobile phase was 75% phosphate buffer (pH4.5, 30mM) added with 25% methanol (v / v); flow rate was 0.8mL / min; detection wavelength was 230nm, and the column oven was kept constant at 25°C.

[0034] Example 2 Construction of a single-site saturation mutant library of penicillin acylase

[0035] 1. Construction of mutation library

[0036] Using penicillin acylase L1 amino acid as a template, SWISS-MODEL was used to build a model. After the modeling, the protein and the product molecule cephradine were inserted into the model. The action site was analyzed by docking. According to the results, the substrate molecule was selected. The sites that interact with the substrate in and around the region (A72, A109, M168, R171, F172, D174, S175, T176, S290, Q312, F313, G314, Y316, S356, T357, R358, F360, R409, T465, I466, Y469, H481, N530, A single-site saturation mutation library was constructed using the following sequence analysis: Y537, L542, F543, A544, F545, L546, R552, Y628, A651, G664, Y665, E666, P672, T673, G674, S675, L676, N677, I678, S679, V680, K683, F748, and T770).

[0037] Using SEQ ID NO.1 as the sequence, primers were designed to construct the corresponding single-site saturation mutation library acylase gene fragment, which was further constructed into the pET24a expression vector through gene recombination.

[0038] The above-mentioned pET24a vector containing the acylase was transformed into Escherichia coli BL21 (DE3) by electroporation, and the electroporated Escherichia coli BL21 (DE3) bacterial solution was evenly spread on a kanamycin-resistant LB plate, and cultured at 37°C for 14 hours to grow a single colony, which is the genetic mutant engineered strain of penicillin acylase SEQ ID NO.1.

[0039] Example 3 Single point mutation library screening and activity determination

[0040] The single colonies in the mutant library were inoculated in 96 deep-well plates, added LB medium, and cultured at 37°C for 8-12h. A negative control group was set up at the same time, and then the seed liquid was inoculated in TB medium. Induce expression at 18°C ​​for 16h, and collect the cell bodies by centrifugation. Add potassium phosphate buffer, resuspend the bacteria, and incubate at 37°C for 1h. Add substrates 7-ADCA and DHME to the cell lysis solution, and react at 37°C for 4-6h. Transfer the reaction solution to a new 96-well plate, add the stop reaction solution, and centrifuge for 10min. Take the supernatant and add the colorimetric agent. After reacting at room temperature for 10min, detect the absorbance at 415nm.

[0041] Table 2. Relative 7-ADCA conversion rates of single point mutant strains

[0042] Mutation site Conversion rate (%) Mutation site Conversion rate (%) Mutation site Conversion rate (%) WT 20 S356K 22 I466W 21 A72S 50 S175D 56 R171F 21 A72F 25 S290M 21 R171Q 28 A72M 26 S290Y 21 R171M 23 A109L 56 S290K 0 F172V 23 M168F 55 Y316S 52 F360G 22 M168N 23 Y316C 60 F360N 22 F313A 49 Y316Q 47 F360S 20 F313G >99 L542Q 58 R409H 42 H481G 54 F545Q 59 R409C 43 Y537N 49 F545E 79 F748K 26 F543T 53 F545R 61 F748D 20 Y628D 46 L546Q 57 F748S 30 A651N 47 A544I 21 E666W 26 T770A 65 A544M 26 I678N 27 A544P 25 G664L 21 E679R 23 L676A 23

[0043] Example 4 Construction of acylase combination mutants

[0044] Based on the highly active single-point mutants screened in Example 2, the following combination mutants were constructed: M168F / F313G, M168G / Y316C, M168G / F360G, M168G / R409H, M168G / F545G, M168G / F748S, M168G / T770A, F313G / Y316C, F313G / F360G, F313G / R409H, F313G / F545E, F313G / F748S, and F313G / T77 0A, Y316C / F360G, Y316C / R409H, Y316C / F545E, Y316C / F748S, Y316C / T770A, F360G / R409H, F360G / F545E, F360 G / F748S, F360G / T770A, R409H / F545E, R409H / F748S, R409H / T770A, F545E / F748S, F545E / T770A, F748S / T770A.

[0045] Primers designed for constructing combined mutants are shown in Table 3.

[0046] Table 3. Primers for site-directed mutagenesis

[0047]

[0048]

[0049] The specific construction method is as follows:

[0050] 1) PCR

[0051] Take M168F-F / R, F313G-F / R, Y316C-F / R, F360G-F / R, R409H-F / R, F545E-F / R, F748S-F / R, and T770A-F / R in Table 3 as upstream and downstream primers to amplify mutants M168F / F313G, F313G / Y316C, F313G / F360G, F313G / R409H, F313G / F545E, F313G / F748S, F313G / T770A, M168G / Y316C, Y316C / F360G, Y316C / R409H , Y316C / F545E, Y316C / F748S, Y316C / T770A, M168G / F360G, F360G / R409H, F360G / F545E, F360G / F748S, F360G / T770A, M168G / R409H , R409H / F545E, R409H / F748S, R409H / T770A, M168G / F545G, F545E / F748S, F545E / T770A, M168G / F748S, F748S / T770A, M168G / T770A.

[0052] 2) Construction of genetic mutant engineering strains

[0053] The obtained PCR product was subjected to the following operation: Dpn I was added to the PCR product to digest the plasmid template, and the product was treated at a constant temperature of 37°C for 3 hours. The PCR product was electrotransformed into Escherichia coli BL21 (DE3), and the electrotransformed Escherichia coli BL21 (DE3) bacterial solution was evenly spread on a kanamycin-resistant LB plate, and a single colony was grown after culturing at a constant temperature of 37°C for 14 hours, which was the mutant strain of the gene of acylase SEQ ID NO.1.

[0054] Example 5 Synthesis of Cefuroxime by Whole Cell Catalysis of Penicillin Acylase Combination Mutants

[0055] 1. Cell culture

[0056] The single clones of the combined mutants constructed in Example 4 were respectively transferred to a test tube containing 5 mL of LB medium and cultured at 37°C, 220 rpm overnight; then inoculated into 100 mL of TB medium and cultured at 37°C, 220 rpm for 3 h. 600 When the value reaches 0.6-0.8, add IPTG and continue shaking culture at 18°C ​​and 220rpm for 16h; collect the bacteria by centrifugation at 4°C and 4000rpm for 10min; wash the bacteria with phosphate buffer, centrifuge, discard the supernatant, and obtain wet bacteria.

[0057] 2. Whole cell reaction

[0058] 10mL system, add 5mL crude enzyme solution, 50mM substrate 7-ADCA and DHME, 20℃, 220rpm, react for 0.5h. Add methanol to terminate the reaction, detect the generated cephradine in the same manner as in Example 1, and obtain the conversion rate of 7-ADCA of the combined mutants, see Table 4.

[0059] Table 4 Conversion efficiency of combined mutants to 7-ADCA

[0060] Mutation site Conversion rate (%) WT 20 M168F / F313G 66 M168G / Y316C 26 M168G / F360G 24 M168G / R409H 21 M168G / F545G 20 M168G / F748S 25 M168G / T770A 58 F313G / Y316C 32 F313G / F360G 43 F313G / R409H 67 F313G / F545E 26 F313G / F748S 54 F313G / T770A 23 Y316C / F360G 22 Y316C / R409H 23 Y316C / F748S 28 Y316C / T770A 55 F360G / R409H 25 F360G / F545E 28 F360G / F748S 26 F360G / T770A 26 R409H / F545E 28 R409H / F748S 29 R409H / T770A 43 F545E / F748S 26 F545E / T770A 21 F748S / T770A 59

[0061] After single-point and combined mutation screening (Table 4), penicillin acylase mutants such as M168F / F313G, M168G / T770A, F313G / R409H, F313G / F748S, R409H / T770A, and F748S / T770A were obtained, which had high conversion rates for 7-ADCA to cephradine.

Claims

1. A penicillin acylase or a mutant thereof, Features The enzyme comes from Klebsiella Klebsiella sp. WP8-S18-ESBL-06, The penicillin acylase has an amino acid sequence as shown in SEQ ID NO.1; the mutant has substitution mutations at the following amino acid positions in the amino acid sequence as shown in SEQ ID NO.1: S356, I466, A72, S175, R171, A72, S290, R171, A72, S290, R171, A109, S290, F172, M168, Y316, F360, M168, Y316, F360, F313 Y316, F360, F313, L542, R409, H481, F545, R409, Y537, F545, F748, F543, F545, F748, Y6 28. L546, F748, A651, L546, 544H, T770, L546, 679E, 544H, 664C, 679C, 544H, 666H, 679A.

2. The penicillin acylase or a mutant thereof according to claim 1, It is characterized in that The mutant has substitution mutations at the following amino acid positions in the amino acid sequence shown in SEQ ID NO. 1: M168F / F313G, M168G / Y316C, M168G / F360G, M168G / R409H, M168G / F545G, M168G / F748S, M168G / T770A, F313G / Y316C, F313G / F360G, F313G / R409H, F313G / F545E, F313G / F748S, F31 3G / T770A, Y316C / F360G, Y316C / R409H, Y316C / F748S, Y316C / T770A, F360G / R409H, F360G / F545E, F360G / F 748S, F360G / T770A, R409H / F545E, R409H / F748S, R409H / T770A, F545E / F748S, F545E / T770A, F748S / T770A.

3. A nucleic acid encoding the penicillin acylase or a mutant thereof according to any one of claims 1 to 2, wherein the nucleic acid is DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid is RNA, such as mRNA or hnRNA.

4. A recombinant vector comprising the encoding nucleic acid of claim 3.

5. The recombinant vector according to claim 4, It is characterized in that The starting vector is pET-28a.

6. A recombinant strain comprising the recombinant vector according to claim 4 or 5.

7. The recombinant strain according to claim 6, It is characterized in that It is Escherichia coli, more specifically, the expression vector constructed by the encoding nucleic acid of claim 3 and the vector pET-28a is transformed into Escherichia coli BL21 (DE3).

8. Use of a penicillin acylase or a mutant thereof according to claim 1 or 2 in the synthesis of β-lactam antibiotics, such as cephradine.

9. The use according to claim 8, It is characterized in that The penicillin acylase synthesizes cephalosporin antibiotics with 6-APA, 7-ACA or 7-ADCA as the parent nucleus.

10. A method for preparing β-lactam antibiotics, It is characterized in that The method comprises: using β-lactam antibiotics 6-APA, 7-ACA, and 7-ADCA as substrates, and utilizing cells of the recombinant strain according to claim 6 or their lysates to perform a catalytic reaction to obtain β-lactam antibiotics; The catalytic reaction temperature is 10-40 ° C, specifically 15 ° C; the reaction is carried out in a phosphate buffer with a pH of 6.5 to 9.5, specifically pH 6.5 to 7.5, more specifically pH 7.0; The reaction time is 0.5-24 hours, specifically 1 hour.

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