Epoxide hydrolase, gene, vector, engineering bacterium and application

Abstract

The invention relates to the technical field of bioengineering and fine chemical engineering, in particular to epoxide hydrolase, a gene, a carrier, engineering bacteria and application, and the amino acid sequence of the epoxide hydrolase is shown as SEQ ID NO.3. The epoxide hydrolase disclosed by the invention has the advantages that the epoxide hydrolase is Sphingopyis macrogoltabia EY-1, and the structural formula of the epoxide hydrolase is as shown in the specification. According to the invention, a recombinant plasmid pET-28a-smh containing an optimized gene and a genetic engineering bacterium E.coli / smh are constructed, the engineering bacterium is induced to over-express the recombinant epoxide hydrolase SmEH, and the recombinant epoxide hydrolase SmEH has catalytic activity. According to the present invention, the E.coli / smh whole cell is adopted to respectively catalyze the hydrolysis reaction of the (R)-styrene oxide (SO) and the (S)-styrene oxide (SO) so as to obtain the (R)-styrene oxide (SO) and the (S)-1, 2-phenyl glycol (1-Phenyl-1, 2-ethanediol, PED) with high optical purity, and the application of the E.coli / smh whole cell to respectively catalyze the hydrolysis reaction of the (R)-styrene oxide (SO) and the (S)-1, 2-phenyl glycol (1-Phenyl-1, 2-ethanediol, PED) is provided;

Description

Technical Field

[0001] The present invention relates to the technical fields of bioengineering and fine chemical engineering, and in particular to an epoxide hydrolase, a gene, a vector, an engineering bacterium and applications. Background Art

[0002] Chiral vicinal diols are a class of high-value-added synthetic building blocks containing adjacent hydroxyl groups. They can be used as drug intermediates and catalyst intermediates and are widely used in the fields of medicine, pesticides, material synthesis, and fine chemicals. Among them, the common 1,2-phenylethylenediol (PED) serves as a key chiral intermediate and is directly involved in the preparation of a variety of highly active drugs. For example, (R)-PED is used in the synthesis of β-blockers. It is also used in the synthesis of triazole antifungal drugs such as fluconazole and anti-HIV nucleoside analogs. By precisely controlling the stereoconfiguration of the sugar ring or active center, the drug's targeting and safety can be improved.

[0003] However, existing technologies for synthesizing chiral vicinal diols still face challenges such as high costs, reliance on toxic heavy metal catalysts, numerous byproducts, poor stereoselectivity, and harsh reaction conditions. Epoxide hydrolases (EC 3.3.2.-) asymmetrically hydrolyze epoxides to their corresponding vicinal diols. They offer advantages such as broad availability, high enantioselectivity and regioselectivity, the absence of cofactors, mild reaction conditions, and environmental friendliness. They are considered biocatalysts with great industrial potential. Therefore, enzymatic conversion of styrene oxide to 1,2-phenylethylene glycol (PED) is environmentally friendly, efficient, and cost-effective. With the rise of sustainable development and the development of biotechnology, coupled with rapid advances in bioinformatics, the accumulation of gene sequence information, and genetic engineering, the rapid identification of new epoxide hydrolase genes has become possible. Therefore, it is imperative to expand the pool of identified epoxide hydrolases and identify new ones through molecular modification or genome mining. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an epoxide hydrolase, gene, vector, engineered bacteria and application, to obtain a new epoxide hydrolase for catalyzing the production of a chiral intermediate 1,2-phenylethylene glycol.

[0005] Based on the above purpose, the present invention provides an epoxide hydrolase, the amino acid sequence of which is shown in SEQ ID NO. 3. The epoxide hydrolase of the present invention is Sphingopyxis macrogoltabida EY-1.

[0006] The amino acid sequence is obtained by replacing, adding, or losing one or more amino acid residues, and encodes a protein derived from the amino acid sequence having epoxide hydrolase activity. The epoxide hydrolase of the present application comprises: a protein having ≥90% homology with the defined amino acid sequence and having epoxide hydrolase activity.

[0007] The present invention also provides a gene encoding the epoxide hydrolase, the nucleotide sequence of which is shown in SEQ ID NO. 2. The nucleotide sequence of the gene is a codon-optimized sequence of the nucleotide sequence shown in SEQ ID NO. 1.

[0008] The invention also provides an expression vector and an engineering bacterium containing the gene.

[0009] The engineered bacteria are constructed and expressed using fungi or bacteria as hosts.

[0010] The present invention also provides a method for preparing the engineered bacteria, comprising the following steps:

[0011] Step 1: codon optimization was performed based on the codon preference of E. coli BL21 series strains to obtain the nucleotide sequence of the smeh gene as shown in SEQ ID NO. 2;

[0012] Step 2: PCR amplification was performed using the nucleotide sequence of the smeh gene obtained in step 1 as a template, and the amplified product was connected to the expression vector pET-28a to obtain the recombinant expression plasmid pET-28a-smeh;

[0013] Step 3: Introduce the recombinant expression plasmid pET-28a-smeh into E. coli BL21 to obtain the engineered bacteria E. coli / smeh to be expressed.

[0014] The present invention also provides the use of the epoxide hydrolase, a gene encoding the epoxide hydrolase according to claim 1, an expression vector containing the gene, and an engineered bacterium containing the gene in chiral biocatalysts.

[0015] Preferably, the application is to use the epoxide hydrolase and / or the engineered bacteria expressing the epoxide hydrolase as a catalyst, (R)- and (S)-epoxystyrene as substrates, and carry out an asymmetric catalytic reaction at 20-35°C and pH 5.5-8.0 to obtain the products (R)- and (S)-1,2-phenylethylene glycol.

[0016] Preferably, the reaction temperature is 30° C., and the pH is adjusted to 7.0 using Na 2 HPO 4 -NaH 2 PO 4 buffer.

[0017] The reaction method is to prepare a 50 mg / mL uniform cell enzyme suspension using pure enzyme or fresh E. coli / smeh cells resuspended in Na2HPO4-NaH2PO4 phosphate buffer (100 mmol / L, pH 7.0), add epoxide hydrolase to it to make the final concentration reach 20 mmol / L, and place the system in an environment of 20-35°C and pH 5.5-8.0 for asymmetric catalysis.

[0018] 20-50 mg / mL E. coli / smeh lyophilized whole cells are used to catalyze the hydrolysis reaction of (R)- and / or (S)-epoxystyrene at a concentration within 20 mmol / L to obtain the product (R)- and / or (S)-1,2-phenylethylene glycol.

[0019] Beneficial effects of the present invention: The present invention provides a novel epoxide hydrolase derived from Sphingopyxis macrogoltabida EY-1, designated SmEH, and its corresponding gene, designated smeh. When whole cells of E. coli / smeh expressing recombinant SmEH catalyze the hydrolysis of (R)- or (S)-epoxystyrene oxide (SO) at concentrations of 20 mmol / L or less, (R)- or (S)-PED can be obtained. Therefore, SmEH and its recombinant engineered strain, E. coli / smeh, possess significant application potential and socioeconomic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is the nucleic acid electrophoresis diagram of the bacterial solution PCR identification of the recombinant SmEH engineering bacteria E. coli / smeh of the present invention; wherein, lane M: DNA marker; lanes 1 to 4: PCR template is E. coli / smeh;

[0022] Figure 2 This is a schematic diagram of the recombinant plasmid pET-28a-smeh of the epoxide hydrolase SmEH of the present invention;

[0023] Figure 3The figure is an SDS-PAGE image of the engineered bacteria E. coli / smeh expressing recombinant SmEH of the present invention; wherein, lane M: protein indicator band marker; lane 1: E. coli / pET-28a cell lysate; lane 2: E. coli / smeh cell lysate; lane 3: E. coli / smeh cell lysate supernatant; lane 4: purified E. coli / smeh cell lysate;

[0024] Figure 4 The GC detection spectra before and after 10 minutes of the reaction using E. coli / smeh freeze-dried cells (50 mg / mL) to catalyze 20 mmol / L (R)- or (S)-SO in the present invention.

[0025] Figure 5 This is the hydrolysis process of (R)- or (S)-SO in the present invention. DETAILED DESCRIPTION

[0026] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to specific embodiments.

[0027] It should be noted that, unless otherwise defined, technical or scientific terms used in this invention should have the same general meaning as those generally understood by persons of ordinary skill in the art to which this invention belongs. The terms "first," "second," and similar expressions used in this invention do not denote any order, quantity, or importance, but are simply used to distinguish different components. The terms "include" or "comprising" and similar expressions mean that the element or object preceding the word includes the elements or objects listed after the word and their equivalents, without excluding other elements or objects.

[0028] Example 1

[0029] (1) Culture medium and phosphate buffer

[0030] LB liquid medium (g / L): peptone 10, yeast extract 5, NaCl 10.

[0031] LB solid medium (g / L): peptone 10, yeast extract 5, NaCl 10, agar powder 20.

[0032] Phosphate buffer solution (NaH2PO4-Na2HPO4, 100 mmol / L, pH 7.0): Weigh 4.99 g NaH2PO4·2H2O and 60.2 g Na2HPO4·12H2O, dissolve in deionized water and dilute to 1 L. Store at 4°C until ready to use.

[0033] (2) Gene manipulation steps

[0034] Codon optimization of the smeh gene and construction of its expression plasmid

[0035] Gene analysis software revealed that the GC content of the original smeh gene (shown in SEQ ID NO. 1) was 65.27%. Codon optimization was performed using the jcat (http: / / www.jcat.de / ) online website based on the codon preferences of E. coli BL21(DE3) strains. The optimized sequence is shown in SEQ ID NO. 2. This optimized sequence was synthesized, and a pair of specific primers for smeh PCR reactions was designed based on this optimized sequence. The upstream and downstream primers (smeh-F and smeh-R) have sequences shown in SEQ ID NOs. 4 and 5, respectively, and contain restriction sites for Nde I and Xho I, respectively.

[0036] PCR amplification was performed using a synthetic, codon-optimized smeh nucleotide as a template. The conditions were: 94°C denaturation for 5 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for 2 minutes, with a final extension at 72°C for 10 minutes. PCR products were analyzed by 1% agarose gel electrophoresis, and the desired bands were recovered by gel excision. The recovered product was ligated with pMD 18-T at 17°C for 10 hours. The ligation system consisted of: pMD 18-T (1.0 μL), recovered product (1.0 μL), sterile water (3.0 μL), and a DNA Ligation Kit (5.0 μL). The ligation product was transformed into E. coli JM109. After selection on ampicillin (Amp)-resistant plates, positive transformants were selected and cultured in 2 mL of Amp-resistant LB (Amp / LB) liquid medium for 4 hours. The culture was then identified by PCR. Strains with correct identification and sequencing results were saved, and the recombinant clone plasmid was named pMD 18-T-smeh. The recombinant clone plasmid was extracted, and pMD 18-T-smeh and the expression vector pET-28a were treated with restriction endonucleases Nde I and Xho I, respectively. The double enzyme digestion system consisted of: 10× H Buffer (2 μL), pET-28a or pMD18-T-smeh (10 μL), ultrapure water (6 μL), Nde I (1 μL), and Xho I (1 μL). After 4 hours in a 37°C water bath, agarose gel electrophoresis analysis was performed, and the enzyme-digested smeh and pET-28a were recovered from the gel, and a connection system was constructed and placed at 17°C for connection. The connection system was: gene fragment after double enzyme digestion (3.0 μL), pET-28a after double enzyme digestion (5.0 μL), T4 DNA Ligase (1.0 μL), 10×T4 DNA Ligase Buffer (1.0 μL). The recombinant expression plasmid was introduced into E. coli BL21 (DE3), cultured at 37°C on a kanamycin sulfate (Kan) resistance plate for 4 hours, and several colonies were picked and inoculated into 2 mL Kan / LB liquid medium for 4 hours, and then the bacterial solution was verified by PCR ( Figure 1 ), and the recombinant expression plasmid with the correct bacterial solution PCR verification and sequencing results was named pET-28a-smeh( Figure 2 ), and the corresponding engineered strain was named E. coli / smeh. Using the same method, the empty plasmid pET-28a, which did not contain the target gene, was transformed into E. coli to obtain the empty engineered strain E. coli / pET-28a, which was used as a blank control in subsequent experiments.

[0037] Inducible Expression of SmEH Recombinant Protein in E. coli BL21(DE3)

[0038] Pick a single colony of E.coli / smeh and inoculate it into 5mL of Kan / LB liquid medium, and culture it at 37℃ with shaking for 12h. Transfer 1mL of culture solution (inoculation volume is 1%, v / v) to 100mL of the same medium, culture it at 37℃ with shaking for 4-5h, add IPTG (isopropyl-β-D-thiogalactopyranoside) (final concentration 0.5mmol / L), and induce it at 25℃ for 12h. Collect the bacteria by centrifugation (8,000×g, 5min), wash them twice with deionized water, and freeze-dry them to obtain whole-cell lyophilized powder. Induce E.coli / pET-28a in the same way as a blank control. Resuspend the lyophilized bacterial powder in deionized water to prepare a 50 mg / mL bacterial suspension. Pipette 1 mL of the E. coli / smeh whole-cell bacterial suspension into a centrifuge tube. Use an ultrasonic disruptor to treat the remaining E. coli / smeh and the strain E. coli / pET-28a containing the pET-28a empty vector. Centrifuge the disrupted bacterial solution at 10,000 × g for 5 minutes. Place the supernatant into a centrifuge tube and prepare a sample for SDS-PAGE. The results are as follows: Figure 3 shown.

[0039] Application of E. coli / smeh in (R)- or (S)-SO hydrolysis

[0040] During the reaction, whole cells were selected for the reaction because whole cells are easier to obtain than crude cell extracts or purified enzyme preparations and have higher tolerance and stability in buffer solutions containing substrates.

[0041] To understand the specifics of the hydrolysis reaction of E. coli / smeh, we first identified the substrates of E. coli / smeh. Next, we prepared stock solutions of (R)- and (S)-SO (final concentration of 200 mmol / L) in methanol and stored sealed at -20°C. E. coli / smeh stem cells were resuspended in phosphate buffer (pH 7.0) to create a uniform 50 mg / mL cell enzyme suspension. The substrate (R)- or (S)-SO was added to the suspension to a final concentration of 20 mmol / L. The suspension was then added to the E. coli / smeh suspension and incubated at 30°C on a shaker for 10 min. Sample preparation and detection by GC were as follows: 1 mL of ethyl acetate (containing 1 mmol / L n-hexane as an internal standard) was added to a 1.5 mL centrifuge tube. 200 μL of the reaction solution was extracted with ethyl acetate. The extract was centrifuged using a microvortex mixer, and the supernatant was dried over anhydrous MgSO₄ and filtered through a 0.22 μm organic filter. The specific conditions of GC detection were referred to the relevant literature (J. Agri. Food Chem. 2017, 65: 9861–9870; Appl. Microbiol. Biot. 2020, 104: 6199–6210; Enzyme. Microb. Tech. 2023, 166: 110228). The R and S configurations of each compound were determined by comparing the chromatographic retention times. Figure 4 As shown, the retention time of (R)-SO was 5.162 min, the retention time of (S)-SO was 5.294 min, the retention time of (R)-PED was 17.863 min, and the retention time of (S)-PED was 17.674 min. The results showed that SmEH is an epoxide hydrolase that maintains a single configuration.

[0042] Those skilled in the art will understand that the discussion of any of the above embodiments is merely illustrative and is not intended to limit the scope of the present invention to these examples. Within the spirit and principles of the present invention, the technical features of the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and many other variations exist for the various aspects of the present invention described above, which are not provided in detail for the sake of clarity. Any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. An epoxide hydrolase, characterized in that The amino acid sequence of the epoxide hydrolase is shown in SEQ ID NO.

3.

2. A gene encoding the epoxide hydrolase according to claim 1, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. An expression vector containing the gene according to claim 2.

4. An engineered bacterium containing the gene according to claim 2.

5. The engineered bacteria according to claim 4, characterized in that The engineered bacteria are constructed and expressed using fungi or bacteria as hosts.

6. A method for producing the engineered bacteria according to claim 4, characterized in that: The steps include: Step 1: codon optimization was performed based on the codon preference of E. coli BL21 series strains to obtain the nucleotide sequence of the smeh gene as shown in SEQ ID NO. 2; Step 2: PCR amplification was performed using the nucleotide sequence of the smeh gene obtained in step 1 as a template, and the amplified product was connected to the expression vector pET-28a to obtain the recombinant expression plasmid pET-28a-smeh; Step 3: Introduce the recombinant expression plasmid pET-28a-smeh into E. coli BL21 to obtain the engineered bacteria E. coli / smeh to be expressed.

7. Use of the epoxide hydrolase according to claim 1, a gene encoding the epoxide hydrolase according to claim 1, an expression vector containing the gene, or an engineered bacterium containing the gene in chiral biocatalysis.

8. The application according to claim 7, characterized in that: The application is to use the epoxide hydrolase and / or the engineered bacteria expressing the epoxide hydrolase as a catalyst, (R)- and (S)-epoxystyrene as substrates, and carry out an asymmetric catalytic reaction at 20-35° C. and pH 5.5-8.0 to obtain the products (R)- and (S)-1,2-phenylethylene glycol.

9. The application according to claim 8, characterized in that: The reaction temperature was 30°C, and the pH was adjusted to 7.0 using Na2HPO4-NaH2PO4 buffer.

10. The use according to claim 8, characterized in that: 20-50 mg / mL E. coli / smeh lyophilized whole cells are used to catalyze the hydrolysis reaction of (R)- and / or (S)-epoxystyrene at a concentration within 20 mmol / L to obtain the product (R)- and / or (S)-1,2-phenylethylene glycol.

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