Plant-derived leuco-anthocyanidin dioxygenase mutant and application thereof in synthesis of chiral drug intermediate

Abstract

The invention discloses a plant source leuco-anthocyanidin dioxygenase mutant and application thereof in synthesis of chiral drug intermediates, and belongs to the technical field of enzymology engineering. According to the invention, leuco-anthocyanidin dioxygenase (LDOX) derived from arabidopsis thaliana is modified through site-specific mutagenesis, an F304L / T239S double mutant (LDOXLS) is obtained, the (R)-phenylglycine analogue can be synthesized with the yield of 1.69 g / L, the yield of 81% and the enantiomeric excess ratio of 97: 3, and different substituent derivatives of the (R)-phenylglycine analogue can be generated. Compared with the traditional method, the method for synthesizing the (R)-phenylglycine analogue and the derivative thereof by using the enzyme has the advantages of wider substrate range, higher stereoselectivity, more environment-friendly requirement, simplicity and convenience in operation and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to a plant source leucoanthocyanidin dioxygenase mutant and its application in the synthesis of chiral drug intermediates, belonging to the technical field of enzyme engineering. BACKGROUND

[0002] Leucoanthocyanidin dioxygenase (LDOX) is a kind of dioxygenase derived from Arabidopsis thaliana. It catalyzes the generation of colored anthocyanidins from colorless proanthocyanidins in vivo. The current research on leucoanthocyanidin dioxygenase is limited to its gene sequence. The reported modification research on leucoanthocyanidin dioxygenase is only focused on the catalytic mechanism of its natural substrate (colorless proanthocyanidin), and there is no literature record of its application in unnatural substrates (aromatic amino acid analogs).

[0003] Phenylglycine analogs and their derivatives are a class of very important pharmaceutical intermediates, mainly used for the synthesis of penicillin, cephalosporin raw materials or intermediates, such as cefadroxil, cefoperazone, etc. In addition, they can also be used for the synthesis of new drugs. They have extremely important application value in the construction of organic molecules and natural products containing biological activity. The traditional preparation methods of phenylglycine analogs and their derivatives include Strecker synthesis, aminoization of halogen benzeneacetic acid, etc. However, these methods have more or less certain defects in product purity, yield, selectivity, environmental protection, etc. Therefore, finding a synthesis strategy of phenylglycine analogs and their derivatives with higher catalytic efficiency and safety and stability has become the key to developing their application potential.

[0004] Different from traditional chemical synthesis methods, the use of biological enzyme catalysis for the asymmetric synthesis of phenylglycine analogs and their derivatives has the advantages of high yield, high enantiomeric purity of products, green and sustainable, etc. However, the reported biological enzyme catalysis products are limited to the biosynthesis of p-hydroxyphenylglycine (Chen Jianbo, Xu Yi. Study on the preparation of D-p-hydroxyphenylglycine by biological enzyme method [J]. Journal of Biology, 2007, (05): 8-11). For phenylglycine analogs with different substituents, there is still a lack of efficient and selective enzyme catalysis system. It is a technical problem encountered in existing researches to have high catalytic capacity while having substrate universality. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a mutant enzyme for catalytically producing (R)-phenylglycine analogs and their derivatives. R )-phenylglycine analogs and their derivatives.

[0006] This invention provides a colorless anthocyanin dioxygenase mutant (LDOX_LS) modified by site-directed mutagenesis, which can synthesize phenylglycine analogs and their derivatives with high yield and enantioselectivity, solving the problems of poor stereoselectivity, high cost and serious environmental pollution of traditional chemical synthesis methods.

[0007] This invention provides a colorless anthocyanin dioxygenase mutant, which uses the amino acid sequence shown in SEQ ID No.1 as the starting sequence, mutates phenylalanine at position 304 to leucine and threonine at position 239 to serine, to obtain the F304L / T239S double mutant LDOX_LS, while the histidine residues in the enzyme active site remain unchanged.

[0008] In one embodiment, the amino acid sequence of the mutant is shown in SEQ ID No. 3.

[0009] The present invention also provides a gene encoding the mutant.

[0010] In one embodiment, the nucleotide sequence of the gene is shown in SEQ ID No. 4.

[0011] The present invention also provides a recombinant vector carrying the gene.

[0012] The present invention also provides recombinant microbial cells expressing the mutant, containing the gene, or containing the recombinant vector.

[0013] The present invention also provides recombinant Escherichia coli expressing the mutant, using Escherichia coli BL21(DE3) as the host and pET22b(+) as the vector.

[0014] The present invention also provides a synthesis ( R A method for producing phenylglycine analogues and their derivatives involves adding the mutant or the lysate of the recombinant Escherichia coli to a reaction system containing a phenylacetylamine substrate for reaction.

[0015] In one embodiment, the lysis buffer is the product of lysing the recombinant Escherichia coli.

[0016] In one embodiment, the lysis buffer is prepared by freezing the recombinant Escherichia coli at -80 °C for 6-8 h to lyse the cells, and then resuspending them in KPI buffer.

[0017] In one embodiment, the derivative includes ( R )-p-methylphenylglycine analogues, ( R )-o-methoxyphenylglycine analogues, ( R )-m-fluorophenylglycine analogues.

[0018] In one embodiment, the phenylacetamide substrate includes methyl (2-phenylacetoxy)carbamate, methyl (2-(4-methylphenyl)acetoxy)carbamate, methyl (2-(2-methoxyphenyl)acetoxy)carbamate, and methyl (2-(3-fluorophenyl)ethoxycarbonylcarbamate).

[0019] In one embodiment, the reaction system further contains ferrous ions and L-ascorbic acid.

[0020] In one embodiment, the reaction system contains 2.5–7.5 mM ferrous ions (final concentration), 2.5–5 mM L-ascorbic acid (final concentration), and 10–20 mM substrate (final concentration).

[0021] This invention also provides the mutant, or the recombinant microbial cell, or the recombinant Escherichia coli in the preparation of (… R Application in products containing phenylglycine analogues and their derivatives, the derivatives including ( R )-p-methylphenylglycine analogues, ( R )-o-methoxyphenylglycine analogues, ( R )-m-fluorophenylglycine analogues.

[0022] Beneficial effects: This invention modifies the colorless anthocyanin dioxygenase (LDOX) from Arabidopsis thaliana through site-directed mutagenesis to obtain the F304L / T239S double mutant (LDOX_LS), which can synthesize ( ) with an 81% yield and a 97:3 enantiomeric excess ratio. R )-Phenylglycine analogues; synthesized in 71% yield and with an enantiomeric excess ratio of 90:10. R )-p-methylphenylglycine analogues; synthesized in 44% yield and with an enantiomeric excess ratio of 86:14. R )-o-methoxyphenylglycine analogues; synthesized in 80% yield and with an enantiomeric excess ratio of 98:2. R The synthesis efficiency of )-m-fluorophenylglycine analogues is significantly higher than that of wild-type colorless anthocyanin dioxygenase. Compared with traditional methods, the synthesis of ( R )-Phenylglycine analogs and their derivatives have advantages such as a wider substrate range, higher stereoselectivity, more environmentally friendly requirements, and simpler operation. Attached Figure Description

[0023] Figure 1 Preparation of the colorless anthocyanin dioxygenase double mutant (LDOX_LS) of the present invention ( R The reaction formula for )-phenylglycine analogues.

[0024] Figure 2This is a gel image of the colorless anthocyanin dioxygenase double mutant (LDOX_LS) described in this invention.

[0025] Figure 3 The substrates used in each embodiment are: a is the substrate used in embodiment 3, b is the substrate used in embodiment 4, c is the substrate used in embodiment 5, and d is the substrate used in embodiment 6.

[0026] Figure 4 The high-performance liquid chromatography (HPLC) chromatogram of a phenylglycine analogue standard is shown.

[0027] Figure 5 The product of enzyme reaction ( R High-performance liquid chromatography (HPLC) chromatograms of phenylglycine analogues.

[0028] Figure 6 This is a high-performance liquid chromatography (HPLC) chromatogram of a standard sample containing a p-methylphenylglycine analogue.

[0029] Figure 7 The product of enzyme reaction ( R High-performance liquid chromatography (HPLC) chromatograms of )-p-methylphenylglycine analogues.

[0030] Figure 8 High-performance liquid chromatography (HPLC) chromatograms of o-methoxyphenylglycine analogue standards.

[0031] Figure 9 The product of enzyme reaction ( R High-performance liquid chromatography (HPLC) chromatograms of )-o-methoxyphenylglycine analogues.

[0032] Figure 10 High-performance liquid chromatography (HPLC) chromatograms of m-fluorophenylglycine analogue standards.

[0033] Figure 11 The product of enzyme reaction ( R High-performance liquid chromatography (HPLC) chromatograms of )-m-fluorophenylglycine analogues. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0035] The Escherichia coli involved in the following examples Escherichia coli BL21(DE3) was purchased from Sangon Biotech (Shanghai) Co., Ltd., and the pET 22b(+) plasmid containing colorless anthocyanin dioxygenase was purchased from Suzhou Genewiz Biotechnology Co., Ltd.

[0036] The culture media involved in the following examples are as follows: LB medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L; TB medium: tryptone 12 g / L, yeast extract 24 g / L, glycerol 4 g / L, KH2PO4 2.31 g / L, K2HPO4·3H2O 16.42 g / L.

[0037] The yields mentioned in the following examples are the ratios of actual yield to theoretical yield, where the yield is calculated using an internal standard curve method, with 1,3,5-trimethoxybenzene as the internal standard. Specifically, using 1,3,5-trimethoxybenzene as the internal standard, a series of standard samples of known concentrations are prepared, and a standard curve is established between the product concentration and the ratio of the product peak area to the internal standard peak area. Based on the peak area ratio of the product to the internal standard in the samples, the actual concentration of the product is calculated, thus yielding the yield.

[0038] The enzyme activity mentioned in the following examples refers to the ability of an enzyme to catalyze a chemical reaction under specific conditions. It is calculated as the ratio (U / mL) of the amount of enzyme (U) that converts 1 micromolar of substrate per minute to the volume of the enzyme solution under the conditions of this invention.

[0039] Example 1: Construction of mutants Site-directed mutagenesis was performed at the F304 and T239 sites of the colorless anthocyanin dioxygenase (LDOX) gene. The primers used are shown in the table below.

[0040] Table 1: Primer sequences for LDOX site-directed mutagenesis

[0041] The specific steps are as follows: First, the colorless anthocyanin dioxygenase gene with the nucleotide sequence shown in SEQ ID No. 2 is ligated to the pET22b(+) vector polyclonal restriction site. Nde I and Xho Between I and II, the wild-type plasmid pET22b(+)-LDOX was obtained; then, using plasmid pET22b(+)-LDOX as a template, the enzyme gene was amplified twice at two sites using site-directed mutagenesis primers to obtain the mutant enzyme plasmid pET22b(+)-LDOX_LS with double mutations of F304L and T239S.

[0042] The PCR reaction system and reaction conditions are as follows: PCR amplification system: 25 μL of DNA polymerase; Upstream primer (100 μg / μL) 1 μL; Downstream primer (100 μg / μL) 1 μL; Plasmid template (100 μg / μL) 1 μL; ddH2O2 2 μL.

[0043] The PCR amplification reaction conditions were as follows (30 cycles): 98℃, pre-denaturation for 30 s; 98℃, denaturation for 10 seconds; Anneal at 65℃ for 5 seconds; 72℃, extended for 1 minute and 50 seconds.

[0044] PCR products were used Dpn After digestion, the transformants were transferred into *E. coli* BL21(DE3). Once single colonies grew, the corresponding transformants were selected for sequencing verification, yielding correctly verified positive transformants BL21(DE3)-LDOX_LS.

[0045] Following the same method, the plasmid pET22b(+)-LDOX carrying the unmutated colorless anthocyanin dioxygenase gene was transformed into Escherichia coli and verified to obtain wild-type recombinant strain BL21(DE3)-LDOX.

[0046] Example 2: Expression of the colorless anthocyanin dioxygenase double mutant (LDOX_LS) The *E. coli* strain BL21(DE3)-LDOX_LS expressing the colorless anthocyanin dioxygenase double mutant (LDOX_LS) obtained in Example 1 and the wild-type recombinant strain BL21(DE3)-LDOX were respectively inoculated into liquid LB. Amp After growing in the culture medium for 8-10 hours, inoculate the seeds into TB at an inoculum rate of 5% (v / v). Amp Liquid fermentation medium; shake flasks were incubated at 37 ℃ and 220 rpm for 2-2.5 h until the OD reached 0.6-0.8. IPTG was added to a final concentration of 0.1 mM and the culture was induced at 20.5 ℃ for 24 h. The resulting bacterial culture was centrifuged at 8000 rpm for 20 min, the supernatant was discarded, and the bacterial cells were collected. Cells were lysed by freezing at -80 ℃ for 6-8 h. The cells were resuspended in KPI (50 mM, pH 7.4) buffer and the OD was adjusted to the specified value. 600 = After 50 minutes, it becomes the crude enzyme solution. Testing showed that the enzyme protein content in the crude enzyme solution was 8-12 μM. The protein gel image of the crude enzyme solution is shown below. Figure 2 As shown.

[0047] Example 3: ( R Biocatalytic preparation method of phenylglycine analogues The colorless anthocyanin dioxygenase double mutant (LDOX_LS) and wild-type expression strains were constructed according to the method shown in Example 1, and expressed and cultured according to the method of Example 2 to obtain crude enzyme solutions of recombinant colorless anthocyanin dioxygenase double mutant (LDOX_LS) and wild-type strains.

[0048] ( R Biocatalytic preparation of 2-phenylglycine analogues: Take 50 mL of crude enzyme solution and add 2.5 mL of ferrous ammonium sulfate aqueous solution (final concentration 5 mM), 1.25 mL of L-ascorbic acid aqueous solution (final concentration 2.5 mM), and 1.25 mL of methyl (2-phenylacetoxy)carbamate (final concentration 10 mM) sequentially to it in an anaerobic glove box. After sealing, incubate the reaction solution at room temperature with shaking for 4 h. The schematic diagram of the reaction process is shown below. Figure 1 As shown.

[0049] After the reaction was completed, high-performance liquid chromatography (HPLC) was used to detect (…). R The yield and er value of the )-phenylglycine analogue were determined using the following analytical methods: Yield detection conditions: Column type: Shimnex cs C18 (4.6×250 mm, 5 μm); Detection wavelength: 210 nm; Column temperature: room temperature; Mobile phase: Mobile phase A (methanol): Mobile phase B (ddH2O, 0.5‰ formic acid) = 95:5; Flow rate: 0.4 mL / min.

[0050] Chiral detection conditions: Column type: CHIRALPAK® IC (4.6 × 250 mm, 5 mic); Detection wavelength: 210 nm; Column temperature: room temperature; Mobile phase A (n-hexane): Mobile phase B (isopropanol, 0.1% trifluoroacetic acid) = 80:20; Flow rate: 1 mL / min.

[0051] The detection chromatogram of the non-amino acid products generated in this embodiment is as follows: Figure 5 As shown, the retention time of the product prepared using the colorless anthocyanin dioxygenase double mutant (LDOX_LS) in this example is similar to that of the phenylglycine analog obtained by chemical methods. Figure 4 The same as the product obtained by chiral separation was used to determine that the product was ( ). R )-Phenylglycine analogues. Upon testing, after the above reaction was completed, the colorless anthocyanin dioxygenase double mutant (LDOX_LS) corresponding to ( RThe yield of the α-phenylglycine analog was 1.69 g / L, with a yield of 81%, an enantiomeric ratio of 97:3 er, and an enzyme activity of 0.4 U / mL; while the wild-type LDOX enzyme solution yielded 0.82 g / L, with a yield of 39%, an enantiomeric ratio of 92:8 er, and an enzyme activity of 0.19 U / mL.

[0052] Example 4: ( R Biocatalytic preparation method of )-p-methylphenylglycine analogues The colorless anthocyanin dioxygenase double mutant (LDOX_LS) and wild-type expression strains were constructed according to the method shown in Example 1, and expressed and cultured according to the method of Example 2 to obtain crude enzyme solutions of recombinant colorless anthocyanin dioxygenase double mutant (LDOX_LS) and wild-type strains.

[0053] ( R Biocatalytic preparation of 2-(4-methylphenylglycine) analogues: 50 mL of crude enzyme solution was taken and, in an anaerobic glove box, 2.5 mL of ferrous ammonium sulfate aqueous solution (final concentration 5 mM), 1.25 mL of L-ascorbic acid aqueous solution (final concentration 2.5 mM), and 1.25 mL of methyl (2-(4-methylphenyl)acetoxy)carbamate (final concentration 10 mM) were added sequentially. After sealing, the reaction solution was incubated at room temperature with shaking for 4 h.

[0054] After the reaction was completed, high-performance liquid chromatography (HPLC) was used to detect (…). R The yield and product er value of the )---methylphenylglycine analogue were determined, and the specific analytical methods were the same as in Example 3.

[0055] The detection chromatogram of the non-amino acid products generated in this embodiment is as follows: Figure 7 As shown, the retention time of the product prepared using the colorless anthocyanin dioxygenase double mutant (LDOX_LS) in this example is similar to that of the p-methylphenylglycine analog obtained by chemical methods. Figure 6 The same as the product obtained by chiral separation was used to determine that the product was ( ). R )-p-methylphenylglycine analogue. After the above reaction was completed, the colorless anthocyanin dioxygenase double mutant (LDOX_LS) was detected. R The yield of the p-methylphenylglycine analogue was 1.58 g / L, with a yield of 71% and an enantiomeric ratio of 90:10 er.

[0056] Example 5: ( R Biocatalytic preparation method of )-o-methoxyphenylglycine analogues The colorless anthocyanin dioxygenase double mutant (LDOX_LS) and wild-type expression strains were constructed according to the method shown in Example 1, and expressed and cultured according to the method of Example 2 to obtain crude enzyme solutions of recombinant colorless anthocyanin dioxygenase double mutant (LDOX_LS) and wild-type strains.

[0057] ( R Biocatalytic preparation of 2-(2-methoxyphenylglycine) analogues: 50 mL of crude enzyme solution was taken and, in an anaerobic glove box, 2.5 mL of ferrous ammonium sulfate aqueous solution (final concentration 5 mM), 1.25 mL of L-ascorbic acid aqueous solution (final concentration 2.5 mM), and 1.25 mL of methyl (2-(2-methoxyphenyl)acetoxy)carbamate (final concentration 10 mM) were added sequentially. The mixture was sealed and incubated at room temperature with shaking for 4 h.

[0058] After the reaction was completed, high-performance liquid chromatography (HPLC) was used to detect (…). R The yield and er value of the )-o-methoxyphenylglycine analogue were determined, and the specific analytical methods were the same as in Example 3.

[0059] The detection chromatogram of the non-amino acid products generated in this embodiment is as follows: Figure 9 As shown, the retention time of the product prepared using the colorless anthocyanin dioxygenase double mutant (LDOX_LS) in this embodiment is similar to that of the o-methoxyphenylglycine analog obtained by chemical methods. Figure 8 The same as the product obtained by chiral separation was used to determine that the product was ( ). R )-o-methoxyphenylglycine analogue. Upon testing, after the above reaction was completed, the colorless anthocyanin dioxygenase double mutant (LDOX_LS) was found to contain... R The yield of the 1-o-methoxyphenylglycine analog was 1.05 g / L, with a yield of 44% and an enantiomeric ratio of 86:14.

[0060] Example 6: ( R Biocatalytic preparation method of )-m-fluorophenylglycine analogues The colorless anthocyanin dioxygenase double mutant (LDOX_LS) and wild-type expression strains were constructed according to the method shown in Example 1, and expressed and cultured according to the method of Example 2 to obtain crude enzyme solutions of recombinant colorless anthocyanin dioxygenase double mutant (LDOX_LS) and wild-type strains.

[0061] ( RBiocatalytic preparation of 2-(3-fluorophenyl)ethoxycarbonylcarbamate analogues: Take 50 mL of crude enzyme solution and add 2.5 mL of ferrous ammonium sulfate aqueous solution (final concentration 5 mM), 1.25 mL of L-ascorbic acid aqueous solution (final concentration 2.5 mM), and 1.25 mL of methyl(2-(3-fluorophenyl)ethoxycarbonylcarbamate (final concentration 10 mM)) sequentially to the solution in an anaerobic glove box. After sealing, incubate the reaction solution at room temperature with shaking for 4 h.

[0062] After the reaction was completed, high-performance liquid chromatography (HPLC) was used to detect (…). R The yield and product er value of the )-m-fluorophenylglycine analogue were determined, and the specific analytical method was the same as in Example 3.

[0063] The detection chromatogram of the non-amino acid products generated in this embodiment is as follows: Figure 11 As shown, the retention time of the product prepared using the colorless anthocyanin dioxygenase double mutant (LDOX_LS) in this example is similar to that of the m-fluorophenylglycine analog obtained by chemical methods. Figure 10 The same as the product obtained by chiral separation was used to determine that the product was ( ). R )-m-Fluorophenylglycine analogue. Upon testing, after the above reaction was completed, the colorless anthocyanin dioxygenase double mutant (LDOX_LS) corresponding to ( R The yield of the 1-m-fluorophenylglycine analogue was 1.81 g / L, with a yield of 80% and an enantiomeric ratio of 98:2.

[0064] Comparative Example 1: Other Mutation Sites The mutant was constructed and crude enzyme solution was obtained according to Examples 1 and 2. The difference was that the mutation sites were changed to N215G and F304I. The primers used are shown in the table below.

[0065] Table 2: Primer sequences for LDOX site-directed mutagenesis

[0066] Synthesized according to the method of Example 3 ( R The results showed that the LDOX_N215G mutant could only synthesize the desired product at a yield of 1.09 g / L, a 52% yield, a 94:6 enantiomeric ratio, and an enzyme activity of 0.26 U / mL; while the LDOX_F304I mutant could only synthesize the desired product at a yield of 1 g / L, a 48% yield, a 94:6 enantiomeric ratio, and an enzyme activity of 0.24 U / mL. Both mutants exhibited inferior catalytic activity and enantioselectivity compared to the LDOX_LS mutant reported in the examples.

[0067] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone 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 colorless anthocyanin dioxygenase mutant, characterized in that, Starting with the amino acid sequence shown in SEQ ID No. 1, phenylalanine at position 304 was mutated to leucine, and threonine at position 239 was mutated to serine.

2. The gene encoding the mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. Recombinant microbial cells expressing the mutant of claim 1, or containing the gene of claim 2, or containing the recombinant vector of claim 3.

5. A recombinant *E. coli* expressing the mutant of claim 1, characterized in that, The study used Escherichia coli BL21(DE3) as the host and pET22b(+) as the vector.

6. A synthesis R A method for producing phenylglycine analogues and their derivatives, characterized in that... The lysate of the mutant of claim 1 or the recombinant Escherichia coli of claim 5 is added to a reaction system containing a phenylacetamide substrate for reaction.

7. The method according to claim 6, characterized in that, The derivatives include ( R )-p-methylphenylglycine analogues, ( R )-o-methoxyphenylglycine analogues, ( R )-m-fluorophenylglycine analogues.

8. The method according to claim 6 or 7, characterized in that, The reaction system also contains ferrous ions and L-ascorbic acid.

9. The method according to any one of claims 6 to 8, characterized in that, The reaction system contains, at a final concentration, 2.5–7.5 mM ferrous ions, 2.5–5 mM L-ascorbic acid, and 10–20 mM substrate.

10. The mutant of claim 1, the recombinant microbial cell of claim 4, or the recombinant Escherichia coli of claim 5, in the preparation of... R The application of phenylglycine analogues and their derivatives in products, characterized by... The derivatives include ( R )-p-methylphenylglycine analogues, ( R )-o-methoxyphenylglycine analogues, ( R )-m-fluorophenylglycine analogues.

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