P-hydroxymandelic acid synthetase, gene, mutant and application of P-hydroxymandelic acid synthetase in asymmetric synthesis of (S)-mandelic acid

Abstract

The invention provides p-hydroxymandelic acid synthetase, a gene, a mutant of the p-hydroxymandelic acid synthetase and application of the p-hydroxymandelic acid synthetase in asymmetric synthesis of (S)-mandelic acid. The p-hydroxymandelic acid synthetase disclosed by the invention is derived from actinomycetes Actinokineospora auranticola, the amino acid sequence of the p-hydroxymandelic acid synthetase is as shown in SEQ ID NO.1, and a mutant of the p-hydroxymandelic acid synthetase is obtained by performing single-point mutation or multi-point combined mutation on glutamic acid at the 187th site, isoleucine at the 189th site and threonine at the 211th site of the amino acid sequence as shown in SEQ ID NO.1. The p-hydroxymandelic acid synthetase and the mutant thereof can convert phenylpyruvic acid into (S)-mandelic acid with high optical purity, have the advantages of mild reaction conditions, simplicity and convenience in operation, safety, environment friendliness and the like, and have a very good industrial application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and particularly relates to a p-hydroxymandelic acid synthase, a gene and a mutant thereof, and their application in the asymmetric synthesis of (S)-mandelic acid. Background Art

[0002] Mandelic acid (MA), also known as amygdalic acid, phenylhydroxyacetic acid, α-hydroxyphenylacetic acid. Chiral mandelic acid has a wide range of applications in organic synthesis and pharmaceutical production. For example, (R)-mandelic acid is widely used in the synthesis of various drugs such as cephalosporins and penicillins. (S)-Mandelic acid is a precursor raw material for the synthesis of (S)-oxybutynin, which is used to treat diseases such as urinary urgency, frequent urination, and urinary incontinence.

[0003] The traditional preparation process of (S)-mandelic acid is to chemically synthesize a racemic product and then perform chiral chemical or biological resolution. This method has disadvantages such as expensive resolution reagents, environmental pollution, and low purity. There are currently two main approaches for the biocatalytic preparation of (S)-mandelic acid. One is the fermentation method. For example, Sun Zhoutong et al. obtained optically pure mandelic acid from glucose by gene modification of the phenylalanine pathway in Escherichia coli, and obtained a yield of 0.092 g / L of (S)-mandelic acid (Sun, Z., Ning, Y., Liu, L. et al. Metabolic engineering of the L-phenylalanine pathway in Escherichia coli for the production of S- or R-mandelic acid. Microb Cell Fact 10, 71 (2011)). The other is the nitrilase-catalyzed method. For example, Sun Huihui et al. modified the nitrilase by directed evolution to obtain (S)-mandelic acid with an enantiomeric excess value of 91% (Sun H, Wang H, Gao W, et al. Directed evolution of nitrilase PpL19 from Pseudomonas psychrotolerans L19 and identification of enantiocomplementary mutants toward mandelonitrile. Biochem Biophys Res Commun. 2015 Dec 25;468(4):820-5.). However, the methods reported so far all have problems such as long reaction time and low conversion rate, and need to be further solved.

[0004] 4-Hydroxymandelate synthase is also applied in the synthesis of (S)-mandelic acid. The key factor affecting the substrate catalyzed by 4-hydroxymandelate synthase is the spatial structure of the substrate binding region. By using protein engineering techniques to reasonably modify the substrate binding region, the catalytic performance of 4-hydroxymandelate synthase towards the substrate can be effectively improved, thereby broadening the application scope of 4-hydroxymandelate synthase in the field of green synthesis of mandelic acid. Therefore, it is of great significance to develop a new 4-hydroxymandelate synthase to catalyze the synthesis of (S)-mandelic acid and promote its industrial application. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a 4-hydroxymandelate synthase, gene and mutant thereof and their application in the asymmetric synthesis of (S)-mandelic acid in view of the deficiencies in the above-mentioned prior art. The present invention provides an (S)-selective 4-hydroxymandelate synthase mutant with significantly improved catalytic activity through enzyme engineering transformation to solve the problem of low activity in the synthesis of mandelic acid by catalyzing 4-hydroxymandelate synthase in the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The present invention provides a 4-hydroxymandelate synthase, which is derived from Actinokineospora auranticolor, and its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0008] The present invention also provides a mutant of p-hydroxymandelic acid synthase, and the amino acid sequence of the mutant is an amino acid sequence obtained by mutating one or more sites among glutamic acid at position 187, isoleucine at position 189, and threonine at position 211 in the amino acid sequence shown in SEQ ID NO.1. The mutation at least includes one of the following mutation sites: E187A, E187C, E187D, E187F, E187G, E187H, E187I, E187K, E187L, E187M, E187N, E187P, E187Q, E187R, E187S, E187T, E187V, E187W, E187Y, I189A, I189C, I189D, I189E, I189F, I189G, I189H, I189K, I189L, I189M, I189N, I189P, I189Q, I189R, I189S, I189T, I189V, I189W, I189Y, T211A, T211C, T211D, T211E, T211F, T211G, T211H, T211I, T211K, T211L, T211M, T211N, T211P, T211Q, T211R, T211S, T211V, T211W, T211Y, E187A+I189C+T211V, E187C+I189C+T211V, E187Q+I189C+T211V, E187R+I189C+T211V, E187N+I189C+T211V, E187A+I189S+T211V, E187C+I189S+T211V, E187Q+I189S+T211V, E187R+I189S+T211V, E187N+I189S+T211V, E187A+I189T+T211V, E187C+I189T+T211V, E187Q+I189T+T211V, E187R+I189T+T211V, E187N+I189T+T211V; wherein, the meanings of amino acid abbreviations are as follows: G-glycine, A-alanine, V-valine, L-leucine, I-isoleucine, F-phenylalanine, W-tryptophan, Y-tyrosine, D-aspartic acid, H-histidine, N-asparagine, E-glutamic acid, K-lysine, Q-glutamine, M-methionine, R-arginine, S-serine, T-threonine, P-proline, C-cysteine.

[0009] The present invention also provides a coding gene, and the coding gene is a coding gene for p-hydroxymandelic acid synthase or a mutant of p-hydroxymandelic acid synthase whose amino acid sequence is as shown in SEQ ID NO.1.

[0010] The present invention also provides a recombinant expression vector, which contains the above-mentioned coding gene.

[0011] The present invention also provides a recombinant plasmid containing the above gene, and the plasmid vector of the recombinant plasmid is pET-24a(+).

[0012] The present invention also provides a genetically engineered bacterium, which is obtained by transforming the recombinant expression vector of claim 4 or 5 into a host microorganism, and the host microorganism is Escherichia coli E. coli BL21.

[0013] The present invention also provides an application of the above-mentioned genetically engineered bacterium in the synthesis of (S)-mandelic acid. The wet bacteria obtained by fermentation culture of the genetically engineered bacterial colony are used as a biocatalyst. Using phenylpyruvic acid as a substrate, FeSO4·7H2O and ascorbic acid are added, and a buffer solution with a pH of 6-9 is used as a reaction medium to form a reaction system, and the reaction is carried out at 25-60°C and 800-1200 rpm. After the reaction is completed, the reaction solution is separated and purified to obtain (S)-mandelic acid.

[0014] Furthermore, the dosage of the biocatalyst is 5-50 g / L of the reaction system based on the weight of the wet bacteria, the final concentration of the substrate is 5-50 mM, the final concentration of FeSO4·7H2O is 30-80 μM, and the final concentration of ascorbic acid is 0.1-1 mM.

[0015] Preferably, the reaction conditions are 25°C, pH 7.5, and 1000 rpm.

[0016] Furthermore, the wet bacteria are prepared as follows: Inoculate the engineered bacterium containing the recombinant p-hydroxymandelic acid synthase or its mutant coding gene into an LB medium containing 50 μg / mL kanamycin sulfate, culture at 37°C for 10-12 h to obtain a seed solution, inoculate the seed solution into a fresh TB medium containing 50 μg / mL kanamycin sulfate at an inoculation amount of 2% (v / v), culture at 37°C for 2 h, then add IPTG with a final concentration of 0.2 mM, induce at 18°C for 24 h to obtain an induced culture solution, and then centrifuge the induced culture solution at 4°C and 7000 rpm for 10 min, discard the supernatant, and collect the wet bacteria.

[0017] The present invention also provides a high performance liquid chromatography (HPLC) detection method during the biocatalysis process.

[0018] Yield detection: Chromatographic column: C18 column; Mobile phase: 0.1% phosphoric acid aqueous solution / methanol (55:45, v / v); Flow rate: 0.8 mL / min; Detection wavelength: 210 nm; Column temperature: 30°C.

[0019] Enantiomeric excess detection: Chromatographic column: AD-H column; Mobile phase: n-hexane / isopropanol / trifluoroacetic acid (90∶10∶0.1); Flow rate: 0.8 mL / min; Detection wavelength: 210 nm; Column temperature: 40 °C.

[0020] The beneficial effects of the present invention are as follows:

[0021] (1) The p-hydroxymandelic acid synthase provided by the present invention was first discovered and experimentally confirmed in the genus ActinokineoSpora. Its amino acid sequence homology with the p-hydroxymandelic acid synthase derived from Amycolatopsis orientalis is 80%, and its homology with other experimentally confirmed p-hydroxymandelic acid synthases is even lower. The present invention provides a new p-hydroxymandelic acid synthase.

[0022] (2) The present invention provides a p-hydroxymandelic acid synthase derived from ActinokineoSpora auranticolor. By constructing a highly expressed recombinant engineering bacterium, it was found that this enzyme can efficiently catalyze phenylpyruvic acid to generate (S)-mandelic acid.

[0023] (3) The present invention mutates the amino acids of the wild-type p-hydroxymandelic acid synthase (SEQ ID NO.1) derived from ActinokineoSpora auranticolor through single-point mutation or combined mutation. The activity of the obtained mutant in catalyzing the synthesis of (S)-mandelic acid from phenylpyruvic acid is significantly higher than that of the wild-type before mutation (abbreviated as WT). This provides a more environmentally friendly and sustainable method for the production of (S)-mandelic acid and has important application value for the industrial preparation of (S)-mandelic acid. Detailed implementation manners

[0024] The following examples facilitate a better understanding of the present invention, but are not limited to the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from regular biochemical reagent stores unless otherwise specified. For the quantitative tests in the following examples, three repeated experiments are set, and the results are averaged. For the chromatograms of different reactions under the same condition parameters, there will be a certain error range for the retention time of the target peak. Generally, a difference within 0.1 min can be regarded as an error and can be considered as the same target substance.

[0025] The present invention will be described in more detail below with specific examples.

[0026] The culture media involved in the following examples are as follows:

[0027] LB solid medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, 15 g / L agar, and 50 mg / L kanamycin.

[0028] LB liquid medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, and 50 mg / L kanamycin.

[0029] TB liquid medium: 24 g / L yeast extract, 12 g / L tryptone, 4 g / L glycerol, 12.54 g / L K2HPO4, 2.31 g / L KH2PO4, and 50 mg / L kanamycin.

[0030] The high performance liquid chromatography (HPLC) detection method involved in the following examples.

[0031] Yield detection: Chromatographic column: C18 column; Mobile phase: 0.1% phosphoric acid aqueous solution / methanol (55:45, v / v); Flow rate: 0.8 mL / min; Detection wavelength: 210 nm; Column temperature: 30 °C.

[0032] Enantiomeric excess detection: Chromatographic column: AD-H column; Mobile phase: n-hexane / isopropanol / trifluoroacetic acid (90:10:0.1); Flow rate: 0.8 mL / min; Detection wavelength: 210 nm; Column temperature: 40 °C.

[0033] Example 1

[0034] Construction, cultivation, and induced expression of the genetically engineered Escherichia coli strain with high expression of p-hydroxymandelic acid synthase AaHmas

[0035] (1) The protein sequence PPK68823.1 of p-hydroxymandelic acid synthase from Actinokineospora auranticolor obtained from the NCBI database was codon-optimized to SEQ ID NO.1 (the nucleotide sequence is shown in SEQ ID NO.2), synthesized by Beijing Tsingke Biotechnology Co., Ltd., and cloned between the NdeI and Hind III restriction enzyme sites of the pET-24a(+) vector, thereby obtaining the pET-24a(+)-AaHmas recombinant plasmid.

[0036] (2) The obtained recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells.

[0037] (3) Pick a single colony and inoculate it into a 5 mL LB test tube (kanamycin sulfate resistant) and culture for 12 h. Then transfer it to TB medium containing 50 μg / mL kanamycin sulfate at an inoculation amount of 2%, add the inducer IPTG to a final concentration of 0.2 mmol / L at 37 °C and continue to culture at 18 °C for 24 h to end the fermentation. Centrifuge at 7000 rpm at 4 °C for 10 min to collect the thalli for standby.

[0038] Example 2

[0039] Wild-type p-hydroxymandelic acid synthase AaHmas was used in the synthesis reaction system of (S)-mandelic acid: 10 mg / mL wet thalli (thalli prepared in Example 1), 10 mM phenylpyruvic acid, 0.5 mM L-ascorbic acid, 1% DMSO, 50 μM FeSO4·7H2O. Reaction conditions: 25 °C, pH 7.5, 1000 rpm, 8 h. After HPLC detection, the yield of (S)-mandelic acid was 71.69%.

[0040] Example 3

[0041] Wild-type p-hydroxymandelic acid synthase AaHmas was used in the synthesis of (S)-mandelic acid (changing the concentration of phenylpyruvic acid)

[0042] Reaction system: 10 mg / mL wet thalli (thalli prepared in Example 1), 20 mM phenylpyruvic acid, 0.5 mM L-ascorbic acid, 1% DMSO, 50 μM FeSO4·7H2O. Reaction conditions: 25 °C, pH 7.5, 1000 rpm, 8 h. After HPLC detection, the yield of (S)-mandelic acid was 21.98%.

[0043] Comparing Example 2 and 3, it can be found that the higher the concentration of the reaction substrate phenylpyruvic acid, the lower the reaction yield. In order to further improve the reaction yield, the p-hydroxymandelic acid synthase AaHmas was further modified.

[0044] Example 4

[0045] Construction, culture and induction expression of saturation mutation plasmids of E187, I189, T211 in the amino acid sequence of p-hydroxymandelic acid synthase AaHmas

[0046] (1) According to the amino acid sequence SEQ ID NO.1 of p-hydroxymandelic acid synthase AaHmas, perform protein homology modeling analysis on the alphafold3 website, and then perform molecular docking of the simulated structure with the substrate

[0047] Using AutoDock 4.2.6 software, the substrate-binding pocket was analyzed, and it was found that the positions of E187, I189, and T211 were relatively close to the active center. It was speculated that these sites might affect the catalytic activity of AaHmas towards the substrate.

[0049] (2) Using the wild-type plasmid pET-24a(+)-AaHmas as a template, whole-plasmid single-point mutation PCR was performed to construct single-point mutation recombinant plasmids containing the coding genes in which the sites of AaHmas-E187, AaHmas-I189, and AaHmas-T211 were respectively mutated into 19 other amino acids (A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / T / V / W / Y). After correct sequencing, the plasmids were extracted and stored (the specific mutation sites are shown in Tables 1 and 2).

[0050] (3) The obtained plasmids were transformed into Escherichia coli BL21(DE3) competent cells.

[0051] (4) Single colonies were picked and inoculated into a 5 mL LB test tube (kanamycin sulfate resistance) and cultured for 12 h. Then, they were transferred to a TB medium containing 50 μg / mL kanamycin sulfate at an inoculation amount of 2%, cultured at 37 °C for 2 h, and then IPTG inducer was added to a final concentration of 0.2 mmol / L, and the culture was continued at 18 °C for 24 h to end the fermentation.

[0052] Centrifuge at 7000 rpm at 4 °C for 10 min to collect the bacterial cells for standby.

[0053] Example 5

[0054] Wild-type p-hydroxymandelic acid synthase AaHmas and its saturation mutants at the E187, I189, and T211 sites were used in the synthesis reaction system of (S)-mandelic acid: 10 mg / mL wet bacterial cells (the bacterial cells prepared in Example 4), 20 mM phenylpyruvic acid, 0.5 mM L-ascorbic acid, 1% DMSO, 50 μM FeSO4·7H2O. Reaction conditions: 25 °C, pH 7.5, 1000 rpm, 8 h. After HPLC detection, only the T211V mutant at the AaHmas-T211 site had activity, with a yield of 70.93% and an ee value of 99%. The reaction data of the mutants at the AaHmas-E187 and AaHmas-I189 sites are shown in Tables 1-2 below. n.c. = no conversion, n.d. = not determined. According to the test results in Tables 1-2, the mutant sites with higher yields and ee values were screened for combinatorial mutation.

[0055] Table 1 Summary of the effects of mutants of the 187th glutamate of p-hydroxymandelic acid synthase on the synthesis of (S)-mandelic acid

[0056]

[0057]

[0058] Table 2 Summary of the effect of the isoleucine mutant at position 189 of p - hydroxymandelic acid synthase on the synthesis of (S) - mandelic acid

[0059] AaHmas mutant Yield(%) ee(%)(S) WT 21.98 98 I189A 28.72 99 I189C 64.26 97 I189D 59.18 96 I189E 28.41 96 I189F n.c. n.d. I189G 44.62 99 I189H 82.48 88 I189K 80.48 93 I189L 22.02 94 I189M n.c. n.d. I189N 29.55 97 I189P n.c. n.d. I189Q n.c. n.d. I189R n.c. n.d. I189S 61.03 96 I189T 50.94 96 I189V 33.13 99 I189W n.c. n.d. I189Y n.c. n.d.

[0060] Example 6

[0061] Reaction system of wild - type p - hydroxymandelic acid synthase AaHmas and its triple - point combination mutant I187 + EI189 + T211 for the synthesis of (S) - mandelic acid: 10 mg / mL wet cells (the operation process for obtaining wet cells is the same as that in Example 4, the difference is that the mutation sites in step (2) are multi - point combination mutations), 20 mM phenylpyruvic acid, 0.5 mM L - ascorbic acid, 1% DMSO, 50 μM FeSO4·7H2O. Reaction conditions: 25 °C, pH 7.5, 1000 rpm, 5 h. After HPLC detection, the mutant reaction data are shown in Table 3 below. Combining the yield and ee value, it can be seen that the mutant AaHmas E187Q + I189T + T211V has the highest yield, and the mutant E187N + I189S + T211V has the highest ee value.

[0062] Table 3 Effect of multi - point mutants of p - hydroxymandelic acid synthase on the synthesis of (S) - mandelic acid

[0063] AaHmas mutant Yield(%) ee(%)(S) WT 21.98 98 E187A+I189C+T211V 81.61 99 E187C+I189C+T211V 87.44 97 E187Q+I189C+T211V 71.66 99 E187R+I189C+T211V 85.52 99 E187N+I189C+T211V 94.46 98 E187A+I189S+T211V 91.82 98 E187C+I189S+T211V 91.35 97 E187Q+I189S+T211V 90.39 97 E187R+I189S+T211V 88.77 97 E187N+I189S+T211V 97.89 99 E187A+I189T+T211V 91.06 98 E187C+I189T+T211V 95.83 95 E187Q+I189T+T211V 98.46 97 E187R+I189T+T211V 83.61 96 E187N+I189T+T211V 96.02 98

[0064] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A p-hydroxymandelic acid synthase, characterized in that, It is derived from Actinokineospora auranticolor, and its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.

2.

2. A mutant of the hydroxymandelic acid synthase as described in claim 1, characterized in that, The amino acid sequence of the mutant is an amino acid sequence obtained by mutating one or more of the 187th glutamate, 189th isoleucine, and 211th threonine in the amino acid sequence shown in SEQ ID NO.

1. The mutations at least include one of the following mutation sites: E187A, E187C, E187D, E187F, E187G, E187H, E187I, E187K, E187L, E187M, E187N, E187P, E187Q, E187R, E187S, E187T, E187V, E187W, E187Y, I189A, I189C, I189D, I189E, I189F, I189G, I189H, I189K, I189L, I189M, I189N, I189P, I189Q, I189R, I189S, I189T, I189V, I189W, I189Y, T211A, T211C, T211D, T211E, T211F, T211G, T211H, T211I, T211K, T211L, T211M, T211N, T211P, T211Q, T211R, T211S, T211V, T211W, T211Y, E187A+I189C+T211V, E187C+I189C+T211V, E187Q+I189C+T211V, E187R+I189C+T211V, E187N+I189C+T211V, E187A+I189S+T211V, E187C+I189S+T211V, E187Q+I189S+T211V, E187R+I189S+T211V, E187N+I189S+T211V, E187A+I189T+T211V, E187C+I189T+T211V, E187Q+I189T+T211V, E187R+I189T+T211V, E187N+I189T+T211V; wherein, the meanings of amino acid abbreviations are as follows: G-glycine, A-alanine, V-valine, L-leucine, I-isoleucine, F-phenylalanine, W-tryptophan, Y-tyrosine, D-aspartic acid, H-histidine, N-asparagine, E-glutamic acid, K-lysine, Q-glutamine, M-methionine, R-arginine, S-serine, T-threonine, P-proline, C-cysteine.

3. A coding gene, characterized in that, The coding gene is the coding gene of the p-hydroxymandelic acid synthase as described in claim 1 or the p-hydroxymandelic acid synthase mutant as described in claim 2.

4. A recombinant expression vector, characterized in that, The vector contains the coding gene as described in claim 3.

5. A recombinant plasmid comprising the gene according to claim 3, characterized in that, The plasmid vector of the recombinant plasmid is pET-24a(+).

6. A genetically engineered bacterium, characterized in that, The genetically engineered bacterium is obtained by transforming the recombinant expression vector of claim 4 or 5 into a host microorganism, and the host microorganism is Escherichia coli BL21.

7. Use of the genetically engineered bacterium according to claim 6 in the synthesis of (S)-mandelic acid, characterized in that, The wet bacterial cells obtained by fermentation culture of the genetically engineered bacterial colonies are used as a biocatalyst. Using phenylpyruvic acid as a substrate, FeSO4·7H2O and ascorbic acid are added, and a buffer solution with a pH of 6-9 is used as the reaction medium to form a reaction system, and the reaction is carried out at 25-60°C and 800-1200 rpm. After the reaction is completed, the reaction solution is separated and purified to obtain (S)-mandelic acid.

8. The application according to claim 7, characterized in that, The dosage of the biocatalyst is 5-50 g / L of the reaction system based on the weight of the wet bacterial cells, the final concentration of the substrate is 5-50 mM, the final concentration of FeSO4·7H2O is 30-80 μM, and the final concentration of ascorbic acid is 0.1-1 mM.

9. The application according to claim 7, wherein The reaction conditions are 25°C, pH 7.5, and 1000 rpm.

10. The application according to claim 7, characterized in that, The wet bacterial cells are prepared as follows: Inoculate the engineering bacteria containing the recombinant p-hydroxymandelic acid synthase or its mutant encoding gene into an LB medium containing 50 μg / mL kanamycin sulfate, culture at 37°C for 10-12 h to obtain a seed solution, inoculate the seed solution into a fresh TB medium containing 50 μg / mL kanamycin sulfate at an inoculation amount of 2% (v / v), culture at 37°C for 2 h, then add IPTG with a final concentration of 0.2 mM, induce at 18°C for 24 h to obtain an induced culture solution, and then centrifuge the induced culture solution at 4°C and 7000 rpm for 10 min, discard the supernatant, and collect the wet bacterial cells.

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