Method for synthesizing non-natural chiral alpha-amino acid through one-pot double-enzyme tandem catalytic synthesis

Abstract

The invention relates to the technical field of gene engineering and enzyme catalysis, and discloses a method for synthesizing non-natural chiral alpha-amino acid through one-pot double-enzyme tandem catalysis. According to the method provided by the invention, decarboxylase (AnFdcUbix), ammonia lyase (LsPAL), ammonium carbamate and ammonium bicarbonate are added into a water phase, so that substrate olefin is subjected to carboxylation under the catalysis of the decarboxylase (AnFdcUbix), and alpha-amino acid with an ee value of 99% is obtained through the catalysis of the ammonia lyase (LsPAL). According to the one-pot method, the step of separating an intermediate product can be avoided, meanwhile, the generated intermediate is converted into the product alpha-amino acid, and the problem of product inhibition is avoided. The alpha-amino acid prepared by the method has high yield. In addition, the method can be applied to synthesis of L-phenylpropionic acid and derivatives thereof, and has great application value and market prospect.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering and enzyme catalysis, and in particular to a method for synthesizing non-natural chiral α-amino acids through double-enzyme tandem catalysis in one pot. Background Art

[0002] With the rapid growth of consumption of chiral drugs in recent years, chiral non-natural α-amino acids have played an increasingly important role in biochemistry, synthetic biology, medicinal chemistry and materials science. For example, L-phenylalanine is one of the eight essential amino acids in the human body and is widely used in the food and pharmaceutical industries. It is an intermediate or raw material for the production of vitamin B6, thyroxine, melanin and other drugs. It is an important precursor of the low-calorie sweetener aspartame and an important carrier of anticancer drugs. For another example, D-phenylglycine is an important pharmaceutical intermediate for the synthesis of β-lactam antibiotics and broad-spectrum antibiotics. It has been widely used in the production of ampicillin, piperacillin, cephalexin, cefotaxime and the like. In the field of peptide drugs, non-natural amino acids are expected to further enrich the diversity of peptide molecules and obtain peptide drugs with better activity and pharmacokinetic properties. In addition, some chiral auxiliaries and catalysts can also be efficiently converted through chiral non-natural amino acids and thus applied in the field of asymmetric synthesis. Therefore, it is of great significance to develop efficient and practical methods to obtain non-natural chiral α-amino acids.

[0003] Taking L-phenylalanine as an example, the traditional preparation methods include protein hydrolysis and chemical synthesis, but their yields are low and the process is complicated. With more research and development, the current industrial production of L-phenylalanine is mainly obtained by enzymatic method and microbial fermentation.

[0004] The enzymatic method uses aminolyase or transaminase to catalyze L-phenylalanine precursors such as glutamine, phenylpyruvate or trans-cinnamic acid to finally obtain L-phenylalanine. The process is relatively simple, but is limited by the cost of precursor preparation, the stability of the enzyme itself and other issues.

[0005] The microbial fermentation method uses glucose as a substrate and is obtained by fermentation of Escherichia coli. The supply of PEP and P4P in the central metabolism and the negative feedback inhibition of PheA and AroG by the product L-phenylalanine have a great influence on the synthesis of L-phenylalanine. Therefore, the L-phenylalanine obtained by this method has problems such as low production efficiency, metabolic blockage and plasmid carrying, which increases the production cost, is not conducive to engineering production and application, and cannot synthesize L-phenylalanine derivatives.

[0006] In summary, it is necessary to develop a synthesis method for non-natural chiral α-amino acids that can replace microbial fermentation, has low raw material prices, is more environmentally friendly, and is easy to industrialize. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a one-pot method for synthesizing non-natural chiral α-amino acids by dual enzyme tandem catalysis, with mild and safe conditions. The present invention adopts a one-pot preparation process, with simple steps, and avoids the problem of intermediate product separation and product inhibition of the enzyme catalytic system. The substrate is a styrene derivative with lower cost. The chiral α-amino acid is prepared by enzymatic reaction under mild, controllable and green conditions, and the ee value reaches 99%, which can solve the problems of low yield, high cost and inability to synthesize α-amino acid derivatives in the existing microbial fermentation method.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] A one-pot dual-enzyme tandem catalytic method for synthesizing unnatural chiral α-amino acids, the specific method comprising: Ubix In the crude enzyme solution, the induced expression of ammonia lyase LsPAL genetic engineering bacteria, ammonium bicarbonate and vinyl aromatic substrate solubilized with methanol were added, and the substrate was first catalyzed by the decarboxylase AnFdc Ubix The unnatural chiral α-amino acid is obtained by carboxylation under the catalysis of , and then catalyzed by ammonia lyase LsPAL;

[0010] The decarboxylase AnFdc Ubix AnFdc Ubix The genetically engineered bacteria were resuspended in ammonium carbamate as a buffer and then lysed and separated;

[0011] The AnFdc Ubix The genetically engineered bacteria have a recombinant plasmid for expressing ferulic acid decarboxylase AnFdc and a recombinant plasmid for expressing flavin isopentenyl transferase Ubix;

[0012] The ammonia lyase LsPAL genetic engineering bacteria has a recombinant plasmid for expressing the ammonia lyase LsPAL.

[0013] Preferably, the coding gene sequence of the ferulic acid decarboxylase AnFdc is shown as SEQ ID No.1, and the amino acid sequence of the ferulic acid decarboxylase AnFdc is shown as SEQ ID No.2.

[0014] Preferably, the coding gene sequence of the flavin isopentenyltransferase Ubix is ​​shown as SEQ ID No.3, and the amino acid sequence of the flavin isopentenyltransferase Ubix is ​​shown as SEQ ID No.4.

[0015] Preferably, the AnFdc UbixIn genetically engineered bacteria, a recombinant plasmid expressing ferulic acid decarboxylase AnFdc is obtained by inserting a coding gene sequence shown in SEQ ID No.1 between NdeI and XhoI sites of an expression vector pET30a, and a recombinant plasmid expressing flavin isopentenyl transferase Ubix is ​​obtained by inserting a coding gene sequence shown in SEQ ID No.3 between NdeI and XhoI sites of an expression vector pET21b, and the two recombinant plasmids are simultaneously introduced into competent Escherichia coli BL21 (DE3) cells by a heat shock transformation method, and single colonies are obtained by recovery culture and resistance screening.

[0016] Preferably, the coding gene sequence of the ammonia lyase LsPAL is shown as SEQ ID No.5, and the amino acid sequence of the ammonia lyase LsPAL is shown as SEQ ID No.6.

[0017] Preferably, in the ammonia lyase LsPAL genetically engineered bacteria, the recombinant plasmid expressing the ammonia lyase LsPAL is obtained by inserting the coding gene sequence shown in SEQ ID No.5 between the EcoRI and HindIII sites of the expression vector pET28a, and the recombinant plasmid is simultaneously introduced into Escherichia coli BL21 (DE3) competent cells by heat shock transformation method, and a single colony is obtained by recovery culture and resistance screening.

[0018] Preferably, the structural formula of the vinyl aromatic substrate is:

[0019] Where: R is R' is H, p-OH, p-CH3, p-Br, p-OCH3.

[0020] Preferably, the concentration of the ammonium carbamate buffer is 480-520 M, and the pH is 7.8-8.2.

[0021] Preferably, the amount of ammonium bicarbonate added is 5 to 50 molar equivalents of the vinyl aromatic substrate.

[0022] Preferably, the one-pot dual-enzyme tandem catalysis is achieved by an oscillating reaction at 180-220 rpm in a closed headspace bottle, the reaction temperature is 20-22° C., and the reaction time is 12-24 hours.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] The present invention adds decarboxylase (AnFdc Ubix ), ammonia lyase (LsPAL), ammonium carbamate, ammonium bicarbonate, so that the substrate olefin is decarboxylase (AnFdc Ubix) is carboxylated under the catalysis of ammonia lyase (LsPAL), and then catalyzed by ammonia lyase (LsPAL) to obtain an α-amino acid with an ee value of 99%. The one-pot method of the present invention can avoid the step of separating the intermediate product, and at the same time, the intermediate produced is immediately converted into the product α-amino acid, avoiding the problem of product inhibition. The method of the present invention has a high yield for preparing α-amino acids. In addition, the method of the present invention can be applied to the synthesis of L-phenylpropionic acid and derivatives, and has great application value and market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the one-pot dual-enzyme tandem catalytic synthesis of unnatural chiral α-amino acids;

[0026] Figure 2 This is a schematic diagram of the complete sequence of the AnFdc recombinant plasmid;

[0027] Figure 3 This is a schematic diagram of the complete sequence of the Ubix recombinant plasmid;

[0028] Figure 4 This is a schematic diagram of the complete sequence of the LsPAL recombinant plasmid;

[0029] Figure 5 For AnFdc Ubix and purified protein gel images of LsPAL;

[0030] Figure 6 For AnFdc Ubix Gas chromatogram of decarboxylation reaction of purified crude enzyme solution;

[0031] Figure 7 It is the liquid phase chromatogram of the amino reaction of the crude enzyme solution purified by LsPAL;

[0032] Figure 8 For AnFdc Ubix -Liquid chromatogram of one-pot dual-enzyme catalytic synthesis of L-phenylalanine by LsPAL;

[0033] Fig. 9 For AnFdc Ubix - Gas chromatogram of the methyl esterification of L-phenylalanine catalyzed by one-pot dual enzymes of LsPAL;

[0034] Fig.10 For AnFdc Ubix -Liquid chromatogram of the one-pot tandem synthesis of 3-(2-thienyl)-L-alanine by LsPAL. DETAILED DESCRIPTION

[0035] In order to make the above-mentioned purpose, features and advantages of the present invention more obvious and easy to understand, the specific implementation mode of the present invention is described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The technical features in each embodiment of the present invention can be combined accordingly without conflicting with each other.

[0036] The present invention uses ferulic acid decarboxylase AnFdc Ubix / ammonia lyase LsPAL one-pot dual enzyme tandem catalysis synthesis of α-amino acids. The decarboxylase is a ferulic acid decarboxylase AnFdc (Ferulicacid decarboxylase) derived from Aspergillus niger, and its amino acid sequence is shown in SEQ ID No. 2 (PDB number: 4ZA4, NCBI accession number: XP_001390534.1). In addition, ferulic acid decarboxylase AnFdc is a prenylated flavin-dependent decarboxylase, belonging to the UbiD family of enzymes, and the flavin-dependent prenyltransferase Ubix catalyzes the mature prenylated flavin mononucleotide (prFMN), and the amino acid sequence of Ubix is ​​shown in SEQ ID No. 4 (NCBI accession number: AJF45212), which is derived from Escherichia coli. The ammonia lyase is ammonia lyase LsPAL derived from lettuce (Lactuca sativa L.), and its amino acid sequence is shown in SEQ ID No.6 (NCBI accession number: XP_023767814). The nucleotide sequences of ferulic acid decarboxylase AnFdc, flavin isopentenyl transferase Ubix, and ammonia lyase LsPAL are shown in SEQ ID No.1, SEQ ID No.3, and SEQ ID No.5, respectively, and the encoded amino acid sequences are shown in SEQ ID No.2, SEQ ID No.4, and SEQ ID No.6, respectively. The route of the synthesis process of the present invention is as shown in Figure 1 shown.

[0037] In the present invention, the preparation of genetically engineered bacteria and the enzymatic synthesis of chiral α-amino acids are as follows:

[0038] 1.AnFdc Ubix Gene synthesis of engineered bacteria and preparation of genetically engineered bacteria

[0039] 1.1 Synthesis of ferulic acid decarboxylase AnFdc gene

[0040] According to the existing publicly available gene data (including databases and published literature), the coding protein sequence of ferulic acid decarboxylase AnFdc SEQ ID No.2 was determined, and then the codon was optimized to SEQ ID No.1, so that it was easy to express in the Escherichia coli engineering strain (BL21 (DE3)). The coding gene sequence of ferulic acid decarboxylase AnFdc was synthesized according to SEQ ID No.1, and the synthesized gene was constructed into the expression vector pET30a plasmid, and the gene insertion sites were NdeI and XhoI, and finally the ferulic acid decarboxylase AnFdc recombinant plasmid was obtained, and its full sequence is as follows Figure 2 shown.

[0041] 1.2 Synthesis of flavin isopentenyltransferase Ubix gene

[0042] According to the existing publicly available gene data (including databases and published literature), the coding protein sequence of flavin isopentenyltransferase Ubix, SEQ ID No.4, was determined, and the codons were optimized to SEQ ID No.3, so that it was easy to express in the Escherichia coli engineering strain (BL21 (DE3)). The coding gene sequence of flavin isopentenyltransferase Ubix was synthesized according to SEQ ID No.3, and the synthesized gene was constructed into the expression vector pET21b plasmid, and the gene insertion sites were NdeI and XhoI. Finally, the recombinant plasmid of flavin isopentenyltransferase Ubix was obtained, and its full sequence is as follows Figure 3 shown.

[0043] 1.3AnFdc Ubix Transformation of recombinant plasmids into engineered bacteria

[0044] (1) Take 1uL of ferulic acid decarboxylase AnFdc recombinant plasmid and 1uL of flavin isopentenyl transferase Ubix recombinant plasmid and add them to 500uL of Escherichia coli BL21 (DE3) competent cells, mix gently, and let stand on ice for 30 minutes.

[0045] (2) Heat shock at 42°C in a water bath for 75 seconds, then quickly transfer to an ice bath and let stand for 5 minutes.

[0046] (3) Add 600 μL of LB liquid culture medium without antibiotics and revive at 200 rpm at 37°C for 60 min.

[0047] (4) Take 50uL of bacterial solution and spread it on the surface of LB solid culture medium containing ampicillin (50mg / L) and kanamycin (50mg / L), turn the plate upside down, and culture at 37℃ for 12-16h until a single colony appears, which is AnFdc Ubix Genetically engineered bacteria.

[0048] 2.AnFdc UbixInducible expression of genetically engineered bacteria

[0049] Pick AnFdc with an inoculating loop Ubix A single colony of genetically engineered bacteria was inoculated into 5 mL of culture medium, which was LB liquid culture medium (formula: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl). The inoculated bacterial solution was cultured at 37°C, 200 rpm for 12-16 hours. The cultured seed solution was transferred to TB culture medium (formula: 12 g / L tryptone, 24 g / L yeast extract, 9.4 g / L K2HPO4, 2.2 g / L KH2PO4, 4 mL / L glycerol) at an inoculation ratio of 10%, cultured at 37°C, 200 rpm for 3 hours, and then 200 mg / L isopropylthio-β-galactoside was added, and cultured at 18°C, 200 rpm for 20 hours. The culture solution was centrifuged at 4°C, 8000 rpm for 30 minutes in a low-temperature centrifuge, the supernatant was discarded, and the bacterial cells were collected, which were the induced expression AnFdc used in the subsequent steps. Ubix Genetically engineered bacteria.

[0050] 3. Gene synthesis of ammonia lyase LsPAL engineered bacteria and preparation of genetically engineered bacteria

[0051] 3.1 Synthesis of ammonia lyase LsPAL gene

[0052] According to the existing public gene data, including databases and published literature, based on its coding protein sequence SEQ ID No.6, the codon was optimized to SEQ ID No.5, so that it can be easily expressed in the Escherichia coli engineering strain (BL21 (DE3)), and the synthesized gene was constructed into the expression vector pET28a, with the gene insertion sites EcoRI and HindIII, and finally the LsPAL recombinant plasmid was obtained, and its full sequence is shown as follows Figure 4 shown.

[0053] 3.2 Transformation of recombinant plasmid of ammonia lyase LsPAL engineered bacteria

[0054] (1) Take 1uL of ammonia lyase LsPAL recombinant plasmid and add it to 500uL of Escherichia coli BL21 (DE3) competent cells, mix gently, and let it stand on ice for 30 minutes.

[0055] (2) Heat shock at 42°C in a water bath for 75 seconds, then quickly transfer to an ice bath and let stand for 5 minutes.

[0056] (3) Add 1 mL of LB liquid culture medium without antibiotics and incubate at 200 rpm at 37°C for 60 min.

[0057] (4) Take 50uL of the bacterial solution and spread it on the surface of LB solid culture medium containing kanamycin (50mg / L), invert the plate, and culture at 37°C for 12-16h until a single colony appears, which is the ammonia lyase LsPAL genetically engineered bacterium.

[0058] 4. Inducible expression of ammonia lyase LsPAL engineered bacteria

[0059] A single colony of ammonia lyase LsPAL genetically engineered bacteria was picked up with an inoculation loop and inoculated into 5 mL of culture medium, the culture medium being LB liquid culture medium (formula: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl). The inoculated seed liquid was cultured at 37°C, 200 rpm for 12-16 hours. The cultured bacterial liquid was transferred to TB culture medium (formula: 12 g / L tryptone, 24 g / L yeast extract, 9.4 g / L K2HPO4, 2.2 g / L KH2PO4, 4 mL / L glycerol) at an inoculation ratio of 10%, cultured at 37°C, 200 rpm for 3 hours, and then 200 mg / L of isopropylthio-β-galactoside was added, and cultured at 18°C, 200 rpm for 20 hours. The culture solution was centrifuged at 8000 rpm for 30 min at 4°C in a low-temperature centrifuge, the supernatant was discarded, and the bacteria were collected, which were the "Escherichia coli wet cells expressing induced ammonia lyase LsPAL" used in the subsequent steps.

[0060] 5. Protein Expression and Purification

[0061] 5.1AnFdc Ubix Protein purification and characterization of LsPAL

[0062] According to the characteristics of the two engineering bacteria, 50mM Tris-HCL, pH=8.0 is preferably used as a lysis buffer in the present invention, 8g of bacteria are added to 32mL of lysis buffer, and after being fully lysed by an ultrasonic lysator, all the lysate is loaded on a nickel column of a protein purification column in an ice bath. After the system is stable, 50mM Tris-HCL, pH=8.0, 50mM Tris-HCL, pH=8.0, 50mM imidazole, 50mM Tris-HCL, pH=8.0, 100mM imidazole, 50mM Tris-HCL, pH=8.0 are used in sequence until the absorbance of the system is leveled, and finally 150mM imidazole 50mM Tris-HCL is used to flush out the protein, and ultrafiltration and concentration are performed using a low-temperature centrifuge to finally obtain 500ul of purified protein.

[0063] Take 100uL of protein diluent, add 25uL of protein loading buffer (5X), shake evenly and centrifuge at 1000rpm for 1min, place in a 100℃ metal bath for 10min, cool to room temperature and centrifuge at 8000rpm for 10min, take 10uL of supernatant and load it on the protein gel channel, electrophoresis at 200V for 60min, stop electrophoresis, clear the protein gel, and stain until the protein gel bands are clear. Ubix The purified protein gel of LsPAL was Figure 5 shown.

[0064] 5.2AnFdc Ubix Characterization of Decarboxylation Reaction of Engineered Bacteria

[0065] According to the above “2.AnFdc Ubix The method of "inducible expression of genetically engineered bacteria" was used to induce the expression of AnFdc Ubix Genetically engineered bacteria, E. coli cells were harvested after centrifugation. Take 1g AnFdc Ubix Add 4 mL of 50 mM Tris-HCL, pH = 8.0 lysis buffer to the cells, and lyse them in an ice bath with ultrasonic lysis until complete lysis. Add 2 mg of 2-thiophene acrylic acid (100 uL of dimethyl sulfoxide to assist dissolution), and place in a closed system for shaking reaction at 30 ° C for 24 hours. After the reaction is completed, dodecane is used as an internal standard and the yield (decarboxylation yield) of 2-vinylthiophene is detected by gas phase. AnFdc Ubix The gas chromatogram of the decarboxylation reaction of the purified crude enzyme solution is shown in Figure 6 shown.

[0066] 5.3 Characterization of amino groups on LsPAL engineered bacteria

[0067] According to the method in "4. Induced expression of ammonia-lyase LsPAL engineered bacteria", wet cells of Escherichia coli (1 g) induced to express ammonia-lyase LsPAL were resuspended in 500 M NH2COONH4, and 5 mg of cinnamic acid was added. The cells were placed in a sealed headspace bottle at 20°C and shaken at 200 rpm for 12-24 hours.

[0068] After the reaction, the mixture was acidified to pH = 2.0 with concentrated hydrochloric acid, filtered through a 0.22um aqueous filter, and the yield was detected by liquid chromatography. Liquid chromatography analysis method: chromatographic column: CROWNPAK CR(+) column (5μm, 4.0×150mm, Daicel); detection wavelength: 254nm; mobile phase: 0.1% trifluoroacetic acid aqueous solution (pH≈2) / methanol = 95 / 5; flow rate: 0.3ml / min; column temperature: 25℃; injection volume: 20uL. The liquid chromatogram of the amino reaction of the LsPAL purified crude enzyme solution is shown in the figure below: Figure 7 shown.

[0069] In addition, in addition to the above-mentioned substrate cinnamic acid, the extended substrate spectrum of LsPAL purified crude enzyme solution and the respective reaction equations can also be expressed as follows:

[0070]

[0071] Therefore, based on the above constructed AnFdc Ubix Genetically engineered bacteria and ammonia lyase LsPAL genetically engineered bacteria, the present invention can provide a one-pot double enzyme tandem catalytic synthesis of non-natural chiral α-amino acids, the specific method is: in the decarboxylase AnFdc Ubix In the crude enzyme solution, the induced expression of ammonia lyase LsPAL genetic engineering bacteria, ammonium bicarbonate and vinyl aromatic substrate solubilized with methanol were added, and the substrate was first catalyzed by the decarboxylase AnFdc Ubix The decarboxylase AnFdc is then catalyzed by ammonia lyase LsPAL to obtain a non-natural chiral α-amino acid. Ubix AnFdc Ubix The genetically engineered bacteria are resuspended in ammonium carbamate as a buffer and then lysed and separated.

[0072] The structural formula of the vinyl aromatic substrate in the present invention is:

[0073]

[0074] Wherein: R group can be phenyl, pyridyl, thienyl, naphthalene structure group, the structural formula is respectively The R' in the phenyl group can be H, p-OH for hydroxyl, p-CH3 for methyl, p-Br for bromo, or p-OCH3 for methoxy.

[0075] The present invention further demonstrates the specific catalytic synthesis method and effect through experiments below.

[0076] 6. One-pot dual enzyme tandem catalysis of decarboxylase / ammonia lyase to synthesize α-amino acids

[0077] 6.1 Obtaining AnFdc Ubix Genetically engineered bacteria crude enzyme solution

[0078] The AnFdc expressed by the method in 2 above is called UbixGenetically engineered bacteria, Escherichia coli cells were harvested after centrifugation, 1 g of bacteria were taken and resuspended with 4 mL of 500 M NH2COONH4 (prepared with NH2COONH4 and HCL, pH = 8.0), the concentration of the bacterial suspension was adjusted to OD600≈30, the bacterial suspension was broken with an ultrasonic lysator (breaking time 20 min) until the lysis was complete, and then centrifuged in a low-temperature centrifuge at 4°C, 8000 rpm for 30 min to separate the supernatant crude enzyme solution.

[0079] 6.2 One-pot dual-enzyme tandem catalytic reaction

[0080] To the supernatant of the crude enzyme solution in 6.1, add the same gram weight of E. coli wet cells (1 g) induced to express ammonia lyase LsPAL according to the method in 4 above, add 20 eq NH4HCO3, add substrate (dissolved with methanol), place in a sealed headspace bottle at 20°C, and shake at 200 rpm for 12-24 hours.

[0081] 6.3 Post-reaction treatment and analysis

[0082] After the reaction, the mixture was acidified to pH = 2.0 with concentrated hydrochloric acid, filtered through a 0.22um aqueous phase filter, and the yield was detected by liquid chromatography. Liquid chromatography analysis method: chromatographic column: CROWNPAK CR (+) column (5μm, 4.0×150mm, Daicel); detection wavelength: 254nm; mobile phase: 0.1% trifluoroacetic acid aqueous solution (pH≈2) / methanol = 95 / 5; flow rate: 0.3ml / min; column temperature: 25°C; injection volume: 20uL.

[0083] The catalytic results of different styrene substituent groups are as follows:

[0084]

[0085] The following two examples further demonstrate the specific implementation of the above one-pot dual-enzyme tandem catalytic synthesis of non-natural chiral α-amino acids on the substrates styrene and 2-vinylthiophene.

[0086] Example 1: One-pot dual-enzyme catalytic synthesis of L-phenylalanine

[0087] In this embodiment, the biocatalytic route of L-phenylalanine based on the substrate styrene is as follows:

[0088]

[0089] The specific method is: the induced expression of AnFdc UbixGenetically engineered bacteria were resuspended in ammonium carbamate as a buffer and then lysed and separated to obtain the decarboxylase AnFdc Ubix The crude enzyme solution was then added to the decarboxylase AnFdc Ubix The crude enzyme solution was added with the inducible expression of ammonia lyase LsPAL genetic engineering bacteria (AnFdc Ubix The reaction enzyme ratio of LsPAL is controlled at a certain ratio, recorded as wetcell weight Fdc / LsPAL, unit: g / g), ammonium bicarbonate and the substrate styrene (Styrene) solubilized with methanol, and the substrate is first decarboxylated by the decarboxylase AnFdc through a one-pot double enzyme cascade catalysis. Ubix The product is then carboxylated by ammonia lyase LsPAL to obtain unnatural chiral L-phenylalanine.

[0090] In this example, based on this catalytic route, the optimal values ​​of a series of parameters in the reaction were explored, as follows:

[0091] 1) Explore the optimal AnFdc Ubix The reaction enzyme ratio of LsPAL was 500 molar equivalents of NH2COONH4 and 20 molar equivalents of NH4HCO3 were added to the reaction based on the substrate. The yield was detected after shaking at 20°C for 24 hours. The reaction results are as follows:

[0092]

[0093] 2) Explore the optimal conditions for reaction time and substrate amount:

[0094]

[0095] 3) Explore the optimal conditions for the type and concentration of the reaction carboxyl source:

[0096]

[0097] In summary, in this embodiment, AnFdc Ubix :LsPAL=3:1, reaction time is 24h, carboxyl source is ammonium bicarbonate and equivalent is 50eq, the reaction yield (Yield / %) is the highest. Taking styrene as the reaction substrate, under the optimal reaction conditions, AnFdc Ubix -LsPAL one-pot series liquid phase diagram Figure 8 shown.

[0098] In addition, after the enzyme reaction solution was quenched with hydrochloric acid, the water was evaporated and the amino acid was methylated. The gas chromatogram of the reaction with the L-phenylalanine standard methyl ester was as follows: Fig. 9 shown.

[0099] Example 2: One-pot dual-enzyme synthesis of 3-(2-thienyl)-L-alanine

[0100] In this embodiment, the biocatalytic route of 3-(2-thienyl)-L-alanine based on the substrate 2-vinylthiophene is as follows:

[0101]

[0102] The specific method is: the induced expression of AnFdc Ubix Genetically engineered bacteria were resuspended in ammonium carbamate as a buffer and then lysed and separated to obtain the decarboxylase AnFdc Ubix The crude enzyme solution was then added to the decarboxylase AnFdc Ubix The crude enzyme solution was added with the induced expression of ammonia lyase LsPAL genetic engineering bacteria, ammonium bicarbonate and the substrate 2-vinylthiophene (2-vinylthiophene) solubilized with methanol. The substrate was first catalyzed by the decarboxylase AnFdc through a one-pot double enzyme cascade. Ubix The unnatural chiral 3-(2-thienyl)-L-alanine was obtained by carboxylation under the catalysis of , and then catalyzed by ammonia lyase LsPAL.

[0103] In this example, based on this catalytic route, the optimal values ​​of a series of parameters in the reaction were explored, as follows:

[0104] 1) Optimal AnFdc Ubix The reaction enzyme ratio of LsPAL was 500 molar equivalents of NH2COONH4 and 20 molar equivalents of NH4HCO3 were added to the reaction based on the substrate. The yield was detected after shaking at 20°C for 24 hours. The reaction results are as follows:

[0105]

[0106] 2) Explore the optimal conditions for reaction time:

[0107]

[0108] Using 2-vinylthiophene as the reaction substrate, AnFdc Ubix -The liquid phase diagram of the one-pot series reaction of LsPAL is shown in Fig.10 shown.

[0109] In summary, in this embodiment, AnFdc Ubix : LsPAL = 3:1, reaction time is 24h, and the reaction yield of 3-(2-thienyl)-L-alanine is the highest.

[0110] Compared with the prior art, the present invention has the following beneficial effects: 1. The one-pot method for synthesizing α-amino acids of the present invention has simple steps and is easy to operate; 2. The process of the present invention is aqueous phase, normal pressure, mild reaction, environmentally friendly and safe; 3. The process of the present invention has a high conversion rate, the product is easy to obtain, the total yield is high, and the cost is low. 4. The catalytic route design of the present invention is novel, and has the advantages of simple operation, short production cycle, low production cost, high yield, low environmental pressure, and suitability for large-scale industrial production.

[0111] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A one-pot dual-enzyme tandem catalytic method for synthesizing unnatural chiral α-amino acids, characterized in that: In the decarboxylase AnFdc Ubix In the crude enzyme solution, the induced expression of ammonia lyase LsPAL genetic engineering bacteria, ammonium bicarbonate and vinyl aromatic substrate solubilized with methanol were added, and the substrate was first catalyzed by the decarboxylase AnFdc Ubix The unnatural chiral α-amino acid is obtained by carboxylation under the catalysis of , and then catalyzed by ammonia lyase LsPAL. The decarboxylase AnFdc Ubix AnFdc Ubix The genetically engineered bacteria were resuspended in ammonium carbamate as a buffer and then lysed and separated; The AnFdc Ubix The genetically engineered bacteria have a recombinant plasmid for expressing ferulic acid decarboxylase AnFdc and a recombinant plasmid for expressing flavin isopentenyl transferase Ubix; The ammonia lyase LsPAL genetic engineering bacteria has a recombinant plasmid for expressing the ammonia lyase LsPAL.

2. The one-pot dual-enzyme tandem catalytic synthesis of unnatural chiral α-amino acids according to claim 1, characterized in that: The coding gene sequence of the ferulic acid decarboxylase AnFdc is shown as SEQ ID No.1, and the amino acid sequence of the ferulic acid decarboxylase AnFdc is shown as SEQ ID No.

2.

3. The one-pot dual-enzyme tandem catalytic synthesis of unnatural chiral α-amino acids according to claim 1, characterized in that: The coding gene sequence of the flavin isopentenyl transferase Ubix is ​​shown in SEQ ID No.3, and the amino acid sequence of the flavin isopentenyl transferase Ubix is ​​shown in SEQ ID No.

4.

4. The one-pot dual-enzyme tandem catalytic synthesis of unnatural chiral α-amino acids according to claim 1, characterized in that: The AnFdc Ubix In genetically engineered bacteria, a recombinant plasmid expressing ferulic acid decarboxylase AnFdc is obtained by inserting a coding gene sequence shown in SEQ ID No.1 between NdeI and XhoI sites of an expression vector pET30a, and a recombinant plasmid expressing flavin isopentenyl transferase Ubix is ​​obtained by inserting a coding gene sequence shown in SEQ ID No.3 between NdeI and XhoI sites of an expression vector pET21b, and the two recombinant plasmids are simultaneously introduced into competent Escherichia coli BL21 (DE3) cells by a heat shock transformation method, and single colonies are obtained by recovery culture and resistance screening.

5. The one-pot dual-enzyme tandem catalytic synthesis of unnatural chiral α-amino acids according to claim 1, characterized in that: The coding gene sequence of the ammonia lyase LsPAL is shown in SEQ ID No.5, and the amino acid sequence of the ammonia lyase LsPAL is shown in SEQ ID No.

6.

6. The one-pot dual-enzyme tandem catalytic synthesis of unnatural chiral α-amino acids according to claim 1, characterized in that: In the ammonia lyase LsPAL genetically engineered bacteria, a recombinant plasmid expressing the ammonia lyase LsPAL is obtained by inserting the coding gene sequence shown in SEQ ID No. 5 between the EcoRI and HindIII sites of the expression vector pET28a, and the recombinant plasmid is introduced into Escherichia coli BL21 (DE3) competent cells by heat shock transformation, and a single colony is obtained by recovery culture and resistance screening.

7. The one-pot dual-enzyme tandem catalytic synthesis of unnatural chiral α-amino acids according to claim 1, characterized in that: The structural formula of the vinyl aromatic substrate is: Where: R is R' is H, p-OH, p-CH3, p-Br, p-OCH3.

8. The one-pot dual-enzyme tandem catalytic synthesis of unnatural chiral α-amino acids according to claim 1, characterized in that: The concentration of the ammonium carbamate buffer is 480-520 M, and the pH is 7.8-8.

2.

9. The one-pot dual-enzyme tandem catalytic synthesis of unnatural chiral α-amino acids according to claim 1, characterized in that: The amount of ammonium bicarbonate added is 5 to 50 molar equivalents of the vinyl aromatic substrate.

10. The one-pot dual-enzyme tandem catalytic synthesis of unnatural chiral α-amino acids according to claim 1, characterized in that: The one-pot dual-enzyme tandem catalysis is achieved in a closed headspace bottle by an oscillating reaction at 180-220 rpm, with a reaction temperature of 20-22° C. and a reaction time of 12-24 hours.

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