Method for synthesizing unnatural chiral alpha-amino acids by one-pot two-enzyme cascade catalysis

Abstract

The present application relates to the field of genetic engineering and enzyme catalysis, and discloses a one-pot two-enzyme tandem catalytic synthesis method of unnatural chiral alpha-amino acid. The present application provides a method, which adds decarboxylase (AnFdc Ubix ), ammonia-lyase (LsPAL), ammonium carbamate and ammonium bicarbonate into an aqueous phase, so that a substrate olefin is carboxylated under the catalysis of decarboxylase (AnFdc Ubix ), and then alpha-amino acid with 99% ee value is obtained through the catalysis of ammonia-lyase (LsPAL). The one-pot method can avoid the step of intermediate product separation, and the produced intermediate is converted into the product alpha-amino acid, thereby avoiding the problem of product inhibition. The method provided by the present application has high yield in preparing alpha-amino acid. In addition, the method can be applied to the synthesis of L-phenylalanine and derivatives, and has great application value and market prospect.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme catalysis, specifically to a one-pot method for the tandem synthesis of non-natural chiral α-amino acids using two enzymes. Background Technology

[0002] With the rapid growth in the consumption of chiral drugs in recent years, chiral non-natural α-amino acids are playing an increasingly important role in fields such as biochemistry, synthetic biology, medicinal chemistry, and materials science. For example, L-phenylalanine, one of the eight essential amino acids in the human body, is widely used in the food and pharmaceutical industries as an intermediate or raw material in the production of drugs such as vitamin B6, thyroxine, and melanin. It is also an important precursor to the low-calorie sweetener aspartame and a crucial carrier for anticancer drugs. D-phenylglycine, for instance, is an important pharmaceutical intermediate in the synthesis of β-lactam antibiotics and broad-spectrum antibiotics, and is widely used in the production of ampicillin, piperazine, cephalexin, and cefotaxime. In the field of peptide drugs, non-natural amino acids hold promise for further enriching the diversity of peptide molecules and obtaining peptide drugs with better activity and pharmacokinetic properties. Furthermore, some chiral auxiliaries and catalysts can be efficiently converted from chiral non-natural amino acids, thus enabling their application in asymmetric synthesis. Therefore, developing efficient and practical methods for obtaining non-natural chiral α-amino acids is of great significance.

[0003] Taking L-phenylalanine as an example, traditional methods for its preparation include protein hydrolysis and chemical synthesis, but these methods have low yields and complex processes. With further research and development, the industrial production of L-phenylalanine is now mainly achieved through enzymatic and microbial fermentation methods.

[0004] Enzymatic methods use aminohydrolases or transaminases to catalyze L-phenylalanine precursors, such as glutamine, phenylpyruvic acid, or transcinnamic acid, to ultimately obtain L-phenylalanine. The process is relatively simple, but it is limited by issues such as the cost of precursor preparation and the stability of the enzyme itself.

[0005] Microbial fermentation, using glucose as a substrate, yields L-phenylalanine through fermentation with *E. coli*. However, the supply of PEP and P4P, the central metabolic byproducts, and the negative feedback inhibition of PheA and AroG by L-phenylalanine significantly impact L-phenylalanine synthesis. Therefore, this method suffers from low production efficiency, metabolic blockage, and plasmid contamination, increasing production costs, hindering engineering applications, and preventing the synthesis of L-phenylalanine derivatives.

[0006] In summary, there is a need to develop a method for synthesizing non-natural chiral α-amino acids that can replace microbial fermentation, use cheaper raw materials, be more environmentally friendly, and be easier to industrialize. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a one-pot method for the synthesis of non-natural chiral α-amino acids using a dual-enzyme tandem catalysis, under mild and safe conditions. This invention employs a one-pot preparation process, which is simple in steps and avoids the problems of intermediate product separation and product inhibition by the enzyme catalysis system. The substrate is a lower-cost styrene derivative. Chiral α-amino acids are obtained through enzymatic reaction under mild, controllable, and green conditions, achieving an ee value of 99%. This method solves the problems of low yield, high cost, and inability to synthesize α-amino acid derivatives by existing microbial fermentation methods.

[0008] To achieve the aforementioned objective, the technical solution adopted by the present invention is as follows:

[0009] A one-pot, two-enzyme tandem catalytic method for the synthesis of non-natural chiral α-amino acids, the specific steps of which are as follows: [The text abruptly shifts to a different topic] ...decarboxylase AnFdc Ubix In the crude enzyme solution, genetically engineered bacteria expressing the induced aminopyrate lyase LsPAL, ammonium bicarbonate, and a vinyl aromatic substrate dissolved in methanol were added. A one-pot, two-enzyme tandem catalysis was then employed, causing the substrate to first react with the decarboxylase AnFdc. Ubix Carboxylation under the catalysis of [catalyst name], followed by catalysis by amino acid lyase LsPAL to obtain non-natural chiral α-amino acids;

[0010] The decarboxylase AnFdc Ubix The crude enzyme solution was composed of induced AnFdc. Ubix The genetically engineered bacteria were obtained by resuspending them in ammonium carbamate buffer and then lysing and separating them.

[0011] The AnFdc Ubix The genetically engineered bacteria contain recombinant plasmids for expressing ferulic acid decarboxylase AnFdc and recombinant plasmids for expressing flavin isopentenyltransferase Ubix.

[0012] The LsPAL gene-engineered bacteria contain a recombinant plasmid for expressing the LsPAL ammonia lyase.

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

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

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

[0016] Preferably, the gene sequence encoding 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.

[0017] Preferably, in the LsPAL genetically engineered bacteria, the recombinant plasmid expressing 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, and single colonies are obtained by resuscitation culture and resistance screening.

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

[0019]

[0020] In the formula: R is R' can be H, p-OH, p-CH3, p-Br, or p-OCH3.

[0021] Preferably, the concentration of the ammonium carbamate buffer solution is 480–520 M, and the pH is 7.8–8.2.

[0022] Preferably, the amount of ammonium bicarbonate added is 5 to 50 molar equivalents of the vinyl aromatic substrate.

[0023] Preferably, the one-pot dual-enzyme tandem catalysis is achieved by oscillation at 180–220 rpm in a sealed headspace flask, at a reaction temperature of 20–22°C, and for a reaction time of 12–24 hours.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] This invention involves adding a decarboxylase (AnFdc) to the aqueous phase. Ubix ), ammonia lyase (LsPAL), ammonium carbamate, and ammonium bicarbonate, which cause the substrate olefin to undergo decarboxylation by enzyme (AnFdc).Ubix The α-amino acid is carboxylated under the catalysis of [catalyst name missing], and then catalyzed by ammonia lyase (LsPAL) to obtain an α-amino acid with a 99% ee value. This one-pot method avoids the intermediate product separation step, and the generated intermediate is immediately converted into the product α-amino acid, avoiding product inhibition problems. The method of this invention provides high yields of α-amino acids. Furthermore, this method can be applied to the synthesis of L-phenylpropionic acid and its derivatives, possessing significant application value and market potential. Attached Figure Description

[0026] Figure 1 A schematic diagram of the one-pot, two-enzyme tandem catalytic synthesis of non-natural chiral α-amino acids;

[0027] Figure 2 This is a schematic diagram of the full sequence of the AnFdc recombinant plasmid;

[0028] Figure 3 This is a schematic diagram of the full sequence of the Ubix recombinant plasmid.

[0029] Figure 4 This is a schematic diagram of the full sequence of the LsPAL recombinant plasmid;

[0030] Figure 5 For AnFdc Ubix Gel images of purified proteins from LsPAL;

[0031] Figure 6 For AnFdc Ubix Gas chromatogram of the decarboxylation reaction of the purified crude enzyme solution;

[0032] Figure 7 The liquid chromatogram of the amino group reaction of the crude enzyme solution purified from LsPAL;

[0033] Figure 8 For AnFdc Ubix HPLC chromatogram of L-phenylalanine synthesized by one-pot dual-enzyme catalysis using LsPAL.

[0034] Figure 9 For AnFdc Ubix - LsPAL one-pot dual-enzyme catalytic synthesis of L-phenylalanine methyl esterification gas chromatogram.

[0035] Figure 10 For AnFdc Ubix Liquid chromatogram of one-pot tandem synthesis of 3-(2-thienyl)-L-alanine using 1-LsPAL. Detailed Implementation

[0036] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. Technical features in the various embodiments of the present invention can be combined accordingly without mutual conflict.

[0037] This invention uses ferulic acid decarboxylase AnFdc Ubix The ammonia lyase LsPAL is a one-pot, two-enzyme tandem catalytic synthesis of α-amino acids. The decarboxylase is ferulic acid decarboxylase AnFdc (Ferulicacid decarboxylase) derived from Aspergillus niger, and its amino acid sequence is shown in SEQ ID No. 2 (PDB No.: 4ZA4, NCBI accession number: XP_001390534.1). Additionally, ferulic acid decarboxylase AnFdc is an isopentenylflavin-dependent decarboxylase, belonging to the UbiD family of enzymes. It depends on the flavin isopentenyltransferase Ubix to catalyze the maturation of isopentenylflavin mononucleotide (prFMN). The amino acid sequence of Ubix is ​​shown in SEQ ID No. 4 (NCBI accession number: AJF45212), and it is derived from Escherichia coli. The ammonia lyase is 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 isopentenyltransferase Ubix, and ammonia lyase LsPAL are shown in SEQ ID No. 1, SEQ ID No. 3, and SEQ ID No. 5, respectively, and their encoding amino acid sequences are shown in SEQ ID No. 2, SEQ ID No. 4, and SEQ ID No. 6, respectively. The synthetic route of this invention is as follows... Figure 1 As shown.

[0038] In this invention, the preparation of genetically engineered bacteria and the enzymatic synthesis of chiral α-amino acids are as follows:

[0039] 1.AnFdc Ubix Gene synthesis and preparation of engineered bacteria

[0040] 1.1 Synthesis of the ferulic acid decarboxylase AnFdc gene

[0041] Based on publicly available genetic data (including databases and published literature), the coding protein sequence of ferulic acid decarboxylase AnFdc, SEQ ID No. 2, was determined. The codon was then optimized to SEQ ID No. 1 to facilitate expression in the engineered *E. coli* strain (BL21(DE3)). The coding gene sequence of ferulic acid decarboxylase AnFdc was synthesized according to SEQ ID No. 1. The synthesized gene was constructed into the expression vector pET30a plasmid with NdeI and XhoI insertion sites, resulting in the recombinant plasmid of ferulic acid decarboxylase AnFdc. Its full sequence is shown below. Figure 2 As shown.

[0042] 1.2 Synthesis of the flavin isopentenyltransferase Ubix gene

[0043] Based on publicly available genetic data (including databases and published literature), the coding protein sequence of flavin isopentenyltransferase Ubix was determined to be SEQ ID No. 4. The codon was then optimized to SEQ ID No. 3 to facilitate expression in the engineered *E. coli* strain (BL21(DE3)). The coding gene sequence of flavin isopentenyltransferase Ubix was synthesized according to SEQ ID No. 3. The synthesized gene was constructed into the expression vector pET21b plasmid, with insertion sites at NdeI and XhoI. The final recombinant plasmid of flavin isopentenyltransferase Ubix was obtained, and its full sequence is shown below. Figure 3 As shown.

[0044] 1.3AnFdc Ubix Transformation of recombinant plasmids of engineered bacteria

[0045] (1) Take 1 μL of ferulic acid decarboxylase AnFdc recombinant plasmid and 1 μL of flavin isopentenyltransferase Ubix recombinant plasmid and add them to 500 μL of Escherichia coli BL21(DE3) competent cells, mix gently and let stand on ice for 30 min.

[0046] (2) Heat shock in a 42℃ water bath for 75 seconds, then quickly transfer to an ice bath and let stand for 5 minutes.

[0047] (3) Add 600uL of antibiotic-free LB liquid medium, 200rpm, 37℃ for 60min recovery.

[0048] (4) Spread 50 μL of bacterial culture onto the surface of LB solid medium containing ampicillin (50 mg / L) and kanamycin (50 mg / L). Invert the plate and incubate at 37°C for 12–16 h until single colonies appear, which is AnFdc. Ubix Genetically engineered bacteria.

[0049] 2.AnFdc UbixInduced expression of genetically engineered bacteria

[0050] Use an inoculation loop to pick up AnFdc Ubix A single colony of the genetically engineered bacteria was inoculated into 5 mL of LB liquid medium (formulation: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl). The inoculated bacterial culture was cultured at 37°C and 200 rpm for 12-16 hours. The resulting seed culture was then transferred at a 10% inoculation ratio to TB medium (formulation: 12 g / L tryptone, 24 g / L yeast extract, 9.4 g / L K₂HPO₄, 2.2 g / L KH₂PO₄, 4 mL / L glycerol), and cultured at 37°C and 200 rpm for 3 hours. Then, 200 mg / L isopropyl thio-β-galactoside was added, and the culture was incubated at 18°C ​​and 200 rpm for 20 hours. The culture was centrifuged at 4°C and 8000 rpm for 30 minutes, the supernatant was discarded, and the bacterial cells were collected; this is the induced AnFdc used in subsequent steps. Ubix Genetically engineered bacteria.

[0051] 3. Gene synthesis and preparation of engineered strains of ammonia lysin LsPAL

[0052] 3.1 Synthesis of the LsPAL gene, an ammonia lyase

[0053] Based on publicly available gene data, including databases and published literature, and according to its encoded protein sequence SEQ ID No. 6, the codon was optimized to SEQ ID No. 5 to facilitate expression in the engineered E. coli strain (BL21(DE3)). The synthesized gene was constructed into the expression vector pET28a, with the gene insertion sites being EcoRI and HindIII, ultimately yielding the LsPAL recombinant plasmid, the full sequence of which is shown below. Figure 4 As shown.

[0054] 3.2 Transformation of recombinant plasmids from engineered bacteria containing the ammonia lysin LsPAL

[0055] (1) Take 1 uL of the ammonia lyase LsPAL recombinant plasmid and add it to 500 uL of Escherichia coli BL21(DE3) competent cells. Mix gently and let stand on ice for 30 min.

[0056] (2) Heat shock in a 42℃ water bath for 75 seconds, then quickly transfer to an ice bath and let stand for 5 minutes.

[0057] (3) Add 1 mL of antibiotic-free LB liquid culture medium, 200 rpm, 37°C for 60 min to recover.

[0058] (4) Take 50 μL of bacterial solution and spread it on the surface of LB solid medium containing kanamycin (50 mg / L). Invert the plate and incubate at 37°C for 12–16 h until a single colony appears. This is the LsPAL genetically engineered ammonia lyase.

[0059] 4. Inducible expression of LsPAL, an amino acid lysin, by engineered bacteria

[0060] A single colony of the LsPAL genetically engineered ammonia lyase strain was picked using an inoculation loop and inoculated into 5 mL of LB liquid medium (formulation: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl). The seed culture was incubated at 37°C and 200 rpm for 12-16 hours. The cultured bacterial culture was then transferred at a 10% inoculation ratio to TB medium (formulation: 12 g / L tryptone, 24 g / L yeast extract, 9.4 g / L K₂HPO₄, 2.2 g / L KH₂PO₄, 4 mL / L glycerol), and incubated at 37°C and 200 rpm for 3 hours. Then, 200 mg / L isopropyl thio-β-galactoside was added, and the culture was incubated at 18°C ​​and 200 rpm for 20 hours. The culture medium was centrifuged at 4°C and 8000 rpm for 30 min using a low-temperature centrifuge. The supernatant was discarded, and the bacterial cells were collected, which were the "inducible expression of ammonia lyase LsPAL Escherichia coli wet cells" used in subsequent steps.

[0061] 5. Protein expression and purification

[0062] 5.1AnFdc Ubix Protein purification and characterization of LsPAL

[0063] Based on the characteristics of these two engineered bacteria, 50 mM Tris-HCl at pH 8.0 is preferred as the lysis buffer in this invention. 8 g of bacterial cells are added to 32 mL of lysis buffer and lysed thoroughly using an ultrasonic lysis apparatus. The lysate is then loaded onto a nickel-based protein purification column in an ice-bath environment. After the system stabilizes, it is sequentially flushed with 50 mM Tris-HCl containing 5 mM imidazole at pH 8.0, 50 mM Tris-HCl containing 50 mM imidazole at pH 8.0, 100 mM Tris-HCl containing 100 mM imidazole at pH 8.0 until the absorbance is balanced. Finally, the protein is flushed with 50 mM Tris-HCl containing 150 mM imidazole, concentrated by ultrafiltration using a low-temperature centrifuge, and finally yields 500 μL of purified protein.

[0064] Take 100 μL of protein dilution buffer, add 25 μL of protein loading buffer (5X), vortex to mix, centrifuge at 1000 rpm for 1 min, place in a 100℃ metal bath for 10 min, cool to room temperature, centrifuge at 8000 rpm for 10 min, take 10 μL of supernatant and load it into the protein gel wells, electrophoresis at 200V for 60 min, stop electrophoresis, clear the protein gel, and stain until the protein gel bands are clear. AnFdc Ubix And LsPAL purified protein gel, such as Figure 5 As shown.

[0065] 5.2AnFdc Ubix Characterization of decarboxylation reaction of engineered bacteria

[0066] According to the above "2.AnFdc" Ubix The method described in "Induced Expression of Genetically Engineered Bacteria" induces the expression of AnFdc. Ubix Genetically engineered bacteria were centrifuged to harvest E. coli cells. 1g of AnFdc was used. Ubix The bacterial cells were added to 4 mL of 50 mM Tris-HCl, pH 8.0 lysis buffer, and sonicated in an ice bath until lysis was complete. Then, 2 mg of 2-thiopheneacrylic acid (with 100 μL dimethyl sulfoxide as a solubilizer) was added, and the mixture was placed in a sealed system and reacted at 30 °C with shaking for 24 h. After the reaction, the yield (decarboxylation yield) of 2-vinylthiophene was determined by gas chromatography using dodecane as an internal standard. AnFdc Ubix The gas chromatogram of the decarboxylation reaction of the purified crude enzyme solution is shown below. Figure 6 As shown.

[0067] 5.3 Amino characterization of LsPAL engineered bacteria

[0068] The LsPAL ammonia lyase was induced to be expressed in the above-mentioned "4. Induction and expression of LsPAL by engineered bacteria" Escherichia coli wet cells (1g), resuspended in 500M NH2COONH4, 5mg cinnamic acid was added, and the reaction was carried out in a sealed headspace bottle at 20℃ with shaking at 200rpm for 12-24h.

[0069] After the reaction, the solution was acidified to pH 2.0 with concentrated hydrochloric acid, filtered through a 0.22 μm aqueous filter, and the yield was determined by liquid chromatography (LC). LC analysis method: Column: CROWNPAK CR(+) column (5 μm, 4.0 × 150 mm, Daicel); Detection wavelength: 254 nm; Mobile phase: 0.1% trifluoroacetic acid aqueous solution (pH≈2) / methanol = 95 / 5; Flow rate: 0.3 mL / min; Column temperature: 25℃; Injection volume: 20 μL. The LC chromatogram of the amino group reaction of the purified crude enzyme solution by LsPAL is shown below. Figure 7 As shown.

[0070] In addition to the aforementioned substrate cinnamic acid, the extended substrate spectrum and respective reaction equations of the LsPAL purified crude enzyme solution can also be described as follows:

[0071]

[0072] Therefore, AnFdc is constructed based on the above. Ubix This invention provides a one-pot method for the tandem synthesis of non-natural chiral α-amino acids using genetically engineered bacteria and the amino acid lyase LsPAL. The specific steps are as follows: [The text abruptly shifts to a different topic] ...in the decarboxylase AnFdc... Ubix In the crude enzyme solution, genetically engineered bacteria expressing the induced aminopyrate lyase LsPAL, ammonium bicarbonate, and a vinyl aromatic substrate dissolved in methanol were added. A one-pot, two-enzyme tandem catalysis was then employed, causing the substrate to first react with the decarboxylase AnFdc. Ubix Carboxylation under the catalysis of [catalyst name missing], followed by catalysis by the amino acid lyase LsPAL, yields non-natural chiral α-amino acids. Among these, the aforementioned decarboxylase AnFdc [missing information]. Ubix The crude enzyme solution can be obtained from the induced expression of AnFdc Ubix The genetically engineered bacteria were obtained by resuspending them in ammonium carbamate buffer and then lysing and separating them.

[0073] The structural formula of the vinyl aromatic substrate in this invention is:

[0074]

[0075] In the formula: R group can be phenyl, pyridyl, thiophene, or naphthalene, with the following structural formulas: The R' in the phenyl group can be H, p-hydroxyl p-OH, p-methyl p-CH3, p-bromo group p-Br, or p-methoxy group p-OCH3.

[0076] The present invention will further demonstrate the specific catalytic synthesis method and its effects through experiments below.

[0077] 6. One-pot two-enzyme tandem catalysis for the synthesis of α-amino acids using decarboxylase / ammonia lyase.

[0078] 6.1 Obtaining AnFdc Ubix Crude enzyme solution from genetically engineered bacteria

[0079] The AnFdc expressed according to the method described in section 2 above is called UbixGenetically engineered bacteria were harvested as E. coli cells after centrifugation. 1g of bacterial cells were taken and resuspended in 4mL of 500M NH2COONH4 (prepared with NH2COONH4 and HCl, pH=8.0). The concentration of the bacterial suspension was adjusted to OD600≈30. The bacterial suspension was lysed by ultrasonic lysis (20min) until complete lysis. Then, it was centrifuged at 4℃, 8000rpm for 30min to separate the supernatant crude enzyme solution.

[0080] 6.2 One-pot dual-enzyme tandem catalytic reaction

[0081] Add the same weight of wet E. coli cells (1g) of aminopyrine lyase LsPAL induced and expressed according to the method described in section 4 above to the supernatant of the crude enzyme solution in 6.1, add 20 eq NH4HCO3, add substrate (dissolved with methanol), place in a sealed headspace flask and carry out the reaction at 20°C with shaking at 200 rpm for 12-24 h.

[0082] 6.3 Post-reaction processing and analysis

[0083] After the reaction was complete, the solution was acidified to pH 2.0 with concentrated hydrochloric acid, filtered through a 0.22 μm aqueous filter, and the yield was determined by liquid chromatography. Liquid chromatography analysis method: Column: CROWNPAK CR(+) column (5 μm, 4.0 × 150 mm, Daicel); Detection wavelength: 254 nm; Mobile phase: 0.1% trifluoroacetic acid aqueous solution (pH≈2) / methanol = 95 / 5; Flow rate: 0.3 mL / min; Column temperature: 25 °C; Injection volume: 20 μL.

[0084] The catalytic results for different styrene substituents are as follows:

[0085]

[0086] The following two examples further demonstrate the specific implementation of the above-described one-pot dual-enzyme tandem catalytic synthesis method for non-natural chiral α-amino acids on substrates styrene and 2-vinylthiophene.

[0087] Example 1: One-pot two-enzyme catalyzed synthesis of L-phenylalanine

[0088] In this embodiment, the biocatalytic route for L-phenylalanine based on the substrate styrene is as follows:

[0089]

[0090] The specific method is as follows: induce the expression of AnFdc UbixThe decarboxylase AnFdc was obtained by resuspending the genetically engineered bacteria in ammonium carbamate buffer and then lysing and separating them. Ubix Crude enzyme solution, then decarboxylase AnFdc Ubix Add the genetically engineered strain (AnFdc) containing the induced aminopyase LsPAL to the crude enzyme solution. Ubix The ratio of the reactant amount to LsPAL is controlled at a certain ratio, denoted as Wetcell weight Fdc / LsPAL (in g / g). Ammonium bicarbonate and styrene (co-soluble in methanol) are used in a one-pot, two-enzyme tandem catalysis process, where the substrate is first reacted with the decarboxylase AnFdc. Ubix Carboxylation under the catalysis of [catalyst name], followed by catalysis by the amino acid lyase LsPAL, yields non-natural chiral L-phenylalanine.

[0091] In this embodiment, based on this catalytic route, the optimal values ​​of a series of parameters in the reaction were explored, as follows:

[0092] 1) Investigating the optimal AnFdc Ubix The reaction enzyme ratio with LsPAL was determined by adding 500 molar equivalents of NH₂COONH₄ and 20 molar equivalents of NH₄HCO₃ to the substrate. The reaction was carried out at 20°C in a shaker for 24 hours, and the yield was measured. The reaction results are as follows:

[0093]

[0094] 2) Investigating the optimal conditions for reaction time and substrate amount:

[0095]

[0096] 3) Investigate the optimal conditions for the type and concentration of carboxyl sources in the reaction:

[0097]

[0098] In summary, in this embodiment, AnFdc Ubix The highest yield (Yield / %) was achieved when the ratio of LsPAL to 1 was 3:1, the reaction time was 24 h, the carboxyl source was ammonium bicarbonate with an equivalent of 50 eq. Using styrene as the substrate, under optimal reaction conditions, AnFdc... Ubix -LsPAL single-pot series liquid phase diagram as shown Figure 8 As shown.

[0099] In addition, after quenching the enzyme reaction solution with hydrochloric acid, the water was evaporated by rotary evaporation and amino acid methyl esterification was performed. The gas chromatogram of the reaction with L-phenylalanine standard methyl ester is shown below. Figure 9 As shown.

[0100] Example 2: One-pot two-enzyme catalyzed synthesis of 3-(2-thienyl)-L-alanine

[0101] In this embodiment, the biocatalytic route for 3-(2-thienyl)-L-alanine based on the substrate 2-vinylthiophene is as follows:

[0102]

[0103] The specific method is as follows: induce the expression of AnFdc Ubix The decarboxylase AnFdc was obtained by resuspending the genetically engineered bacteria in ammonium carbamate buffer and then lysing and separating them. Ubix Crude enzyme solution, then decarboxylase AnFdc Ubix In the crude enzyme solution, genetically engineered bacteria expressing the induced ammonia lyase LsPAL, ammonium bicarbonate, and the substrate 2-vinylthiophene (soluble in methanol) were added. A one-pot, two-enzyme tandem catalysis was then employed, causing the substrate to first react with the decarboxylase AnFdc. Ubix Carboxylation under the catalysis of [catalyst name], followed by catalysis by the amino acid lyase LsPAL, yields non-natural chiral 3-(2-thienyl)-L-alanine.

[0104] In this embodiment, based on this catalytic route, the optimal values ​​of a series of parameters in the reaction were explored, as follows:

[0105] 1) Optimal AnFdc Ubix The reaction enzyme ratio with LsPAL was determined by adding 500 molar equivalents of NH₂COONH₄ and 20 molar equivalents of NH₄HCO₃ to the reaction mixture, using the substrate as a reference. The reaction was carried out at 20°C in a shaker for 24 hours, and the yield was measured. The reaction results are shown in the table below:

[0106]

[0107] 2) Investigating the optimal conditions for reaction time:

[0108]

[0109] Using 2-vinylthiophene as the reaction substrate, AnFdc under optimal reaction conditions Ubix The liquid phase diagram of the LsPAL one-pot series reaction is shown below. Figure 10 As shown.

[0110] In summary, in this embodiment, AnFdc Ubix The ratio of LsPAL to 3:1, with a reaction time of 24 h, yielded the highest reaction rate for 3-(2-thienyl)-L-alanine.

[0111] Compared with existing technologies, the present invention has the following advantages: 1. The one-pot synthesis method of α-amino acids of the present invention is simple and convenient to operate; 2. The process of the present invention is aqueous phase, atmospheric pressure, and mild reaction, which is environmentally friendly and safe; 3. The process of the present invention has high conversion rate, easy-to-obtain products, high overall yield, and low cost; 4. The catalytic route design of the present invention is novel, with advantages such as simple operation, short production cycle, low production cost, high yield, low environmental pressure, and suitability for large-scale industrial production.

[0112] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A one-pot method for the tandem synthesis of non-natural chiral α-amino acids using two enzymes, characterized in that, In decarboxylase An Fdc Ubix Add the induced aminopyase to the crude enzyme solution. Ls PAL genetically engineered bacteria, ammonium bicarbonate, and methanol-soluble vinyl aromatic substrates are used in a one-pot, two-enzyme tandem catalysis process, where the substrate is first decarboxylated by an enzyme. An Fdc Ubix Carboxylation under catalysis, followed by amino-lyase reaction. Ls PAL catalysis yields non-natural chiral α-amino acids; The decarboxylase An Fdc Ubix The crude enzyme solution was induced from expression An Fdc Ubix The genetically engineered bacteria were obtained by resuspending them in ammonium carbamate buffer and then lysing and separating them. The An Fdc Ubix Genetically engineered bacteria possess the ability to express ferulic acid decarboxylase. An Recombinant plasmids for Fdc and recombinant plasmids for expressing flavin isopentenyltransferase Ubix; the ferulic acid decarboxylase An The gene sequence encoding Fdc is shown in SEQ ID No. 1, and the gene sequence encoding the flavin isopentenyltransferase Ubix is ​​shown in SEQ ID No. 3; The aminopyrase Ls PAL genetically engineered bacteria contain enzymes for expressing ammonia lysin. Ls Recombinant plasmid of PAL; the amino lyase Ls The coding gene sequence for PAL is shown in SEQ ID No. 5; The structural formula of the vinyl aromatic substrate is: In the formula: R is , R' can be H, p-CH3, p-Br, or p-OCH3; The pH of the ammonium carbamate buffer solution is 7.8~8.2; The amount of ammonium bicarbonate added is 5 to 50 molar equivalents of the vinyl aromatic substrate; The one-pot dual-enzyme tandem catalysis is achieved in a sealed headspace via oscillation at 180-220 rpm, at a reaction temperature of 20-22°C, and for a reaction time of 12-24 hours.

2. The method for one-pot dual-enzyme tandem catalytic synthesis of non-natural chiral α-amino acids as described in claim 1, characterized in that, The An Fdc Ubix In genetically engineered bacteria, ferulic acid decarboxylase is expressed. An The recombinant plasmid for Fdc was obtained by inserting the coding gene sequence shown in SEQ ID No. 1 between the NdeI and XhoI sites of the expression vector pET30a. The recombinant plasmid for expressing flavin isopentenyltransferase Ubix was obtained by inserting the coding gene sequence shown in SEQ ID No. 3 between the NdeI and XhoI sites of the expression vector pET21b. Both recombinant plasmids were simultaneously introduced into Escherichia coli BL21(DE3) competent cells by heat shock transformation, and single colonies were obtained by resuscitation culture and resistance screening.

3. The method for one-pot dual-enzyme tandem catalytic synthesis of non-natural chiral α-amino acids as described in claim 1, characterized in that, The aminopyrase Ls In PAL genetically engineered bacteria, an ammonia lyase is expressed. Ls The recombinant plasmid of PAL was obtained by inserting the coding gene sequence shown in SEQ ID No. 5 between the EcoRI and HindIII sites of the expression vector pET28a. The recombinant plasmid was introduced into Escherichia coli BL21(DE3) competent cells by heat shock transformation, and single colonies were obtained by resuscitation culture and resistance screening.

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