A mutant of an ancestral ω-aminotransferase for efficiently catalyzing the synthesis of (R)-chiral amine compounds and its application

Abstract

The present invention discloses an ω-transaminase ancestral enzyme mutant and application for efficiently catalyzing the synthesis of (R)-chiral amine compounds, relating to the field of molecular biology technology. Obtained by mutation of the ancestral enzyme Ancata-101 of the ω-transaminase from Aspergillus terreus, the amino acid sequence of the ω-transaminase mutant is shown in SEQ ID NO.6. Compared with the ancestral enzyme, the ω-transaminase ancestral enzyme mutant has significantly improved activity on non-natural substrates 1-acetylnaphthalene, N-tert-butyloxycarbonyl-3-piperidone or 3-oxopyrrolidine-1-carboxylic acid tert-butyl ester.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology technology, and in particular to a mutant of an ω-aminotransferase ancestral enzyme obtained based on pocket engineering, and in particular to a mutant of an ω-aminotransferase ancestral enzyme that efficiently catalyzes the synthesis of (R)-chiral amine compounds and its application. Background Art

[0002] Chiral amines are small molecules containing an amino group at the chiral center and are important intermediates in pharmaceutical synthesis. In recent years, with the continued expansion of the chiral drug market, chiral amines and their derivatives have accounted for over 70% of the market share of chiral drugs, including neurological drugs, cardiovascular drugs, antihypertensive drugs, anti-infective drugs, and vaccines. Sitagliptin, the main ingredient in the anti-diabetic drug, is an R-amine. The enormous market demand for chiral amine drugs makes their efficient preparation extremely important.

[0003] Transaminases are key enzymes in the biosynthesis of chiral amines. They reversibly catalyze the transamination reaction between a keto group and an amino group to synthesize amine compounds. The ω-transaminase from Aspergillus terreus uses pyridoxal phosphate (PLP) as a coenzyme to catalyze the transfer of an amino group from an amino donor to a chiral ketone acceptor, generating a chiral amine and a ketone as a byproduct. The catalytic process is shown below:

[0004]

[0005] Although transaminases have good application prospects in synthesizing chiral amines, wild-type enzymes have the disadvantages of poor thermal stability, low activity on non-natural substrates, and a narrow substrate spectrum, which are not conducive to their application in industrial production. In the prior art, patent applications with application numbers CN105441404A and CN105950581A utilize site-directed mutagenesis technology to transform the ω-transaminase wild type, and obtain ω-transaminase mutants with further improved thermal stability. And the invention patent with authorization number CN114134128B utilizes ancestral sequence reconstruction technology to transform the wild-type ω-transaminase from Aspergillus terreus, and obtains an ω-transaminase mutant Ancata-101 with significantly improved thermal stability. However, the activity of the mutant on non-natural substrates does not change significantly compared to the wild-type enzyme.

[0006] The steric constraints of the ω-aminotransferase substrate binding pocket are the main reasons for its narrow substrate spectrum and low activity towards non-natural substrates. Therefore, modifying the substrate binding pocket is one of the methods to enhance the activity of ω-aminotransferase and expand its substrate spectrum.

[0007] To date, there are no reports on pocket modification of the ancestral enzyme of Aspergillus terreus ω-aminotransferase to enhance its activity and expand its substrate range. Summary of the Invention

[0008] The present invention is based on a structure-guided pocket engineering strategy, through which a mutant of the Aspergillus terreus ω-aminotransferase ancestral enzyme with significantly improved enzyme activity towards non-natural substrates is obtained.

[0009] The present invention analyzed the structure of Ancata-101, the ancestral enzyme of Aspergillus terreus ω-aminotransferase, and selected 17 amino acid sites near the substrate binding pocket for saturation iterative mutagenesis. After screening, a mutant with significantly improved activity was obtained:

[0010] The mutant was named Ancata-1016: H55T-E117S-R128M-V150A-L183F-L188F.

[0011] The present invention provides a mutant based on an ω-aminase ancestral enzyme, which is obtained by mutating the ω-aminase ancestral enzyme Ancata-101 from Aspergillus terreus. The amino acid sequence of Ancata-101 is shown in SEQ ID NO.4, and the amino acid sequence of the ω-aminase ancestral enzyme mutant is shown in SEQ ID NO.6.

[0012] The present invention further provides the use of the ω-aminotransferase ancestral enzyme mutant in catalyzing 1-acetylnaphthalene to generate (R)-(+)-1-(1-naphthyl)ethylamine.

[0013] The present invention further provides the use of the ω-transaminase ancestral enzyme mutant in catalyzing N-tert-butyloxycarbonyl-3-piperidone to generate ((R)-1-tert-butyloxycarbonyl-3-aminopiperidine.

[0014] The present invention further provides the use of the ω-transaminase ancestral enzyme mutant in catalyzing the generation of (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine from tert-butyl 3-oxopyrrolidine-1-carboxylate.

[0015] Compared with the ancestral enzyme Ancata-101, the mutant enzyme has better activity towards non-natural substrates and is more suitable for industrial applications.

[0016] The present invention further provides a gene encoding the ω-aminotransferase ancestral enzyme mutant.

[0017] The gene sequence of the mutant is shown in SEQ ID NO.5.

[0018] The present invention further provides application of the gene in catalyzing 1-acetylnaphthalene to generate (R)-(+)-1-(1-naphthyl)ethylamine.

[0019] The present invention further provides application of the gene in catalyzing N-tert-butyloxycarbonyl-3-piperidone to generate ((R)-1-tert-butyloxycarbonyl-3-aminopiperidine.

[0020] The present invention further provides the use of the gene in catalyzing 3-oxopyrrolidine-1-carboxylic acid tert-butyl ester to generate (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine.

[0021] The present invention also provides a recombinant expression plasmid comprising the gene.

[0022] The present invention also provides a genetically engineered bacterium comprising the recombinant expression plasmid.

[0023] The present invention further provides the use of the genetically engineered bacteria in catalyzing 1-acetylnaphthalene to produce (R)-(+)-1-(1-naphthyl)ethylamine.

[0024] The present invention further provides the use of the genetically engineered bacteria in catalyzing N-tert-butyloxycarbonyl-3-piperidone to produce ((R)-1-tert-butyloxycarbonyl-3-aminopiperidine.

[0025] The present invention further provides the use of the genetically engineered bacteria in catalyzing the production of (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine from tert-butyl 3-oxopyrrolidine-1-carboxylate.

[0026] In summary, the present invention provides the use of the ω-aminotransferase ancestral enzyme mutant obtained by the present invention, the gene encoding the mutant, or the above-mentioned genetically engineered bacteria in any of the following catalytic reactions:

[0027] (1) Catalyzes 1-acetylnaphthalene to produce (R)-(+)-1-(1-naphthyl)ethylamine;

[0028] (2) catalyzes N-tert-butyloxycarbonyl-3-piperidone to produce (R)-1-tert-butyloxycarbonyl-3-aminopiperidine;

[0029] (3) Catalyzes tert-butyl 3-oxopyrrolidine-1-carboxylate to generate (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine.

[0030] The present invention also provides a method for catalyzing 1-acetylnaphthalene to produce (R)-(+)-1-(1-naphthyl)ethylamine, wherein 1-acetylnaphthalene and (R)-(+)-α-methylbenzylamine are respectively used as substrates, and the above-mentioned ω-transaminase ancestral enzyme mutant or the above-mentioned genetically engineered bacteria are used to catalyze a transamination reaction to produce (R)-(+)-1-(1-naphthyl)ethylamine.

[0031] The present invention also provides a method for catalyzing N-tert-butoxycarbonyl-3-piperidone to produce (R)-1-tert-butoxycarbonyl-3-aminopiperidine, wherein N-tert-butoxycarbonyl-3-piperidone and (R)-(+)-α-methylbenzylamine are used as substrates, respectively, and the above-mentioned ω-transaminase ancestral enzyme mutant or the above-mentioned genetically engineered bacteria are used to catalyze a transamination reaction to produce (R)-1-tert-butoxycarbonyl-3-aminopiperidine.

[0032] The present invention also provides a method for catalyzing the production of (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine from tert-butyl 3-oxopyrrolidine-1-carboxylate, wherein tert-butyl 3-oxopyrrolidine-1-carboxylate and (R)-(+)-α-methylbenzylamine are used as substrates, respectively, and the above-mentioned ω-transaminase ancestral enzyme mutant or the above-mentioned genetically engineered bacteria are used to catalyze a transamination reaction to produce (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) Compared with the ancestral enzyme Ancata-101, the activity of mutant Ancata-1016 towards 1-acetylnaphthalene, N-tert-butyloxycarbonyl-3-piperidone, and tert-butyl 3-oxopyrrolidine-1-carboxylate was 0.106 U / mg, 0.495 U / mg, and 0.199 U / mg, respectively, while that of the ancestral enzyme Ancata-101 was only 0.016 U / mg, 0.012 U / mg, and ND (not detected). The activity of Ancata-1016 was significantly improved.

[0035] (2) Using structure-guided pocket engineering strategies, we screened for ω-transaminase mutants with significantly enhanced activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the catalytic process of ω-aminotransferase relying on the coenzyme PLP to reversibly catalyze the transfer of amino groups to prochiral ketones to prepare chiral amine compounds.

[0037] Figure 2 The diagram shows the enzyme activity assay results of wild-type ω-transaminase, ancestral enzyme Ancata-101, and mutant Ancata-1016.

[0038] Figure 3 The figure shows the conversion rate determination results using wild-type ω-aminotransferase, ancestral enzyme Ancata-101 and mutant Ancata-1016; among them, a is the conversion rate result of 1-acetylnaphthalene; b is the conversion rate result of N-tert-butyloxycarbonyl-3-piperidone; c is the conversion rate result of tert-butyl 3-oxopyrrolidine-1-carboxylate. DETAILED DESCRIPTION

[0039] The schematic diagram of the catalytic process of the ω-aminotransferase of the present invention to prepare chiral amine compounds by reversibly catalyzing the transfer of amino groups to prochiral ketones based on the coenzyme PLP is shown in FIG. Figure 1 The specific embodiments are as follows.

[0040] Example 1

[0041] The amino acid sequence of the wild-type ω-transaminase (R)-ω-TA from Aspergillus terreus is shown in SEQ ID NO.2, and the gene sequence is shown in SEQ ID NO.1; the amino acid sequence of its ancestral enzyme Ancata-101 is shown in SEQ ID NO.4, and the gene sequence is shown in SEQ ID NO.3.

[0042] The structure of Ancata-101 was analyzed, and amino acid sites near the substrate binding pocket were selected for saturation iterative mutagenesis. Ultimately, a mutant with significantly improved activity was obtained and named Ancata-1016. The mutation sites and sequence are as follows:

[0043] Ancata-1016: H55T-E117S-R128M-V150A-L183F-L188F, the gene sequence is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6.

[0044] Example 2

[0045] (1) Materials and reagents

[0046] The genes for (R)-ω-TA and Ancata-101 were synthesized by Anhui General Biotechnology Co., Ltd. using the pET-28a(+) vector and the E. coli BL21(DE3) expression host strain. The gene for Ancata-1016 was derived from a mutation of Ancata-101. Isopropyl-β-D-thiogalactopyranoside (IPTG), kanamycin sulfate, pyrodoxal-5'-phosphate (PLP), and a modified Bradford protein assay kit were purchased from Shanghai Bioengineering Co., Ltd.; Ni-NTA chromatography medium was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; and dimethyl sulfoxide (DMSO), 1-acetylnaphthalene, N-tert-butyloxycarbonyl-3-piperidone, tert-butyl 3-oxopyrrolidine-1-carboxylate, and (R)-α-methylbenzylamine were purchased from Aladdin Biochemical Technology Co., Ltd.

[0047] (2) Combined mutation

[0048] Saturation mutagenesis was performed on 17 selected sites (M54, H55, Y60, D61, V62, R79, F115, E117, R128, V148, W149, V150, L183, L188, E237, V239, and T240). The primers are shown in Table 1 , where N represents A / T / C / G, K represents G / T, and M represents A / C.

[0049] Table 1 Primers and their sequences

[0050]

[0051] Note: The underline indicates the mutation site. The primer name with -F indicates the upstream primer, and the primer name with -R indicates the downstream primer.

[0052] PCR amplification was performed using the pET-28a-ω-Ancata-101 plasmid as a template. The PCR amplification system (25 μL) contained: 12.5 μL Prime STAR Max DNA Polymerase, 1 μL upstream primer, 1 μL downstream primer, 1 μL plasmid template (100 ng / μL), and autoclaved ultrapure water to a total volume of 25 μL.

[0053] The PCR amplification procedure was as follows: denaturation at 98°C for 5 minutes, followed by 35 cycles of denaturation at 98°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 100 seconds, followed by a final extension at 72°C for 8 minutes. The PCR product was detected by electrophoresis, revealing a single, clear band.

[0054] The resulting site-directed PCR reaction products were enzymatically digested with DpnI at 37°C for 2 h to eliminate the paternal template. The enzymatic hydrolysis products were transformed into chemically competent E. coli BL21 (DE3) cells using the heat shock method. The transformation solution was spread on LB solid plates containing kanamycin (50 μg / mL) and incubated at 37°C for 12 h to obtain a mutant library.

[0055] The forward mutations obtained by screening were iterated to gradually improve the activity of the transaminase.

[0056] (3) Enzyme expression and purification

[0057] 10 μL of wild-type recombinant plasmid solution, Ancata-101 and mutant solution were inoculated into 5 mL of Luria-Bertani liquid medium (LB medium) containing a final concentration of 50 μg / mL Kanamycin, and cultured in a shaking incubator at 37°C and 200 rpm for 12 h. The solution was transferred to 200 mL of LB liquid medium containing a final concentration of 50 μg / mL Kanamycin at a 2% inoculum volume (V / V), and cultured for 2-3 h at 37°C and 200 rpm. When the OD 600 When the p-value reached 0.8, IPTG was added to a final concentration of 0.5 mM and protein expression was induced at 25°C and 150 rpm. After 20 h of induction, the cells were collected by centrifugation at 6000 rpm and 4°C.

[0058] The bacterial cells were washed once with 50mM PBS buffer (50mM sodium dihydrogen phosphate, 50mM sodium hydrogen phosphate, 300mM sodium chloride, pH 8.0) to remove residual culture medium and then resuspended in the above PBS buffer. The bacterial cells were homogenized in an ice bath. The cell lysate was centrifuged at 8000rpm and 4°C for 1h, and the supernatant was collected as the crude enzyme solution containing ω-transaminase. Subsequently, the crude enzyme solution was filtered through a 0.45μm filter membrane and the target protein was isolated and purified using a Ni-NTA affinity chromatography column.

[0059] The purification buffer is as follows:

[0060] 20 mM imidazole wash buffer: 50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 20 mM imidazole, pH 8.0;

[0061] 50 mM imidazole wash buffer: 50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 50 mM imidazole, pH 8.0;

[0062] 250 mM Imidazole Elution Buffer: 50 mM Sodium Dihydrogen Phosphate (NaHPO), 300 mM Sodium Chloride, 250 mM Imidazole, pH 8.0.

[0063] Specific purification steps:

[0064] 1) Equilibrate the Ni-NTA affinity chromatography column: wash sequentially with 20% (v / v) ethanol aqueous solution, deionized water, and 20 mM imidazole wash buffer for 3 column volumes each.

[0065] 2) Sample loading: The crude enzyme solution is taken with a syringe and filtered through a 0.45 μm filter membrane. The target protein with a 6-histidine tag can bind to the filler.

[0066] 3) Washing: Wash with 3 column volumes of 20 mM imidazole washing buffer and 50 mM imidazole washing buffer respectively, and check with Bradford solution to see if the impurities are washed away.

[0067] 4) Elution: Wash with 250 mM imidazole elution buffer and collect 5 mL of flow-through.

[0068] 5) Column storage: wash with 20 mM imidazole washing buffer, deionized water and 20% (v / v) ethanol aqueous solution for 3 column volumes each in sequence, and finally store in 20% (v / v) ethanol aqueous solution.

[0069] (4) Determination of enzyme activity

[0070] 100 μL of pure enzyme was mixed with 400 μL of substrate solution (0.1 mM PLP, 5 mM 1-acetylnaphthalene, N-tert-butyloxycarbonyl-3-piperidone or tert-butyl 3-oxopyrrolidine-1-carboxylate, 5 mM (R)-α-MBA ((R)-(+)-α-methylbenzylamine, 0.20% DMSO, 50 mM PBS, pH 7.5). 8.0) at 20°C for 5 min, then terminated with 50% (v / v) acetonitrile and the product yield was determined by high-performance liquid chromatography. Enzyme activity (U) is defined as the amount of transaminase required per minute to catalyze the transamination reaction between the substrate (1-acetylnaphthalene, N-tert-butyloxycarbonyl-3-piperidone, or tert-butyl 3-oxopyrrolidine-1-carboxylate) and (R)-α-MBA to produce 1 μmol of product ((R)-(+)-1-(1-naphthyl)ethanamine, (R)-1-tert-butyloxycarbonyl-3-aminopiperidine, or (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine) under specific conditions.

[0071] The enzyme activity assay results of wild type (WT), Ancata-101 and mutant (Ancata-1016) are shown in Figure 2 As shown, the enzyme activity of Ancata-1016 was significantly improved compared with that of WT and Ancata-101.

[0072] (5) Determination of conversion rate

[0073] Engineered bacteria harboring recombinant expression plasmids expressing wild-type, Ancata-101, and mutant enzymes were used to catalyze the conversion of 1-acetylnaphthalene, N-tert-butyloxycarbonyl-3-piperidone, or tert-butyl 3-oxopyrrolidine-1-carboxylate to the corresponding products (R)-(+)-1-(1-naphthyl)ethylamine, (R)-1-tert-butyloxycarbonyl-3-aminopiperidine, or (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine. The conversion rates at different reaction times were determined by high-performance liquid chromatography. The reaction system contained 50 mM PBS, 10 mM substrate, 0.1 mM PLP, 20% DMSO, and 2 g of wet bacterial cells in a total volume of 50 mL.

[0074] The results of the conversion rate determination of the three substrates by wild type (WT), Ancata-101 and mutant (Ancata-1016) engineering bacteria are as follows Figure 3 As shown, after 6 hours of reaction, Ancata-1016 achieved conversions of 54.7%, 85.2%, and 66.8% for 1-acetylnaphthalene, N-tert-butyloxycarbonyl-3-piperidone, and tert-butyl 3-oxopyrrolidine-1-carboxylate, respectively. The catalytic efficiency for N-tert-butyloxycarbonyl-3-piperidone reached 81.3% within 40 minutes. In comparison, the catalytic performance of WT and Ancata-101 was poor. After 24 hours of reaction, the conversions of WT for the three substrates were 10.15%, 14.4%, and 3.7%, respectively. Ancata-101 achieved similar conversions of N-tert-butyloxycarbonyl-3-piperidone and tert-butyl 3-oxopyrrolidine-1-carboxylate as WT, but achieved a 45.5% conversion of 1-acetylnaphthalene. In summary, mutant Ancata-1016 exhibits excellent catalytic performance.

Claims

1. A ω-aminotransferase ancestral enzyme mutant, characterized in that The mutant is obtained by mutation of an ancestral enzyme of ω-aminase from Aspergillus terreus. The amino acid sequence of the mutant ancestral enzyme of ω-aminase is shown in SEQ ID NO.

6.

2. A gene encoding the ω-aminotransferase ancestral enzyme mutant according to claim 1.

3. The gene according to claim 2, wherein The gene sequence encoding the ω-aminotransferase ancestral enzyme mutant is shown in SEQ ID NO.

5.

4. A recombinant expression plasmid comprising the gene according to claim 2 or 3.

5. A genetically engineered bacterium comprising the recombinant expression plasmid according to claim 4.

6. Use of the ω-aminotransferase ancestral enzyme mutant according to claim 1, the gene according to claim 2 or 3, or the genetically engineered bacterium according to claim 5 in any of the following catalytic reactions: (1) Catalyzes 1-acetylnaphthalene to produce (R)-(+)-1-(1-naphthyl)ethylamine; (2) catalyzes N-tert-butyloxycarbonyl-3-piperidone to produce (R)-1-tert-butyloxycarbonyl-3-aminopiperidine; (3) Catalyzes tert-butyl 3-oxopyrrolidine-1-carboxylate to generate (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine.

7. A method for producing (R)-(+)-1-(1-naphthyl)ethylamine from 1-acetylnaphthalene, characterized in that: 1-acetylnaphthalene and (R)-(+)-α-methylbenzylamine are used as substrates, and the ω-transaminase ancestral enzyme mutant according to claim 1 or the genetically engineered bacteria according to claim 5 are used to catalyze a transamination reaction to produce (R)-(+)-1-(1-naphthyl)ethylamine and acetophenone.

8. A method for producing (R)-1-tert-butyloxycarbonyl-3-aminopiperidine from N-tert-butyloxycarbonyl-3-piperidone, characterized in that: N-tert-butyloxycarbonyl-3-piperidone and (R)-(+)-α-methylbenzylamine are used as substrates, and the ω-transaminase ancestral enzyme mutant according to claim 1 or the genetically engineered bacteria according to claim 5 is used to catalyze a transamination reaction to produce (R)-1-tert-butyloxycarbonyl-3-aminopiperidine and acetophenone.

9. A method for producing (R)-1-tert-butyloxycarbonyl-3-aminopiperidine from tert-butyl 3-oxopyrrolidine-1-carboxylate, characterized in that: Using tert-butyl 3-oxopyrrolidine-1-carboxylate and (R)-(+)-α-methylbenzylamine as substrates, the ω-transaminase ancestral enzyme mutant according to claim 1 or the genetically engineered bacteria according to claim 5 is used to catalyze a transamination reaction to produce (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine and acetophenone.

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