An aminotransferase mutant with improved (R)-3-aminobutanol production ability and its application

Abstract

The present invention discloses a transaminase mutant with improved ability to produce (R)-3-aminobutanol and its application. The transaminase mutant includes: a mutant L160V obtained by mutating the leucine at position 160 of the amino acid sequence shown in SEQ ID NO.1 to valine; a mutant V162L obtained by mutating the valine at position 162 of the amino acid sequence shown in SEQ ID NO.1 to leucine; a mutant L160V / V162L obtained by mutating the valine at position 162 of the amino acid sequence shown in SEQ ID NO.3 to leucine. The present invention obtains a novel transaminase evolved naturally through soil metagenomic mining technology, and after semi-rational design and modification of the active pocket, a mutant enzyme with further enhanced (R)-3-aminobutanol synthesis ability is obtained, which has important industrial application potential for the green and efficient preparation of (R)-3-aminobutanol.

Description

Technical Field

[0001] The present invention belongs to the technical field of biocatalysis, and relates to a transaminase mutant with improved ability to produce (R)-3-aminobutanol, nucleic acid molecules encoding the same, vectors and cells containing these nucleic acid molecules, and their application in catalyzing the substrate 4-hydroxy-2-butanone to produce (R)-3-aminobutanol. Background Art

[0002] Chiral amines are a class of amine compounds with an amino group at the chiral center, and are important intermediates in the research of chiral drugs, usually widely used in various industries such as medicine, pesticides, and fine chemicals. They can also be used as resolving agents to resolve racemates, and have good application prospects. Among them, (R)-3-aminobutanol is not only an important intermediate for the synthesis of the anti-AIDS drug dolutegravir, an integrase strand transfer inhibitor of human immunodeficiency virus type-1 (HIV-1), but has also been widely used in the organic synthesis of medicine, materials, fine chemicals, and asymmetric catalysis. Therefore, it is urgently necessary to find an economical, efficient, and practical method for synthesizing (R)-3-aminobutanol.

[0003] Compared with other methods, the enzymatic catalysis of chiral amines is the most green and efficient route for synthesizing (R)-3-aminobutanol. There are several enzymes in nature that catalyze the synthesis of chiral amine compounds: transaminases, amine oxidases, imine reductases, and amino acid dehydrogenases. Among them, monoamine oxidase has a 100% theoretical conversion rate, and its substrate specificity is also very wide, and it can convert secondary and tertiary amines. However, few (S)-selective enzymes have been found so far; for imine reductases, its catalytic mechanism is still unclear, and few substrates have been studied, and it is still in the preliminary stage of research; for the asymmetric synthesis of amine dehydrogenases, the theoretical conversion rate of this enzyme can be as high as 100%, and the ammonia donor is very cheap, only ammonia needs to be introduced, but this reaction requires cofactor recycling, and the cost is relatively expensive. Moreover, it is reported that only one natural amine dehydrogenase can accept NADPH, and its enantioselectivity is also small; while the asymmetric synthesis of ω-transaminase has many advantages, such as a wide substrate spectrum, high stereoselectivity, fast reaction rate, and good stability. Due to these characteristics, transaminases have become one of the several industrial enzymes for producing amino acids, non-natural amino acids, chiral amines, amino alcohols, and amino sugars.

[0004] In the past ten-odd years, the use of ω-transaminase has been considered a very effective method for preparing optically pure amines from the corresponding ketones. However, as academic research and practical production work gradually unfold, the challenges faced by ω-transaminase in the application process inevitably emerge, mainly including the thermodynamic equilibrium problem of the reversible reaction, low reaction substrate concentration, high cost of the amino donor, and the influence of the size limitation of the small pocket on the substrate spectrum. The above problems are the research hotspots of many scholars at present and are also the limiting factors affecting the industrial production of transaminases.

[0005] Molecular modification of transaminases can solve many limitations. There are usually traditional directed evolution methods, computer-aided rational design, and semi-rational design that combines the two. Among them, the directed evolution method mimics the characteristics of natural evolution, uses one or more rounds of random mutations, and at the same time selects appropriate high-throughput screening methods to screen out mutant enzymes that meet the expectations. This method is currently the most commonly used, but the workload of this method is large, time-consuming, and inefficient. In contrast, the method of using computer calculation simulation to analyze and speculate on the relationship between the protein structure, function and sequence of enzymes, and thus reasonably select the modification sites is more targeted and greatly speeds up the process of enzyme modification. However, this method can only be carried out with the known structural and functional information of the enzyme, so it is restricted. To sum up, the semi-rational design that combines the two has higher feasibility in practical applications. Summary of the Invention

[0006] The object of the present invention is to provide a transaminase mutant with improved ability to produce (R)-3-amino-1-butanol, a coding gene, a recombinant plasmid, a recombinant genetically engineered bacterium and their application in catalyzing the substrate 4-hydroxy-2-butanone to produce (R)-3-amino-1-butanol, so as to solve the problems of the existing ω-transaminase in the application process, such as the thermodynamic equilibrium problem of the reversible reaction, the low concentration of the reaction substrate, and the high cost of the amino donor.

[0007] In order to solve the above problems, the present invention adopts the following technical solutions:

[0008] According to the first aspect of the present invention, there is provided a transaminase mutant with improved ability to produce (R)-3-amino-1-butanol, and the transaminase mutant is one of the following: 1) the transaminase mutant L160V obtained by mutating the leucine L at position 160 of the amino acid sequence shown in SEQ ID NO.1 to valine V, and the amino acid sequence is as shown in SEQ ID NO.3; 2) the transaminase mutant V162L obtained by mutating the valine V at position 162 of the amino acid sequence shown in SEQ ID NO.1 to leucine L, and the amino acid sequence is as shown in SEQ ID NO.4; 3) the transaminase mutant L160V / V162L obtained by mutating the valine V at position 162 of the amino acid sequence shown in SEQ ID NO.3 to leucine L, and the amino acid sequence is as shown in SEQ ID NO.5.

[0009] According to the second aspect of the present invention, there is also provided a coding gene of the transaminase mutant.

[0010] According to the third aspect of the present invention, there is provided a recombinant vector containing the coding gene of the transaminase mutant.

[0011] According to the fourth aspect of the present invention, there is provided a genetically engineered bacterium prepared by transformation with the recombinant plasmid. The host cell transformed with the recombinant plasmid can be various conventional host cells in the art. Preferably, the host cell is Escherichia coli BL21(DE3).

[0012] According to the fifth aspect of the present invention, there is provided a method for preparing a transaminase mutant, comprising the following steps: 1) culturing the recombinant genetically engineered bacterium and inducing the expression of the transaminase mutant; 2) isolating the transaminase mutant from the culture obtained in step 1).

[0013] According to the sixth aspect of the present invention, there is provided an application of the transaminase mutant in catalyzing the preparation of (R)-3-aminobutanol from the substrate 4-hydroxy-2-butanone.

[0014] The present invention first mined a novel transaminase SITA from the soil metagenomic database, which is naturally evolved from Sinirhodobacter huangdaonensis. The amino acid sequence of this enzyme is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2. Then, the inventors successfully achieved the heterologous expression of transaminase SITA through a series of molecular cloning means. In this molecular modification process, homology modeling was carried out using the crystal structure of the transaminase of Rhodobacter sp.140A (PDB ID: 7DBE) as a template. This enzyme has the highest sequence identity (61%) with the target protein SITA among all the searched templates. After evaluating the rationality of the model, the substrate was docked into the transaminase SITA model. By analyzing the conformation of the substrate-enzyme docking complex, it was found that there are two special amino acids near the catalytic active pocket of SITA. These two residues are relatively close to the carbonyl group of the substrate and may affect the proton transfer between the substrate and PLP. Therefore, the present invention respectively carried out saturation mutations at these two sites (the 160th and 162nd positions). Subsequently, the ability of all mutants to catalyze the formation of (R)-3-aminobutanol was measured using 4-hydroxy-2-butanone as the substrate. Through these protein engineering strategies, the present invention obtained multiple mutants with improved conversion rates of transaminases to produce (R)-3-aminobutanol. The obtaining of these mutants is of great significance for the catalytic synthesis of (R)-3-aminobutanol.

[0015] In summary, according to a transaminase mutant with improved ability to produce (R)-3-aminobutanol, the encoding gene, the recombinant plasmid, the recombinant genetically engineered bacterium, and their application in catalyzing the substrate 4-hydroxy-2-butanone to produce (R)-3-aminobutanol provided by the present invention, compared with the prior art, have the following advantages:

[0016] 1) The present invention obtained a novel transaminase SITA evolved naturally through the method of soil metagenome mining. This enzyme is derived from Sinirhodobacter huangdaonensis and has excellent catalytic performance, enriching the transaminase enzyme library.

[0017] 2) The present invention further carried out molecular modification on SITA through a semi-rational design strategy, constructing a variety of transaminase mutants capable of catalyzing the formation of (R)-3-aminobutanol from 4-hydroxy-2-butanone, which is beneficial to the directional production of the target product (R)-3-aminobutanol by the transaminase mutants. Among them, the forward mutant with the highest amount of (R)-3-aminobutanol produced by catalyzing 4-hydroxy-2-butanone is L160V / V162L, and the conversion rate of the product (R)-3-aminobutanol it catalyzes is 89%, and the ee value is as high as 99.9%.

[0018] 3) The present invention provides a useful reference for the development of a green and efficient synthesis method of (R)-3-aminobutanol. Description of the Drawings

[0019] Figure 1 Shows the structural model of transaminase SITA and the spatial position distribution of its key mutation sites L160 and V162.

[0020] Figure 2 Shows the sequence alignment analysis of transaminases with catalytic activity on the substrate 4-hydroxy-2-butanone. Detailed Embodiments

[0021] The following further describes the present invention in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional operations in the art or experimental methods recommended by the reagent kit and instrument equipment manufacturers.

[0022] The formula of Luria-Bertani (LB) liquid medium is: peptone 10 g / L, sodium chloride 10 g / L, yeast extract 5 g / L. Quantitative deionized water was added to the weighed three substances and stirred evenly. The pH value was natural. The prepared medium was sterilized at 121 °C for 20 min using an autoclave.

[0023] Luria-Bertani (LB) solid medium can be obtained by adding 1.5% agar to the LB liquid medium, and the sterilization conditions are the same as above.

[0024] Construction of Novel Transaminase SITA in Example 1

[0025] (1) Obtaining of the SITA gene

[0026] Combined with the analysis of the NCBI database and the soil metagenomic database, the inventors screened out an ideal transaminase SITA. Using genomic DNA as a template, the gene fragment encoding SITA was amplified with the following primers. Among them, the nucleotide sequence of transaminase SITA is shown in SEQ ID NO.2, and the amino acid sequence it encodes is shown in SEQ ID NO.1.

[0027] SITA-F: CGC GGATCC ATGACCATGAATCAG (SEQ ID NO.6)

[0028] SITA-R: CCG GAATTC TTACAGATATGACACC (SEQ ID NO.7)

[0029] The PCR reaction system is as follows:

[0030]

[0031] The PCR amplification program is as follows:

[0032]

[0033] After the PCR instrument finishes running, the amplified product is loaded onto a nucleic acid gel, and analyzed by agarose gel electrophoresis. Under ultraviolet light irradiation, the bands with the appropriate size are judged, cut, and then subjected to gel extraction to obtain the gene fragment of SITA.

[0034] (2) Construction of SITA

[0035] The gene fragment of SITA obtained in (1) and the pET-28a(+) vector are double digested with BamH I and EcoR I. The enzyme digestion reaction system is as follows:

[0036]

[0037] The enzyme digestion products are loaded onto a nucleic acid gel, analyzed by agarose gel electrophoresis, the correct bands are cut and subjected to gel extraction. The two are ligated according to the ratio of the addition amount of the target gene to the vector of 5:3. The ligated plasmid is introduced into the competent cell Escherichia coli BL21(DE3) to achieve heterologous expression of the amplified gene. Positive single colonies are picked on the transformed plate and sent for sequencing. Those with correct sequencing are considered successfully constructed.

[0038] Selection of Mutation Sites in Example 2

[0039] Homology modeling was performed using SWISS-MODEL (http: / / www.swissmodel.expasy.org / ) to obtain the model of transaminase SITA. The transaminase from Sinirhodobacter huangdaonensis (PDB ID: 7DBE) was selected as the template, which had the highest identity (61%) with the target protein SITA. After the modeling was completed, the obtained structure was downloaded and optimized. Then, the prepared substrate molecules were docked into the model. After the docking was completed, ideal docking complexes were screened and saved according to the binding energy of the complex, the dominant orientation of the substrate, and the distance between the substrate and the catalytic key groups.

[0040] By analyzing the docking structure, the key residues L160 and V162 near the catalytic active pocket of transaminase SITA were screened out. The spatial position distributions of the structural model of transaminase SITA and its key mutation sites L160 and V162 are as Figure 1 shown. The sequence alignment analysis of the transaminase with catalytic activity on the substrate 4-hydroxy-2-butanone is as Figure 2 shown. Subsequently, saturation mutagenesis was performed on these two potential sites to construct a mutant library.

[0041] Construction of Mutants in Example 3

[0042] Escherichia coli carrying the pET-28a(+)-SITA recombinant plasmid was cultured in an LB liquid medium test tube for 12 h, and the plasmid was extracted as the template for subsequent mutant construction. The primers used for mutagenesis are shown in Table 1.

[0043] Table 1 Primer information for mutants

[0044]

[0045] The PCR reaction system (20 μL) is as follows:

[0046]

[0047] PCR reaction conditions: (1) Pre-denaturation at 95 °C for 3 min; (2) Denaturation at 98 °C for 5 s; (3) Annealing at 56 °C for 15 s; (4) Extension at 72 °C for 6 min. Steps (2)–(4) were cycled 30 times; (5) Finally, extension at 72 °C for 5 min and storage at 4 °C.

[0048] After the PCR product was verified as positive by agarose gel electrophoresis, 0.5 μL of restriction endonuclease Dpn I was added, and the template was removed by incubating in a 37 °C water bath for 2.5 h. Subsequently, transformation, colony picking, and sequencing could be carried out.

[0049] Inducible Expression of Transaminase in Example 4

[0050] The correctly sequenced bacterial sample was transferred into a 5 mL LB liquid medium test tube containing 50 μg / mL kanamycin resistance, and cultured in a shaker at 37°C and 200 rpm for about 12 h. Then it was transferred to a conical flask containing 50 mL LB liquid medium and cultured under the same conditions for about 2.5 h until the OD 600 value reached 0.6 - 0.8. When the OD 600 value met the requirements, an inducer IPTG with a final concentration of 0.1 mM was added, and induction was carried out in a shaker at 20°C and 200 rpm for about 16 - 20 h, and then the next experiment could be carried out.

[0051] After the induction culture was completed, the bacteria were collected. The bacterial liquid was poured into a centrifuge tube and deionized water was added to balance it, and then it was placed in a pre-cooled 4°C centrifuge and centrifuged (8,000 rpm, 10 min). After centrifugation, the supernatant was discarded, 20 mL of 0.85% NaCl solution was added to resuspend the bacteria, and after balancing, it was centrifuged again under the same conditions. After centrifugation, the supernatant was removed as much as possible, and then it was placed in a -40°C refrigerator and frozen for more than 3 h, and then freeze-drying could be carried out. The sample was taken out after being placed in a vacuum freeze-dryer for about 10 h, ground evenly, and freeze-dried bacterial powder was obtained.

[0052] Screening of Mutants in Example 5

[0053] Using the freeze-dried bacterial powder of the transaminase prepared in Example 4 as a catalyst, the conversion reaction of the substrate 4-hydroxy-2-butanone was carried out to screen for mutants with improved ability to produce (R)-3-aminobutanol.

[0054] The composition of the 1 mL reaction system: 20 μL of 1 M substrate (dissolved in methanol), 30 μL of 1 M phenylethylamine (dissolved in methanol), 10 mg of freeze-dried bacterial powder, 10 μL of PLP (10 mM), 940 μL of PB buffer (100 mM pH 8.0). After all components were added, they were mixed well, and three parallel samples were made for each reaction.

[0055] The reaction was carried out in a shaker at 30°C and 250 rpm for 12 h. The reaction was terminated by adding 5% (v / v) of 6 M HCl. Then, the supernatant was taken after centrifugation at 14,000 rpm for 10 min, and the conversion rate was analyzed by high performance liquid chromatography HPLC.

[0056] The HPLC detection method when 4-hydroxy-2-butanone was used as the substrate and phenylethylamine was used as the amino donor: Using an ExtendC18 column (5 μm × 4.6 mm × 250 mm) chromatographic column, the flow rate was 1 mL / min, the mobile phase was an aqueous solution containing 0.1% trifluoroacetic acid and 50% methanol, the ultraviolet detection wavelength was 210 nm, the column temperature was 30°C, and the detection duration was 15 min.

[0057] Using 4-hydroxy-2-butanone as the substrate for the catalytic reaction, the constructed mutant library was screened and analyzed by the above method. The mutants with higher (R)-3-aminobutanol production ability than the wild type are shown in Table 2. As can be seen from the table, the mutants with relatively large increases in conversion rate are L160V, L160C, L160A, V162L, and V162I. These mutants were subjected to combined mutagenesis, and there were a total of six double-point mutants. The primers used for mutagenesis (SEQ ID NO.8-19) are shown in Table 3. The constructed double-point mutant library was screened and analyzed by the same method as above. The mutants with higher (R)-3-aminobutanol production ability than the wild type are shown in Table 4. Among them, the conversion rate of the L160V / V162L double-point mutant reached 89%, and the ee value was as high as 99.9%, far higher than that of the wild type.

[0058] Table 2 Conversion rates of transaminase mutants (3 parallels were set for each experiment)

[0059]

[0060] Table 3 Primer information for mutants

[0061]

[0062] Table 4 Conversion rates of transaminase mutants (3 parallels were set for each experiment)

[0063]

[0064]

[0065] As described above, it is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Various changes can be made to the above embodiments of the present invention. That is, all simple, equivalent changes and modifications made according to the claims and the content of the specification of the present invention application fall within the scope of protection of the claims of the present invention patent. The content not described in detail in the present invention is all conventional technical content.

Claims

1. An aminotransferase mutant with improved (R)-3-aminobutanol production ability, characterized in that, The transaminase mutant is one of the following: 1) the transaminase mutant L160V obtained by mutating the leucine L at position 160 in the amino acid sequence shown in SEQ ID NO.1 to valine V, and the amino acid sequence is as shown in SEQ ID NO.3; 2) the transaminase mutant V162L obtained by mutating the valine V at position 162 in the amino acid sequence shown in SEQ ID NO.1 to leucine L, and the amino acid sequence is as shown in SEQ ID NO.4; 3) the transaminase mutant L160V / V162L obtained by mutating the valine V at position 162 in the amino acid sequence shown in SEQ ID NO.3 to leucine L, and the amino acid sequence is as shown in SEQ ID NO.

5.

2. A coding gene of the transaminase mutant according to claim 1.

3. A recombinant plasmid containing the coding gene according to claim 2.

4. A recombinant genetically engineered bacterium containing the recombinant plasmid according to claim 3.

5. The recombinant genetically engineered bacterium according to claim 4, characterized in that, The host cell is Escherichia coli BL21(DE3).

6. A method for preparing a transaminase mutant, characterized in that, It includes the following steps: 1) Culturing the recombinant genetically engineered bacterium according to claim 4 and inducing the expression of the transaminase mutant; 2) Isolating the transaminase mutant according to claim 1 from the culture obtained in step 1).

7. An application of the transaminase mutant according to claim 1 in catalyzing the substrate 4-hydroxy-2-butanone to produce (R)-3-aminobutanol.

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