Beta-amino acid dehydrogenase mutants and their use in the synthesis of beta-amino acid compounds

Abstract

The application provides a beta-amino acid dehydrogenase mutant and application thereof in synthesis of beta-amino acid compounds. The mutant protein has significantly improved activity of catalyzing synthesis of corresponding chiral beta-amino acid from beta-keto acid substrate. The beta-amino acid dehydrogenase mutant with improved activity in the application can improve the reductive amination capacity thereof, and can convert 50-200 g / L substrate to complete conversion to synthesize optically pure beta-amino acid compounds.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and enzyme engineering, and relates to β-amino acid dehydrogenase mutants and their application in the synthesis of chiral β-amino acids. Specifically, it relates to β-amino acid dehydrogenase mutants, recombinant host cells, cell cultures, and methods for the enzymatic synthesis of chiral β-amino acids. Background Technology

[0002] β-Amino acids are a class of non-protein amino acids with the amino group located on the β-carbon atom. They are key structural units constituting various bioactive substances, drug molecules, natural products, and peptide compounds, and have wide applications in medicine, food, feed, cosmetics, and fine chemicals. For example, (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid is a key intermediate in the synthesis of sitagliptin, an important hypoglycemic drug; β-tyrosine-containing jasamide cyclic ester peptides have potent insecticidal and antibacterial activities; and β-alanine, as the only naturally occurring β-amino acid, can be used as a food or feed additive with broad market prospects.

[0003] The biocatalytic synthesis of chiral β-amino acids offers significant advantages, including mild reaction conditions, high stereoselectivity, and environmental friendliness. β-amino acid dehydrogenases (β-AADHs) can directly reduce pre-amination chiral β-keto acids to β-amino acids using inexpensive inorganic ammonia, exhibiting high atom economy and producing no byproducts, thus holding immense potential for application in the asymmetric synthesis of β-amino acids. However, research on the application of β-AADHs is still in its early stages. Currently, only l-erythro-3,5-diaminohexanoate dehydrogenase (3,5-DAHDH) has been discovered, modified, and used for the synthesis of chiral β-amino acids (ACS Catal. 2015, 5, 2220-2224; Angew. Chem. Int. Ed. 2021, 60, 10203-10210; Org. Lett. 2023, 25, 8469-8473). These enzymes exhibit extremely high substrate specificity for their natural substrates, but show almost no or very low activity for non-natural substrates, resulting in a narrow substrate spectrum. Although our research group previously obtained mutants of 3,5-DAHDH with enhanced catalytic activity for linear aliphatic and aromatic β-amino acid substrates through molecular modification (CN 202110133031.4, CN 202210131291.2), the catalytic activity of these mutants for aromatic or branched aliphatic and small molecule β-amino acids remains very low. This limits their practical application and cannot meet the needs of industrial synthesis of β-amino acids.

[0004] Building upon previous research, we identified a 3,5-DAHDH enzyme derived from *Carboxylicivirga sediminis* and molecularly modified it to enhance its catalytic activity. This led to the construction of a new generation of production strains suitable for the synthesis of chiral β-amino acids, achieving highly efficient biosynthesis of important β-amino acids such as 3-aminobutyric acid (GABA) and β-phenylalanine. This invention improves the synthesis efficiency and yield of chiral β-amino acids, reduces production costs, and establishes a more environmentally friendly synthetic process. Summary of the Invention

[0005] This invention provides a β-amino acid dehydrogenase mutant, its encoding gene, and its applications, which improve enzyme catalytic activity and substrate structural diversity, thereby meeting the needs of industrial use.

[0006] This invention involves directed evolution of a β-amino acid dehydrogenase (NCBI: WP_212191963.1) derived from Carboxylicivirga sediminis, resulting in a β-amino acid dehydrogenase mutant with significantly enhanced enzyme activity. This mutant can catalyze the asymmetric synthesis of corresponding chiral β-amino acids from various β-keto acid substrates.

[0007] This invention provides a β-amino acid dehydrogenase mutant, which is obtained by mutating one of the following mutations based on the wild-type amino acid sequence shown in SEQ ID NO:1:

[0008] 1) The 90th position mutates to I.

[0009] 2) The 103rd position mutates to C.

[0010] 3) Mutation number 106 is changed to W.

[0011] 4) The 106th position mutates to H.

[0012] 5) The 151st position mutates to A.

[0013] 6) The 153rd position mutates to V.

[0014] 7) The 153rd position mutates to K.

[0015] 8) The 153rd position mutates to E.

[0016] 9) The 155th position mutates to H.

[0017] 10) The 173rd position mutates to S.

[0018] 11) The 173rd position mutates to H.

[0019] 12) The 305th position mutates to G.

[0020] 13) The 305th position mutates to S.

[0021] 14) The 308th position mutates to S.

[0022] 15) The 308th position mutates to T.

[0023] 16) The 308th position mutates to C.

[0024] 17) The 318th position mutates to S.

[0025] 18) The 318th position mutates to T.

[0026] 19) The 323rd position mutates to S.

[0027] 20) The 323rd position mutates to N.

[0028] 21) The 323rd position mutates to I.

[0029] 22) The 324th position mutates to E.

[0030] 23) The mutation at position 305 is G and the mutation at position 318 is S.

[0031] 24) The 90th position mutates to I, the 305th position mutates to G, and the 318th position mutates to S.

[0032] 25) The 90th position mutates to I, the 173rd position mutates to S, the 305th position mutates to G, and the 318th position mutates to S.

[0033] 26) The 90th position mutates to I, the 173rd position mutates to S, the 305th position mutates to G, the 308th position mutates to S, and the 318th position mutates to S.

[0034] 27) The 90th position mutates to I, the 173rd position mutates to S, the 305th position mutates to G, the 308th position mutates to S, the 318th position mutates to S, and the 323rd position mutates to S.

[0035] 28) The 90th position mutates to I, the 153rd position mutates to V, the 173rd position mutates to S, the 305th position mutates to G, the 308th position mutates to S, the 318th position mutates to S, and the 323rd position mutates to S.

[0036] 29) The 90th position mutates to I, the 153rd position mutates to V, the 173rd position mutates to S, the 305th position mutates to G, the 308th position mutates to S, the 318th position mutates to S, the 323rd position mutates to S, and the 324th position mutates to E.

[0037] The present invention also provides the encoding gene of the β-amino acid dehydrogenase mutant.

[0038] The present invention further provides a recombinant vector carrying the coding gene.

[0039] The present invention also provides genetically engineered bacteria for expressing the mutant. Specifically, *Escherichia coli* is used as the expression host.

[0040] Preferably, the formate dehydrogenase gene is also overexpressed. Specifically, the formate dehydrogenase gene is the formate dehydrogenase mutant gene 131A-160A-184A-228A.

[0041] The present invention also provides the application of the β-amino acid dehydrogenase mutant and the genetically engineered bacteria in catalyzing the synthesis of corresponding chiral β-amino acids from β-keto acid substrates.

[0042] Specifically, the substrate is a β-keto ester, which is hydrolyzed by an esterase to obtain the corresponding β-keto acid, and the chiral β-amino acid is the corresponding β-amino acid product.

[0043] The present invention particularly provides a method for preparing chiral β-amino acids, which involves contacting the β-amino acid dehydrogenase mutant with a β-keto acid substrate to produce the corresponding chiral β-amino acid.

[0044] Specifically, the β-amino acid dehydrogenase mutant participates in the reaction in the form of pure enzyme, crude enzyme solution, or whole cell obtained by fermentation of the genetically engineered bacteria. The substrate is β-keto ester, which is hydrolyzed by esterase to obtain the corresponding β-keto acid. The chiral β-amino acid is the corresponding β-amino acid product.

[0045] Compared with existing technologies, this invention has the following advantages: The inventors, through directed evolution of β-amino acid dehydrogenase derived from *Carboxylicivirga sediminis*, screened and obtained β-amino acid dehydrogenase mutants with significantly improved catalytic activity and high substrate tolerance. Heterologous overexpression of the β-amino acid dehydrogenase mutant gene in *E. coli* yielded a genetically engineered bacterium that efficiently expresses the β-amino acid dehydrogenase mutant. Under conditions of added coenzyme cycling, a biotransformation process for the synthesis of chiral β-amino acids from β-keto acid substrates using this genetically engineered bacterium was established. The reaction conditions are mild, the catalytic efficiency is high, the reaction cycle is short, and the amount of biocatalyst required is small. The yield reaches over 90% with no byproducts generated. It can convert reactions with high substrate concentrations and possesses excellent catalytic performance, saving costs for the synthesis of chiral β-amino acids and showing great promise for industrialization. Attached Figure Description

[0046] Figure 1 The image shows the protein electrophoresis diagram of the wild-type CsAADH enzyme, where M represents the marker, lane 1 is the control without enzyme expression, and lane 2 is the supernatant expression of CsAADH.

[0047] Figure 2 The liquid phase spectrum of 3-aminobutyric acid is shown, where 1 represents the catalytic conversion reaction of β-amino acid dehydrogenase mutant and 2 represents 3-aminobutyric acid standard. Detailed Implementation

[0048] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional techniques and methods. For example, experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the embodiments can be obtained through legitimate commercial channels.

[0049] Example 1: Construction of a β-amino acid dehydrogenase mutant library and screening of mutants

[0050] First round of revitalization

[0051] The mimic protein structure of CsAADH was obtained using Alphafold2 modeling. Saturation mutations were then performed on amino acid residues within a 12 Å region of its substrate binding pocket (positions 90, 92, 103, 105, 106, 115, 117, 151, 152, 153, 154, 155, 167, 170, 173, 174, 305, 306, 307, 308, 318, 319, 323, 324, 325, and 326). Using pET21a-CsAADH as a template, degenerate codon NNK primers were designed, and one-step PCR was performed using the high-fidelity polymerase Super PfxMasterMix.

[0052] (1) Construction of recombinant plasmid pET21a-CsAADH

[0053] The amino acid sequence of the wild-type β-amino acid dehydrogenase is shown in SEQ ID NO:1. After codon optimization based on this protein sequence, the nucleotide sequence was fully synthesized and cloned into the pET21a expression vector. The gene insertion sites were NdeI and XhoI, resulting in the recombinant plasmid pET21a-CsAADH. The recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells using conventional methods. Positive clones were selected to obtain the recombinant engineered bacterium *E. coli* BL21(DE3) / pET21a-CsAADH, which was stored as a glycerol bacterium at -80℃. The target protein in this recombinant bacterium showed good soluble expression, such as... Figure 1 As shown.

[0054] (2) Construction of CsAADH single-point saturation mutant library

[0055] Using recombinant plasmid pET21a-CsAADH as a template, amplification was performed using mutant primers designed with the degenerate codon NNK. The PCR reaction system and reaction conditions are as follows:

[0056] To a 50 μL PCR reaction system, add 50 ng of template, 1 μL (10 μM) of each of a pair of mutant primers, 25 μL of Super Pfx MasterMix, and distilled water to a final volume of 50 μL. The PCR reaction program is as follows: ① 98℃ pre-denaturation for 2 min, ② 98℃ denaturation for 10 sec, ③ 58℃ annealing for 30 sec, ④ 72℃ extension for 4 min, ⑤ 72℃ final extension for 10 min. Perform steps ② through ④ for a total of 25 cycles.

[0057] After the PCR products obtained in the above steps were verified by agarose gel electrophoresis, the products were purified and digested with restriction endonuclease DpnI at 37°C for 2 h. The digested products were transformed into E. coli BL21(DE3) competent cells and plated on plates containing 100 μg / mL ampicillin antibiotic. The plates were then incubated at 37°C for about 12 h until single colonies grew, yielding a strain expressing the β-amino acid dehydrogenase mutant.

[0058] (3) Induced expression of CsAADH mutant

[0059] The single colonies obtained after the above steps were picked and transferred to 0.6 mL of LB liquid medium containing ampicillin (100 μg / mL) (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl), and cultured overnight in a 96-well plate at 37°C and 800 rpm to obtain a seed culture. The seed culture was then inoculated at a rate of 10% (v / v) into fresh LB medium (1 mL), containing 100 μg / mL ampicillin and 0.1 mM IPTG, and induced to grow at 25°C and 800 rpm for 8–12 h. The bacterial cells were collected by centrifugation at 6000 g, and 1 mg / mL lysozyme was added. The cells were lysed at 37°C and 800 rpm for 2 h, and centrifuged again to obtain the crude enzyme solution of the CsAADH mutant for subsequent enzyme activity assays.

[0060] (4) Screening of CsAADH mutants

[0061] The screening method involved detecting the decrease in absorbance of NADH at 340 nm using a microplate reader. Higher enzyme activity indicated faster NADH consumption and a steeper slope in the absorption curve. The enzyme activity assay system (200 μL) consisted of 50 mM β-keto acid substrate, 0.25 mg / mL NADH, 10 μL crude enzyme solution, and 100 mM formate buffer (pH 9.0) to a final volume of 200 μL. The relative activity of wild-type CsAADH was defined as 1.0. Beneficial mutation sites that enhanced enzyme activity were identified through screening: 90, 103, 105, 106, 151, 153, 155, 173, 305, 308, 318, 323, and 324, specifically mutants 1–22, as shown in Table 1.

[0062] 2. Iterative saturation mutation vitality modification

[0063] Based on the results of saturation mutation, a combination mutant with a single mutation site was constructed at the enhanced site. The resulting mutants were picked and cultured in test tubes containing 4 mL of LB medium. The activity of the expressed protein was detected, and the results of significantly enhanced activity are shown in Table 1.

[0064] Using mutant 12, which showed the highest fold increase in activity, as a template, saturation mutation screening was performed at sites 90, 103, 105, 106, 151, 153, 155, 173, 308, 318, 323, and 324 to obtain the optimal mutant 23. Using mutant 23, which showed the highest activity, as a template, saturation mutation screening was performed at sites 90, 103, 105, 106, 151, 153, 155, 173, 308, 318, 323, and 324 to obtain mutant 24, which showed further increased activity. Using mutant 24 as a template, saturation mutation screening was performed at sites 103, 105, 106, 151, 153, 155, 173, 308, 323, and 324 to obtain mutant 25, which showed further increased activity. Using mutant 25 as a template, saturation mutation screening was performed at sites 103, 105, 106, 151, 153, 155, 173, 308, 318, 323, 173, 308, 318, 323, 173, 308, 318, 323, 324 to obtain mutant 25, which showed further increased activity. Saturation mutation screening was performed at sites 55, 308, 323, and 324, yielding mutant 26 with further enhanced activity. Using mutant 26 as a template, saturation mutation screening was performed at sites 103, 105, 106, 151, 153, 155, 323, and 324, yielding mutant 27 with further enhanced activity. Using mutant 27 as a template, saturation mutation screening was performed at sites 103, 105, 106, 151, 153, 155, and 324, yielding mutant 28 with further enhanced activity. Using mutant 28 as a template, saturation mutation screening was performed at sites 103, 105, 106, 151, 155, and 324, yielding mutant 29 with further enhanced activity. Using mutant 29 as a template, no mutants with further enhanced activity at sites 103, 105, 106, 151, and 155 were obtained.

[0065] Table 1. Relative activity of CsAADH and its mutants against substrates

[0066] ;

[0067] Example 2: Synthesis of β-aminobutyric acid catalyzed by crude enzyme solution of mutant.

[0068] Thaw the bacterial strain stored at -80℃, streak it on an agar plate containing ampicillin, and incubate it overnight at 37℃. Select a single colony from the agar plate and inoculate it into 20 mL of LB medium containing ampicillin. Incubate for approximately 12 hours to obtain a seed culture. Inoculate this seed culture into 700 mL of LB medium containing ampicillin at a 1% inoculation rate and incubate at 37℃ on a shaker at 200 rpm until OD (dose retardation). 600 The pH was approximately 0.6-0.8. IPTG was added to a final concentration of 0.1 mM and the cells were induced at 25°C for 12 h. The cells were then collected by centrifugation at 6000 rpm. A portion of the cells was weighed, resuspended in ammonium formate buffer (pH 8.5), and the cells were disrupted using an autoclave or ultrasonic homogenizer to obtain the crude enzyme solution of the CsAADH mutant. The remaining cells can be stored at -20°C as a whole-cell catalyst.

[0069] 6.5 g of ethyl acetoacetate substrate was added to 100 mL of ammonium formate buffer (pH 8.5, 2 M), and 10 g / L esterase was added. The mixture was hydrolyzed at 30 °C for 0.5 h to obtain 3-oxobutyric acid. 20 g / L of crude enzyme solution from the whole-cell lysis of mutant 27 was added as a biocatalyst, along with 0.5 g / L NAD. + 3 U / mL glucose dehydrogenase and 1.5 M glucose were added, and the pH was controlled at 8.0-8.5. The reaction was carried out at 30℃ for 14 h. After the reaction, the yield was 80.5% as determined by HPLC. The (S)-3-aminobutyric acid product was obtained by separation and purification using a strongly acidic cation exchange resin, with a final yield of 78.7% and an ee >99%. The HPLC results are shown below. Figure 2 As shown.

[0070] The reaction conditions for preparing 3-oxobutyric acid were the same as described above, with the addition of 30 g / L crude enzyme solution from the whole-cell lysis of mutant 27 as a biocatalyst, and 0.5 g / L NAD. + 3 U / mL formate dehydrogenase was used, and the pH was controlled at 8.5-9.0. The reaction was carried out at 30℃ for 12 h. After the reaction, the yield was 85.8% as determined by HPLC. The final yield of (S)-3-aminobutyric acid product after separation and purification was 82.5%, and the product ee > 99%.

[0071] The reaction conditions for preparing 3-oxobutyric acid were the same as described above. 30 g / L of crude enzyme solution after whole-cell lysis of mutant 28 was added as a biocatalyst. The coenzyme cycle system and reaction conditions of formate dehydrogenase were as described above. After 11 h of reaction, the yield was 90.3% as determined by HPLC. The final yield of (S)-3-aminobutyric acid product obtained by separation and purification was 87.2%, and the product ee > 99%.

[0072] The reaction conditions for preparing 3-oxobutyric acid were the same as described above. 20 g / L of crude enzyme solution after whole-cell lysis of mutant 29 was added as a biocatalyst. The coenzyme cycle system and reaction conditions of formate dehydrogenase were as described above. After 10 h of reaction, the yield was 91.3% as determined by HPLC. The final yield of (S)-3-aminobutyric acid product obtained by separation and purification was 88.4%, and the product ee > 99%.

[0073] Example 3: Whole-cell catalytic synthesis of β-aminobutyric acid using mutants

[0074] The mutant was induced and expressed according to the method in Example 2. The bacterial cells were collected by centrifugation (6000 rpm), and the obtained whole cells were used as a biocatalyst. Other conditions were the same as in Example 2.

[0075] The reaction conditions and glucose dehydrogenase-coenzyme cycle system for preparing 3-oxobutyric acid were the same as in Example 2. 20 g / L of mutant 27 whole cells were added as a biocatalyst. After 12 h of reaction, the yield was 88.7% as determined by HPLC. The final yield of (S)-3-aminobutyric acid product obtained through separation and purification was 83.5%, as shown in the HPLC results. Figure 2 As shown.

[0076] The reaction conditions and formate dehydrogenase coenzyme cycle system for preparing 3-oxobutyric acid were the same as in Example 2. 30 g / L of mutant 27 whole cells were added as a biocatalyst. After 11 h of reaction, the yield was 90.4% as detected by HPLC. The final yield of (S)-3-aminobutyric acid product obtained by separation and purification was 82.0%, and the product ee > 99%.

[0077] The reaction conditions and formate dehydrogenase coenzyme cycle system for preparing 3-oxobutyric acid were the same as in Example 2. 30 g / L of mutant 28 whole cells were added as a biocatalyst. After 10 h of reaction, the yield was 92.5% as determined by HPLC. The final yield of (S)-3-aminobutyric acid product obtained by separation and purification was 83.6%, and the product ee > 99%.

[0078] The reaction conditions and formate dehydrogenase coenzyme cycle system for preparing 3-oxobutyric acid were the same as in Example 2. 20 g / L of mutant 29 whole cells were added as a biocatalyst. After 11 h of reaction, the yield was 90.8% as determined by HPLC. The final yield of (S)-3-aminobutyric acid product obtained by separation and purification was 82.9%, and the product ee > 99%.

[0079] 10.4 g of ethyl acetoacetate substrate was used to prepare 3-oxobutyric acid. The reaction conditions and formate dehydrogenase coenzyme cycle system were the same as in Example 2. 30 g / L of mutant 29 whole cells were added as a biocatalyst. After 10 h of reaction, the yield was 93.4% as determined by HPLC. The final yield of (S)-3-aminobutyric acid product obtained by separation and purification was 85.2%, and the product ee > 99%.

[0080] Take 13 g of ethyl acetoacetate substrate, add 15 g / L esterase, and prepare 3-oxobutyric acid under the same reaction conditions and formate dehydrogenase coenzyme cycle system as in Example 2. Add 30 g / L of mutant 29 whole cells as a biocatalyst. After reacting for 11 h, the yield was 94.6% as determined by HPLC. The final yield of (S)-3-aminobutyric acid product obtained by separation and purification was 86.3%, and the product ee > 99%.

[0081] 20 g of ethyl acetoacetate substrate was added, and 20 g / L esterase was added. The reaction conditions for preparing 3-oxobutyric acid were the same as in Example 2. 30 g / L of mutant 29 whole cells were added as a biocatalyst, along with 5 U / mL formate dehydrogenase. Other reaction conditions were the same as in Example 2. After reacting for 12 h, the yield was 85.8% as determined by HPLC. The final yield of (S)-3-aminobutyric acid product after separation and purification was 80.6%, with an ee >99%.

[0082] Example 4: Whole-cell catalytic synthesis of β-aminobutyric acid using mutant co-expression strains

[0083] Construction of CsAADH mutant co-expressing strain with formate dehydrogenase: Primers were designed for the CsAADH mutant 27 gene and the formate dehydrogenase mutant 131A-160A-184A-228A gene from Pseudomonas sp. 101 (whose wild-type amino acid sequence is shown in SEQ ID NO:2, Vladimir I. Tishkov and Vladimir O. Popov, Protein engineering of formate dehydrogenase, Biomol. Eng. 2006, 23, 89-110). The gene fragments were amplified by PCR according to the method in Example 1. The fragments were ligated to the Nde I / Xho I and Nco I / Hind III multiple cloning sites of the pRSFDuet-1 plasmid using homologous recombinase. The ligation products were transformed into Escherichia coli BL21(DE3) competent cells. Positive clones were selected to obtain the mutant co-expressing recombinant engineered strain, which was stored as a glycerol strain at -80℃. The construction methods for CsAADH mutants 28 and 29 co-expressing strains with formate dehydrogenase are the same as described above. The co-expressing engineered bacteria are kanamycin resistant, and other conditions are induced according to the method in Example 2 to obtain whole cells of the mutant co-expressing bacteria.

[0084] Take 13 g of ethyl acetoacetate substrate, add 15 g / L esterase, and prepare 3-oxobutyric acid under the same reaction conditions as in Example 2. Add 30 g / L whole cells of mutant 27 co-expressing bacteria as a biocatalyst, and 0.5 g / L NAD. + The pH was controlled at 8.5-9.0, and the reaction was carried out at 30℃ for 10 h. The yield was 88.2% as determined by HPLC. The final yield of (S)-3-aminobutyric acid product obtained by separation and purification was 82.0%, and the product ee > 99%.

[0085] The reaction conditions for preparing 3-oxobutyric acid were the same as described above. 30 g / L of whole cells of mutant 28 co-expressing bacteria were added as a biocatalyst. Other reaction conditions were the same as described above. After 9 h of reaction, the yield was 89.7% as determined by HPLC. The final yield of (S)-3-aminobutyric acid product obtained by separation and purification was 83.5%, and the product ee > 99%.

[0086] The reaction conditions for preparing 3-oxobutyric acid were the same as described above. 30 g / L of whole cells of mutant 29 co-expressing bacteria were added as a biocatalyst. Other reaction conditions were the same as described above. After 8 h of reaction, the yield was 94.3% as determined by HPLC. The final yield of (S)-3-aminobutyric acid product obtained by separation and purification was 89.0%, and the product ee > 99%.

[0087] 20 g of ethyl acetoacetate substrate was added, and 20 g / L of esterase was added. The reaction conditions for preparing 3-oxobutyric acid were the same as in Example 2. 30 g / L of whole cells of mutant 29 co-expressing bacteria were added as a biocatalyst, and other reaction conditions were the same as described above. After reacting for 10 h, the yield was 87.6% as determined by HPLC. The final yield of (S)-3-aminobutyric acid product after separation and purification was 82.9%, with an ee > 99%.

[0088] Example 5: Whole-cell catalytic synthesis of 3-aminovaleric acid using mutant co-expression strains

[0089] Co-expressing bacteria of mutant 29 were obtained by inducing expression according to the method of Example 4. The bacterial cells were collected by centrifugation (6000 rpm) and the obtained whole cells were used as biocatalysts.

[0090] 7.2 g of ethyl propionyl acetate substrate was added to 100 mL of ammonium formate buffer (pH 8.5, 2 M), and 15 g / L of esterase was added. The mixture was hydrolyzed at 30 °C for 0.5 h to obtain 3-oxovalerate. 30 g / L of whole cells of mutant 29 co-expressing bacteria was added as a biocatalyst. Other conditions were the same as in Example 4. After 8 h of reaction, the yield was 93.2% as determined by HPLC. The final yield of (S)-3-aminovalerate product after purification was 88.0%, with an ee > 99%.

[0091] Example 6: Whole-cell catalytic synthesis of 3-aminohexanoic acid using mutant co-expression strains

[0092] 7.9 g of ethyl butyrylamide substrate was added to 100 mL of ammonium formate buffer (pH 8.5, 2 M), and 15 g / L of esterase was added. The mixture was hydrolyzed at 30 °C for 0.5 h to obtain 3-oxohexanoic acid. 30 g / L of whole cells of mutant 29 co-expressing bacteria was added as a biocatalyst. Other reaction conditions were the same as in Example 4. After 6 h of reaction, the yield was 92.5% as determined by HPLC. The (S)-3-aminohexanoic acid product was obtained by separation and purification using a strongly acidic cation exchange resin, with a final yield of 88.1% and an ee > 99%.

[0093] Example 7: Whole-cell catalytic synthesis of 3-amino-3-phenylpropionic acid using mutant co-expression strains

[0094] 5.8 g of ethyl benzoyl peroxide substrate was added to 100 mL of ammonium formate buffer (pH 8.5, 2 M), and 20 g / L esterase was added. The mixture was hydrolyzed at 30 °C for 1 h to obtain 3-oxo-3-phenylpropionic acid. 30 g / L of whole cells of mutant 29 co-expressing bacteria was added as a biocatalyst. Other reaction conditions were the same as in Example 4. After 12 h of reaction, the yield was 91.6% as determined by HPLC. The final yield of (R)-3-amino-3-phenylpropionic acid after purification was 88.4%, with an ee > 99%.

[0095] Example 8: Whole-cell catalytic synthesis of 3-amino-4-phenylbutyric acid using mutant co-expression strains

[0096] 10.3 g of ethyl 3-oxo-4-phenyl-butyrate substrate was added to 100 mL of ammonium formate buffer (pH 8.5, 2 M), and 20 g / L esterase was added. The mixture was hydrolyzed at 30 °C for 1 h to obtain 3-oxo-4-phenylbutyric acid. 30 g / L of whole cells of mutant 29 co-expressing bacteria was added as a biocatalyst. Other reaction conditions were the same as in Example 4. After 9 h of reaction, the yield was 94.3% as determined by HPLC. The final yield of (S)-3-amino-4-phenylbutyric acid product after purification was 90.3%, with an ee > 99%.

[0097] Example 9: Whole-cell catalytic synthesis of 3-amino-4-(2,4,5-trifluorophenyl)-butyric acid using mutant co-expression strain

[0098] 13 g of ethyl 3-oxo-4-(2,4,5-trifluorophenyl)butyrate substrate was added to 100 mL of ammonium formate buffer (pH 8.5, 2 M), and 20 g / L esterase was added. The mixture was hydrolyzed at 30 °C for 1 h to obtain 3-oxo-4-(2,4,5-trifluorophenyl)butyric acid. 30 g / L of whole cells of mutant 29 co-expressing bacteria was added as a biocatalyst. Other reaction conditions were the same as in Example 4. After 10 h of reaction, HPLC analysis showed a yield of 92.2%. The final yield of (S)-3-amino-4-(2,4,5-trifluorophenyl)butyric acid after purification was 89.4%, with an ee > 99%.

Claims

1. A β-amino acid dehydrogenase mutant, characterized in that, It was obtained by performing one of the following mutations based on the wild-type amino acid sequence shown in NCBI accession number WP_212191963.1: Mutant 12: The 305th position is mutated to G. Mutant 13: The 305th position is mutated to S. Mutant 23: The 305th position is mutated to G and the 318th position is mutated to S. Mutant 24: 90th position mutated to I, 305th position mutated to G, and 318th position mutated to S. Mutant 25: The 90th position is mutated to I, the 173rd position to S, the 305th position to G, and the 318th position to S. Mutant 26: 90th position mutated to I, 173rd position mutated to S, 305th position mutated to G, 308th position mutated to S, and 318th position mutated to S. Mutant 27: 90th position mutated to I, 173rd position mutated to S, 305th position mutated to G, 308th position mutated to S, 318th position mutated to S, and 323rd position mutated to S. Mutant 28: 90th position mutated to I, 153rd position mutated to V, 173rd position mutated to S, 305th position mutated to G, 308th position mutated to S, 318th position mutated to S, and 323rd position mutated to S. Mutant 29: The 90th position is mutated to I, the 153rd position to V, the 173rd position to S, the 305th position to G, the 308th position to S, the 318th position to S, the 323rd position to S, and the 324th position to E.

2. The encoding gene of the β-amino acid dehydrogenase mutant as described in claim 1.

3. A recombinant vector carrying the encoding gene as described in claim 2.

4. Genetically engineered bacteria expressing the mutant as described in claim 1.

5. The genetically engineered bacterium according to claim 4, characterized in that, Escherichia coli was used as the expression host.

6. The genetically engineered bacteria as described in claim 4, characterized in that, It also overexpresses the formate dehydrogenase gene.

7. The application of the β-amino acid dehydrogenase mutant as described in claim 1, and the genetically engineered bacteria as described in claim 4, 5, or 6, in catalyzing the synthesis of corresponding chiral β-amino acids from β-keto acid substrates.

8. The application as described in claim 7, characterized in that, The substrate is a β-keto ester, which is hydrolyzed by an esterase to obtain the corresponding β-keto acid, and the chiral β-amino acid is the corresponding β-amino acid product.

9. A method for preparing chiral β-amino acids, characterized in that, The β-amino acid dehydrogenase mutant as described in claim 1 is contacted with a β-keto acid substrate to produce the corresponding chiral β-amino acid.

10. The method as described in claim 9, characterized in that, The β-amino acid dehydrogenase mutant participates in the reaction in the form of pure enzyme, crude enzyme solution, or whole cell form obtained by fermentation of genetically engineered bacteria as described in claim 4, 5, or 6. The substrate is β-keto ester, which is hydrolyzed by esterase to obtain the corresponding β-keto acid. The chiral β-amino acid is the corresponding β-amino acid product.

Images