Method for producing L-2-aminobutyric acid

Abstract

The invention discloses a method for producing L-2-aminobutyric acid, which comprises the following steps of: constructing a recombinant bacterium capable of expressing heterologous aldolase, lactic dehydrogenase and leucine dehydrogenase, taking propionaldehyde and ammonium formate as substrates, and converting the propionaldehyde and the ammonium formate into the L-2-aminobutyric acid by using the strain. The selected substrates propionaldehyde and ammonium formate are wide in source, the preparation process is simple, the price is low, and the production cost can be reduced by applying propionaldehyde and ammonium formate to production of L-2-aminobutyric acid; meanwhile, the selected enzyme has the advantages of high activity, high optical specificity and the like, and the L-2-aminobutyric acid can be efficiently produced by utilizing the enzyme. Therefore, the method for producing the L-2-aminobutyric acid, provided by the invention, has a good industrial application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a method for producing L-2-aminobutyric acid. Background Art

[0002] L-2-Aminobutyric acid, also known as α-aminobutyric acid and homoalanine, is an unnatural chiral amino acid that can be used as an important precursor to synthesize a variety of chiral drugs, including ethambutol and brivaracetam for the treatment of tuberculosis, and the anti-epileptic drug levetiracetam.

[0003] The main methods for preparing L-2-aminobutyric acid are chemical synthesis and biotransformation. The chemical synthesis method has the disadvantages of serious environmental pollution, low chiral purity of the product and harsh reaction conditions, making it difficult to achieve industrial production. The biotransformation method has the advantages of high specificity, green environmental protection, mild reaction conditions and no need for multi-step separation and purification, and has received widespread attention.

[0004] At present, scholars at home and abroad have reported a variety of biological preparation routes for L-2-aminobutyric acid. For example, Rongsheng Tao et al. used L-threonine as a substrate and produced L-2-aminobutyric acid through the catalysis of L-threonine deaminase, L-leucine dehydrogenase and formate dehydrogenase; Yufeng Liu et al. used L-glutamate as a substrate and produced L-2-aminobutyric acid through the action of glutamate mutase and L-aspartate-β-decarboxylase.

[0005] The L-2-aminobutyric acid preparation routes reported above generally have disadvantages such as low yield and high substrate cost. Therefore, it is very necessary to develop a method for preparing L-2-aminobutyric acid with low cost and high production efficiency.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a method for producing L-2-aminobutyric acid, which can efficiently produce L-2-aminobutyric acid by using low-cost propionaldehyde and ammonium formate as substrates.

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

[0009] In a first aspect, the present invention provides a recombinant bacterium for producing L-2-aminobutyric acid, which is capable of expressing heterologous aldolase, lactate dehydrogenase and leucine dehydrogenase;

[0010] Among them, aldolase is derived from Sphaerobacter thermophilus or Lachnellula suecica; lactate dehydrogenase is derived from Aeromonas hydrophila or Staphylococcus argenteus; and leucine dehydrogenase is derived from Thermosediminibacter oceani or Halanaerobium praevalens.

[0011] In a second aspect, the present invention provides a method for preparing the above-mentioned recombinant bacteria, which comprises: inserting the genes of aldolase, lactate dehydrogenase and leucine dehydrogenase into an expression vector to obtain a recombinant vector, and then introducing the recombinant vector into a starting strain to obtain the recombinant bacteria.

[0012] In a third aspect, the present invention provides a whole-cell catalyst comprising the above-mentioned recombinant bacteria.

[0013] In a fourth aspect, the present invention provides the use of the above-mentioned recombinant bacteria in the synthesis of L-2-aminobutyric acid and its downstream products.

[0014] In a fifth aspect, the present invention provides a method for producing L-2-aminobutyric acid, which comprises adding the above-mentioned recombinant bacteria to a reaction system containing propionaldehyde for whole-cell catalysis to obtain L-2-aminobutyric acid.

[0015] The present invention has the following beneficial effects:

[0016] The present invention constructs a recombinant bacterium capable of expressing heterologous aldolase, lactate dehydrogenase and leucine dehydrogenase, and propionaldehyde and ammonium formate are used as substrates, and the strain can be used to convert them into L-2-aminobutyric acid. The substrate propionaldehyde and ammonium formate selected in the present invention have a wide source, a simple preparation process and a low price. Applying them to the production of L-2-aminobutyric acid can reduce production costs; at the same time, the enzyme selected in the present invention has the advantages of high activity and strong optical specificity, and the above enzyme can efficiently produce L-2-aminobutyric acid. Therefore, the method for producing L-2-aminobutyric acid provided by the present invention has good prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 This is the synthetic route of L-2-aminobutyric acid of the present invention. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0020] like Figure 1 As shown, the L-2-aminobutyric acid synthesis pathway adopted by the present invention is: propionaldehyde and ammonium formate are converted into (S)-2-hydroxybutyric acid by aldolase (ALD), (S)-2-hydroxybutyric acid is converted into 2-ketobutyric acid by lactate dehydrogenase (LaDH), and 2-ketobutyric acid is converted into L-2-aminobutyric acid by leucine dehydrogenase (LeuDH). The coenzymes required in the reaction process are provided by glucose metabolism by the bacteria, and the coenzymes required for the reaction mediated by LaDH and LeuDH can complement each other. The present invention is based on Figure 1 The L-2-aminobutyric acid synthesis pathway shown in the figure designs a new recombinant bacterium, which can express the key enzymes of the synthesis pathway, namely ALD, LaDH and LeuDH. Through screening and comparison, the present invention obtains six enzymes with high L-2-aminobutyric acid production efficiency.

[0021] Among them, ALD includes StALD derived from Sphaerobacter thermophilus and LsALD derived from Lachnellulasuecica.

[0022] LaDH includes AhLaDH derived from Aeromonas hydrophila and SaLaDH derived from Staphylococcusargenteus.

[0023] LeuDH includes ToLeuDH derived from Thermosediminibacter oceani and HpLeuDH derived from Halanaerobium praevalens.

[0024] The Genbank number of StALD is ACZ39383.1, its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.3; the Genbank number of LsALD is TVY82384.1, its amino acid sequence is shown in SEQ ID NO.2, and its nucleotide sequence is shown in SEQ ID NO.4.

[0025] The Genbank number of AhLaDH is CAD7560089.1, its amino acid sequence is shown in SEQ ID NO.5, and its nucleotide sequence is shown in SEQ ID NO.7; the Genbank number of SaLaDH is BBD87208.1, its amino acid sequence is shown in SEQ ID NO.6, and its nucleotide sequence is shown in SEQ ID NO.8.

[0026] The Genbank number of ToLeuDH is ADL08309.1, its amino acid sequence is shown in SEQ ID NO.9, and its nucleotide sequence is shown in SEQ ID NO.11; the Genbank number of HpLeuDH is ADO77485.1, its amino acid sequence is shown in SEQ ID NO.10, and its nucleotide sequence is shown in SEQ ID NO.12.

[0027] The method for constructing the above-mentioned recombinant bacteria comprises: connecting the genes of ALD, LaDH and LeuDH to an expression vector, and then introducing the obtained recombinant expression vector into the starting strain, thereby obtaining the recombinant bacteria.

[0028] In some embodiments, the starting strain of the recombinant bacteria is Escherichia coli. In other embodiments, the starting strain of the recombinant bacteria can be other strains, which are not specifically limited in the present invention.

[0029] In some embodiments, the Escherichia coli is selected from any one of Escherichia coli BL21 (DE3), Escherichia coli DH5α, and Escherichia coli XL-Blue.

[0030] In some embodiments, the expression vectors include pCDFDuet-1 plasmid and pET28a(+) plasmid.

[0031] The following recombinant vectors can be constructed using the above expression vectors: pET28a-StALD, pET28a-LsALD, pCDFDuet-AhLaDH-ToLeuDH, pCDFDuet-AhLaDH-HpLeuDH, pCDFDuet-SaLaDH-ToLeuDH and pCDFDuet-SaLaDH-HpLeuDH.

[0032] Preferably, when constructing recombinant Escherichia coli, the combination of pET28a-StALD and pCDFDuet-SaLaDH-ToLeuDH can increase the production of L-2-aminobutyric acid.

[0033] Furthermore, the present invention also provides a whole-cell catalyst based on this, which contains the above-mentioned recombinant bacteria.

[0034] The above-mentioned recombinant bacteria are used as whole-cell catalysts, and the ALD, LaDH and LeuDH expressed by them can efficiently convert propionaldehyde and ammonium formate into L-2-aminobutyric acid.

[0035] The present invention also provides the use of the recombinant bacteria in synthesizing L-2-aminobutyric acid and its downstream products.

[0036] Based on this, the present invention can use the recombinant bacteria to produce L-2-aminobutyric acid, the method is as follows:

[0037] The recombinant bacteria are added into a reaction system containing propionaldehyde and ammonium formate for whole-cell catalysis to obtain L-2-aminobutyric acid.

[0038] In some embodiments, the recombinant bacteria are induced to culture before whole-cell transformation. The induction culture process is as follows: the recombinant bacteria are inoculated into LB medium containing 30-60 mg / L kanamycin and 30-60 mg / L streptomycin, and seed liquid is obtained after culture. The seed liquid is then inoculated into fresh LB medium until the bacterial concentration OD600 is 1.5-2.0. nm When the concentration reaches 0.6-0.8, inducer is added, and wet bacteria are obtained by separation and washing after induction.

[0039] In some embodiments, the culture conditions of the above-mentioned recombinant bacteria are: temperature of 32-38°C, rotation speed of 150-250rpm, and culture time of 10-16h.

[0040] In some embodiments, the culture conditions of the above seed solution are: temperature of 32-38°C and rotation speed of 150-250rpm.

[0041] In some embodiments, the induction conditions are: inducer 0.2-0.6 mM IPTG, temperature 25-30° C., time 10-16 h.

[0042] In some embodiments, the above separation and washing conditions are: a rotation speed of 7500-8500 rpm, a centrifugation time of 5-15 min; and the wet bacteria are washed with saline, and the concentration of the saline is 0.9%.

[0043] In some embodiments, the whole-cell catalyzed reaction system includes: 1-10 g / L of recombinant bacteria wet weight, 1-50 g / L of propionaldehyde, 2-60 g / L of ammonium formate, 5-20 g / L of glucose, and 0.01-0.05 g / L of pyridoxal phosphate.

[0044] In some embodiments, the reaction conditions for whole-cell catalysis are: pH=6.0-9.0, temperature is 15-40°C, and reaction time is 8-24h.

[0045] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0046] (1) Selection of aldolase

[0047] The amino acid sequences of aldolase StALD and LsALD were obtained from the NCBI database, and codon optimization was performed according to the preference of Escherichia coli. Two nucleotide sequences were synthesized by total synthesis through conventional genetic engineering operations, as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. Restriction sites EcoRI and HindIII were added at both ends of the nucleotide sequence.

[0048] (2) Selection of lactate dehydrogenase

[0049] The amino acid sequences of lactate dehydrogenase AhLaDH and SaLaDH were obtained from the NCBI database, and codon optimization was performed according to the preference of Escherichia coli. Two nucleotide sequences were synthesized by total synthesis through conventional genetic engineering operations, as shown in SEQ ID NO.7 and SEQ ID NO.8, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively. Restriction sites EcoRI and HindIII were added at both ends of the nucleotide sequence.

[0050] (3) Selection of leucine dehydrogenase

[0051] The amino acid sequences of leucine dehydrogenase ToLeuDH and HpLeuDH were obtained from the NCBI database, and codon optimization was performed according to the preference of Escherichia coli. Two nucleotide sequences were synthesized by total synthesis through conventional genetic engineering operations, as shown in SEQ ID NO.11 and SEQ ID NO.12, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively. Restriction sites NdeI and XhoI were added at both ends of the nucleotide sequence.

[0052] 3. Construction of the three-enzyme co-expression system and bacterial culture

[0053] Select one enzyme from each of the above selected aldolase, lactate dehydrogenase and leucine dehydrogenase, and co-express the three enzymes. Use pET28a(+) and pCDFDuet-1 double plasmids to co-express the coding genes of the three enzymes; pET28a(+) is loaded with aldolase, and pCDFDuet-1 is loaded with lactate dehydrogenase and leucine dehydrogenase. After obtaining the co-expression recombinant plasmid, the two recombinant plasmids are simultaneously transferred into Escherichia coli BL21 competent cells, and positive transformants are obtained by screening on plates containing kanamycin and streptomycin, that is, recombinant Escherichia coli. The obtained recombinant bacteria are inoculated into fresh liquid culture medium, induced culture, centrifuged, and wet bacteria are obtained.

[0054] 4. Whole-cell conversion of propionaldehyde and ammonium formate to produce L-2-aminobutyric acid

[0055] Conversion system: propionaldehyde 1-50g / L, ammonium formate 2-60g / L, glucose 5-20g / L, pyridoxal phosphate 0.01-0.05g / L, adjust pH between 6.0-9.0, fresh cell volume 1-10g / L, then transform at 15-40°C, 100-250rpm, transform for 8-24h. After the transformation, the yield of L-2-aminobutyric acid was determined by liquid chromatography.

[0056] 5. Sample Detection and Analysis

[0057] Take 800 μL of the appropriately diluted sample, add 200 μL of OPA derivatization agent, mix at 25°C for 1 min, and inject immediately. The conversion solution was analyzed by Shimadzu 2030C high performance liquid chromatograph (HPLC), and the chromatographic conditions were as follows: the mobile phase was methanol: water (v / v = 1:1), using Inertsustain C18 column (4.6×250 mm, 5 μm), flow rate 1 mL / min, column temperature 30°C, injection volume 20 μL, and detection wavelength 333 nm.

[0058] Example 1

[0059] Construction of recombinant Escherichia coli

[0060] The fully synthetic ALD recombinant plasmid and the pET28a(+) vector were double-digested by restriction endonucleases EcoRI and HindIII, and ALD from different sources were connected to the pET28a(+) vector by T4 DNA ligase to obtain recombinant plasmid 1; the fully synthetic LaDH recombinant plasmid and the pCDFDuet-1 vector were double-digested by restriction endonucleases EcoRI and HindIII, and the fully synthetic LeuDH recombinant plasmid and the pCDFDuet-1 vector were double-digested by restriction endonucleases NdeI and XhoI, and LaDH and LeuDH from different sources were connected to the pCDFDuet vector in pairs by T4 DNA ligase to obtain recombinant plasmid 2; different recombinant plasmids 1 and recombinant plasmid 2 were transformed into E.coli BL21(DE3) competent cells in pairs to obtain recombinant Escherichia coli.

[0061] Example 2

[0062] Induction culture of recombinant Escherichia coli

[0063] The recombinant E. coli was inoculated into LB medium containing 50 mg / L kanamycin and 50 mg / L streptomycin, and cultured at 37°C and 200 rpm for 12 hours to obtain seed liquid. The seed liquid was inoculated into fresh LB medium at a 2% inoculum, and cultured at 37°C and 200 rpm until the bacterial concentration OD600nm reached 0.7, 0.5 mM IPTG was added, and after induction at 28°C for 15 hours, the culture was centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the wet bacteria were washed twice with 0.9% physiological saline, centrifuged, and set aside.

[0064] Example 3

[0065] Comparison of transformation ability of various recombinant Escherichia coli

[0066] The collected recombinant E. coli was resuspended in a 50 mL system, with a final cell concentration of 10 g / L, propionaldehyde 50 g / L, ammonium formate 60 g / L, glucose 20 g / L, pyridoxal phosphate 0.05 g / L, pH 8.0, and reacted at 30 ° C, with a shaker speed of 200 rpm and a conversion time of 24 h. After the conversion, the yield of L-2-aminobutyric acid was determined by HPLC.

[0067] Table 1 Comparison of L-2-aminobutyric acid production of various recombinant bacteria

[0068] Recombinant bacteria L-2-Aminobutyric acid (g / L) E.coli BL21(DE3) / pET28a-StALD + pCDFDuet-AhLaDH-ToLeuDH 65.3 E.coli BL21(DE3) / pET28a-StALD + pCDFDuet-AhLaDH-HpLeuDH 58.9 E.coli BL21(DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH 87.1 E.coli BL21(DE3) / pET28a-StALD + pCDFDuet-SaLaDH-HpLeuDH 70.2 E.coli BL21(DE3) / pET28a-LsALD + pCDFDuet-AhLaDH-ToLeuDH 55.7 E.coli BL21(DE3) / pET28a-LsALD + pCDFDuet-AhLaDH-HpLeuDH 48.8 E.coli BL21(DE3) / pET28a-LsALD + pCDFDuet-SaLaDH-ToLeuDH 60.6 E.coli BL21(DE3) / pET28a-LsALD + pCDFDuet-SaLaDH-HpLeuDH 52.7

[0069] Example 4

[0070] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 1g / L, propionaldehyde was 6g / L, ammonium formate was 7g / L, glucose was 5g / L, pyridoxal phosphate was 0.01g / L, pH 8.0, temperature was 35 ° C, the shaking speed was 200rpm, and the conversion time was 24h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 10.4g / L.

[0071] Example 5

[0072] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 3g / L, propionaldehyde was 16g / L, ammonium formate was 18g / L, glucose was 7g / L, pyridoxal phosphate was 0.01g / L, pH 8.0, temperature was 30 ° C, the shaking speed was 200rpm, and the conversion time was 24h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 27.9g / L.

[0073] Example 6

[0074] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 4g / L, propionaldehyde was 21g / L, ammonium formate was 24g / L, glucose was 10g / L, pyridoxal phosphate was 0.03g / L, pH 8.0, temperature was 30 ° C, the shaking speed was 200rpm, and the conversion time was 24h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 36.5g / L.

[0075] Example 7

[0076] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 6g / L, propionaldehyde was 29g / L, ammonium formate was 33g / L, glucose was 12g / L, pyridoxal phosphate was 0.03g / L, pH 8.0, temperature was 30 ° C, shaking speed was 200rpm, and conversion time was 24h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 50.1g / L.

[0077] Example 8

[0078] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 7g / L, propionaldehyde was 36g / L, ammonium formate was 41g / L, glucose was 14g / L, pyridoxal phosphate was 0.05g / L, pH 8.0, temperature was 30 ° C, the shaking speed was 200rpm, and the conversion time was 24h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 63.1g / L.

[0079] Example 9

[0080] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 9g / L, propionaldehyde was 46g / L, ammonium formate was 53g / L, glucose was 18g / L, pyridoxal phosphate was 0.05g / L, pH 8.0, temperature was 30 ° C, shaking speed was 200rpm, and conversion time was 24h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 80.2g / L.

[0081] Example 10

[0082] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 10g / L, propionaldehyde was 25g / L, ammonium formate was 29g / L, glucose was 20g / L, pyridoxal phosphate was 0.05g / L, pH 8.0, temperature was 30 ° C, shaking speed was 200rpm, and conversion time was 12h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 43.2g / L.

[0083] Embodiment 11

[0084] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 10g / L, propionaldehyde was 15g / L, ammonium formate was 17g / L, glucose was 20g / L, pyridoxal phosphate was 0.05g / L, pH 8.0, temperature was 30 ° C, the shaking speed was 200rpm, and the conversion time was 8h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 26.1g / L.

[0085] Example 12

[0086] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 8g / L, propionaldehyde was 17g / L, ammonium formate was 20g / L, glucose was 16g / L, pyridoxal phosphate was 0.05g / L, pH 8.0, temperature was 30 ° C, shaking speed was 200rpm, and conversion time was 10h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 29.7g / L.

[0087] Example 13

[0088] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 7g / L, propionaldehyde was 25g / L, ammonium formate was 28g / L, glucose was 15g / L, pyridoxal phosphate was 0.04g / L, pH 8.0, temperature was 30 ° C, the shaking speed was 200rpm, and the conversion time was 17h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 43.9g / L.

[0089] Embodiment 14

[0090] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 6g / L, propionaldehyde was 14g / L, ammonium formate was 16g / L, glucose was 11g / L, pyridoxal phosphate was 0.04g / L, pH 6.0, temperature was 35 ° C, the shaking speed was 200rpm, and the conversion time was 12h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 24.4g / L.

[0091] Embodiment 15

[0092] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 5g / L, propionaldehyde was 12g / L, ammonium formate was 14g / L, glucose was 10g / L, pyridoxal phosphate was 0.03g / L, pH 7.0, temperature was 35 ° C, the shaking speed was 200rpm, and the conversion time was 12h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 20.7g / L.

[0093] Example 16

[0094] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 4g / L, propionaldehyde was 11g / L, ammonium formate was 13g / L, glucose was 8g / L, pyridoxal phosphate was 0.03g / L, pH 7.5, temperature was 35 ° C, the shaking speed was 200rpm, and the conversion time was 12h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 19.3g / L.

[0095] Embodiment 17

[0096] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 2g / L, propionaldehyde was 6g / L, ammonium formate was 7g / L, glucose was 5g / L, pyridoxal phosphate was 0.02g / L, pH 8.5, temperature was 35 ° C, the shaking speed was 200rpm, and the conversion time was 12h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 10.6g / L.

[0097] Embodiment 18

[0098] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 6g / L, propionaldehyde was 30g / L, ammonium formate was 35g / L, glucose was 13g / L, pyridoxal phosphate was 0.03g / L, pH 7.5, temperature was 15 ° C, the shaking speed was 200rpm, and the conversion time was 24h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 52.5g / L.

[0099] Embodiment 19

[0100] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 4g / L, propionaldehyde was 21g / L, ammonium formate was 25g / L, glucose was 9g / L, pyridoxal phosphate was 0.03g / L, pH 7.5, temperature was 25 ° C, the shaking speed was 200rpm, and the conversion time was 24h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 36.8g / L.

[0101] Embodiment 20

[0102] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 5g / L, propionaldehyde was 36g / L, ammonium formate was 42g / L, glucose was 11g / L, pyridoxal phosphate was 0.03g / L, pH 7.5, temperature was 40 ° C, the shaking speed was 200rpm, and the conversion time was 24h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 62.5g / L.

[0103] Embodiment 21

[0104] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 12g / L, propionaldehyde was 60g / L, ammonium formate was 70g / L, glucose was 25g / L, pyridoxal phosphate was 0.08g / L, pH7.5, temperature was 35 ° C, the shaking speed was 200rpm, and the conversion time was 36h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 15.9g / L.

[0105] Comparative Example 1

[0106] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 1g / L, propionaldehyde was 1g / L, ammonium formate was 2g / L, glucose was 5g / L, pyridoxal phosphate was 0.01g / L, pH 8.0, temperature was 30 ° C, the shaking speed was 200rpm, and the conversion time was 24h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 1.75g ​​ / L.

[0107] Comparative Example 2

[0108] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 2g / L, propionaldehyde was 5g / L, ammonium formate was 6g / L, glucose was 5g / L, pyridoxal phosphate was 0.02g / L, pH 9.0, temperature was 35 ° C, the shaking speed was 200rpm, and the conversion time was 12h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 8.68g / L.

[0109] Comparative Example 3

[0110] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 5g / L, propionaldehyde was 25g / L, ammonium formate was 30g / L, glucose was 10g / L, pyridoxal phosphate was 0.03g / L, pH 5.5, temperature was 35 ° C, the shaking speed was 200rpm, and the conversion time was 36h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 5.77g / L.

[0111] Comparative Example 4

[0112] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 3g / L, propionaldehyde was 20g / L, ammonium formate was 25g / L, glucose was 5g / L, pyridoxal phosphate was 0.03g / L, pH 9.5, temperature was 35 ° C, the shaking speed was 200rpm, and the conversion time was 36h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 6.04g / L.

[0113] Comparative Example 5

[0114] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 4g / L, propionaldehyde was 22g / L, ammonium formate was 27g / L, glucose was 10g / L, pyridoxal phosphate was 0.03g / L, pH 7.5, temperature was 10 ° C, shaking speed was 200rpm, and conversion time was 36h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 7.42g / L.

[0115] Comparative Example 6

[0116] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 6g / L, propionaldehyde was 30g / L, ammonium formate was 36g / L, glucose was 15g / L, pyridoxal phosphate was 0.04g / L, pH 8.0, temperature was 45 ° C, shaking speed was 200rpm, and conversion time was 36h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 6.39g / L.

[0117] Comparative Example 7

[0118] According to the induction expression method described in Example 2, after the induction expression of E.coli BL21 (DE3) / pET28a-StALD + pCDFDuet-SaLaDH-ToLeuDH was completed, the cells were collected, and in a 50mL system, the cell wet weight was 0.1g / L, propionaldehyde was 0.5g / L, ammonium formate was 0.6g / L, glucose was 1g / L, pyridoxal phosphate was 0.005g / L, pH 8.0, temperature was 30 ° C, shaking speed was 200rpm, and conversion time was 36h. HPLC measurement results showed that the yield of L-2-aminobutyric acid was 0.12g / L.

[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A recombinant bacterium for producing L-2-aminobutyric acid, characterized in that: The recombinant bacteria express heterologous aldolase, lactate dehydrogenase and leucine dehydrogenase; The aldolase is derived from Sphaerobacter thermophilus or Lachnellula suecica; the lactate dehydrogenase is derived from Aeromonas hydrophila or Staphylococcus argenteus; and the leucine dehydrogenase is derived from Thermosediminibacter oceani or Halanaerobium praevalens.

2. The recombinant bacterium according to claim 1, characterized in that The aldolase includes StALD with an amino acid sequence as shown in SEQ ID NO.1 and LsALD with an amino acid sequence as shown in SEQ ID NO.2; Preferably, the nucleotide sequence of StALD is shown as SEQ ID NO.3, and the nucleotide sequence of LsALD is shown as SEQ ID NO.

4.

3. The recombinant bacterium according to claim 1, characterized in that The lactate dehydrogenase includes AhLaDH whose amino acid sequence is shown in SEQ ID NO.5 and SaLaDH whose amino acid sequence is shown in SEQ ID NO.6; Preferably, the nucleotide sequence of AhLaDH is shown as SEQ ID NO.7, and the nucleotide sequence of SaLaDH is shown as SEQ ID NO.

8.

4. The recombinant bacterium according to claim 1, characterized in that The leucine dehydrogenase includes ToLeuDH shown in the amino acid sequence SEQ ID NO.9 and HpLeuDH shown in the amino acid sequence SEQ ID NO.10; Preferably, the nucleotide sequence of the ToLeuDH is shown as SEQ ID NO.11, and the nucleotide sequence of the HpLeuDH is shown as SEQ ID NO.

12.

5. The recombinant bacterium according to claim 1, characterized in that The starting strain of the recombinant bacteria includes Escherichia coli; Preferably, the Escherichia coli includes Escherichia coli BL21 (DE3), Escherichia coli DH5α and Escherichia coli XL-Blue.

6. The method for preparing a recombinant bacterium according to any one of claims 1 to 5, characterized in that: include: Inserting the genes of aldolase, lactate dehydrogenase and leucine dehydrogenase into an expression vector to obtain a recombinant vector, and then introducing the recombinant vector into a starting strain to obtain the recombinant bacterium; Preferably, the expression vector includes pCDFDuet-1 and pET28a(+).

7. The preparation method according to claim 6, characterized in that: The preparation method of the recombinant bacteria comprises: inserting gene fragments of StALD, SaLaDH and ToLeuDH into an expression vector to obtain a recombinant vector, then introducing the recombinant vector into a starting strain, and cultivating and screening to obtain a recombinant bacteria producing L-2-aminobutyric acid; Preferably, the recombinant expression vector comprises: pET28a-StALD and pCDFDuet-SaLaDH-ToLeuDH.

8. A whole cell catalyst, characterized in that Contains the recombinant bacteria according to any one of claims 1 to 5.

9. Use of the recombinant bacterium according to any one of claims 1 to 5 in the synthesis of L-2-aminobutyric acid and its downstream products.

10. A method for producing L-2-aminobutyric acid, characterized in that: The method comprises adding the recombinant bacteria according to any one of claims 1 to 5 to a reaction system containing propionaldehyde and ammonium formate for whole-cell catalysis to obtain the L-2-aminobutyric acid; Preferably, the whole-cell catalytic reaction system comprises: 1-10 g / L of recombinant bacteria wet weight, 1-50 g / L of propionaldehyde, 2-60 g / L of ammonium formate, 5-20 g / L of glucose, and 0.01-0.05 g / L of pyridoxal phosphate; Preferably, the reaction conditions of the whole-cell catalysis are: pH=6.0-9.0, temperature 15-40°C, and reaction time 8-24h.

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