A method for producing l-alanine

Abstract

The application discloses a method for producing L-alanine, which is characterized by high production efficiency, high atom utilization rate and low cost, and is realized by converting a substrate acetaldehyde and ammonium formate into L-alanine by using a recombinant bacterium capable of expressing a heterologous aldolase, a lactate dehydrogenase and an alanine dehydrogenase as a whole cell catalyst. Meanwhile, the selected enzymes have the advantages of high activity and strong optical specificity, so that the recombinant bacterium of the application is used to convert and produce L-alanine, and the production efficiency is high, the production is green and environment-friendly, and the cost is low, and the method has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more specifically, to a method for producing L-alanine. Background Technology

[0002] L-alanine, also known as L-α-alanine, is an important natural amino acid. As one of the smallest chiral compounds, it is widely used in food, medicine, and personal care.

[0003] The main methods for preparing L-alanine include extraction, chemical synthesis, and biotransformation. Extraction primarily involves hydrolyzing products with high protein content, such as corn gluten, but this method is costly and unsuitable for industrial production. Chemical synthesis suffers from severe environmental pollution, low product purity, and high costs, making industrial-scale production difficult. Biotransformation, on the other hand, offers advantages such as high specificity, environmental friendliness, mild reaction conditions, and the elimination of the need for multi-step separation and purification, and has thus attracted widespread attention.

[0004] Currently, scholars both domestically and internationally have reported various biological methods for the preparation of L-alanine. For example, Chinese patent CN102690762A uses L-aspartic acid as a substrate and synthesizes L-alanine through the catalysis of *Trichophyton mentagrophytes*, a strain that independently screens high-yield L-aspartate-β-decarboxylase, achieving a conversion rate of over 390% (W / V) and an atom utilization rate of 66.9%. However, these methods generally suffer from drawbacks such as long conversion cycles, high substrate costs, and low atom utilization rates.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for producing L-alanine, which uses recombinant bacteria capable of expressing heterologous aldolase, lactate dehydrogenase and alanine dehydrogenase as whole-cell catalysts to convert the substrates acetaldehyde and ammonium formate into L-alanine. This method has the characteristics of high production efficiency, high atom utilization and low cost.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides a recombinant bacterium for synthesizing L-alanine, which expresses at least aldolase, lactate dehydrogenase and alanine dehydrogenase.

[0009] Secondly, the present invention provides a method for preparing the above-mentioned recombinant bacteria, which includes: inserting the genes of aldolase, lactate dehydrogenase and alanine dehydrogenase into an expression vector to obtain a recombinant vector, and then introducing the recombinant vector into a starting strain to obtain recombinant bacteria.

[0010] Thirdly, the present invention provides applications of the above-mentioned recombinant bacteria in the following aspects:

[0011] (a) Catalytic synthesis of L-alanine and its downstream products;

[0012] (b) Preparation of the product of catalytic synthesis of L-alanine.

[0013] Fourthly, the present invention provides a product for the catalytic synthesis of L-alanine, which contains the above-mentioned recombinant bacteria.

[0014] Fifthly, the present invention provides a method for synthesizing L-alanine, which includes adding the above-mentioned recombinant bacteria to a solution containing acetaldehyde and ammonium formate for whole-cell catalysis to obtain L-alanine.

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

[0016] This invention modifies the starting strain to alter the synthetic route of L-alanine, enabling it to use acetaldehyde and ammonium formate as substrates. The modified recombinant bacteria catalyze the production of L-alanine. The production method of this invention can efficiently convert inexpensive substrates to produce L-alanine, offering advantages such as high yield, wide availability of substrates, simple preparation process, and low cost. Furthermore, the enzyme selected in this invention possesses advantages such as high activity and strong optical specificity. Therefore, the production of L-alanine using the recombinant bacteria of this invention is highly efficient, environmentally friendly, and low-cost, showing promising prospects for industrial application. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is the synthetic route for L-alanine in this invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0020] like Figure 1As shown, the synthetic route of L-alanine in this invention is as follows: acetaldehyde and formic acid are converted into L-lactic acid by aldolase (ALD), pyruvate is obtained under the action of lactate dehydrogenase (LDH), and pyruvate is converted into L-alanine by alanine dehydrogenase (ADH). The coenzymes required in the reaction process are provided by glucose metabolism by the bacteria. The coenzymes required for the LDH and ADH-mediated reactions can complement each other.

[0021] To enable the production of L-alanine via the aforementioned synthetic route, this invention constructs a recombinant bacterium and uses it as a catalyst in a reaction system containing acetaldehyde and formic acid, thereby achieving L-alanine production via a novel route. The recombinant bacterium expresses at least ALD, LDH, and ADH.

[0022] Furthermore, in order to further increase the L-alanine production based on the above-mentioned recombinant bacteria, the present invention compared and screened the sources of ALD, LDH and ADH.

[0023] Specifically, ALDs include MmALD (Genbank ID: XMY63565.1) derived from Methanococcus maripaludis and MkALD (Genbank ID: KEP43241.1) derived from Mycobacterium kansasii, with the amino acid sequence of MmALD as shown in SEQ ID NO.1 and the amino acid sequence of MkALD as shown in SEQ ID NO.3.

[0024] LDH includes BcLDH (Genbank ID: BDI00612.1) derived from Bacillus cereus and MsLDH (Genbank ID: KMQ76928.1) derived from Marinobacter subterrani. The amino acid sequence of BcLDH is shown in SEQ ID NO.5, and the amino acid sequence of MsLDH is shown in SEQ ID NO.7.

[0025] ADH includes BfADH (Genbank ID: OCR36366.1) derived from Bacteroides fragilis and MtADH (Genbank ID: AIB49478.1) derived from Mycobacterium tuberculosis. The amino acid sequence of BfADH is shown in SEQ ID NO.9, and the amino acid sequence of MtADH is shown in SEQ ID NO.11.

[0026] In experiments, this invention found that the combination of ALD, LDH, and ADH from the aforementioned sources resulted in higher L-alanine production efficiency. Furthermore, after obtaining the amino acid sequences of ALD, LDH, and ADH from these sources, codon optimization was performed based on the preferences of the starting strain (e.g., *E. coli*).

[0027] Specifically, the nucleotide sequence of MmALD is shown in SEQ ID NO.2; the nucleotide sequence of MkALD is shown in SEQ ID NO.4; the nucleotide sequence of BcLDH is shown in SEQ ID NO.6; the nucleotide sequence of MsLDH is shown in SEQ ID NO.8; the nucleotide sequence of BfADH is shown in SEQ ID NO.10; and the nucleotide sequence of MtADH is shown in SEQ ID NO.12.

[0028] Correspondingly, the preparation method of the above-mentioned recombinant bacteria includes: inserting the genes of ALD, LDH and ADH into an expression vector to obtain a recombinant vector, and then introducing the recombinant vector into the starting strain to obtain the recombinant bacteria.

[0029] In some embodiments, the expression vector described above includes pET28a(+) and pCDFDuet-1 plasmid.

[0030] 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 bacteria; the present invention does not specifically limit it. Those skilled in the art can perform codon optimization based on different starting strains to adapt to new reaction systems.

[0031] In some embodiments, the above-mentioned Escherichia coli includes Escherichia coli BL21, Escherichia coli DH5α, and Escherichia coli XL-Blue.

[0032] The recombinant Escherichia coli of this invention co-expresses the encoding genes of three enzymes through two plasmids. Optionally, pET28a(+) is loaded with ALD, and pCDFDuet-1 is loaded with LDH and ADH. Then, the two recombinant plasmids are transformed into the starting strain to obtain the recombinant bacteria.

[0033] Based on this, L-alanine can be produced using the above recombinant bacteria by adding the above recombinant bacteria to a solution containing acetaldehyde and ammonium formate for whole-cell catalysis to obtain L-alanine.

[0034] In some embodiments, the recombinant bacteria are induced to undergo culture before whole-cell transformation. This induction culture process involves inoculating the recombinant bacteria into LB medium containing 30-60 mg / L kanamycin and 30-60 mg / L streptomycin, culturing to obtain a seed culture, and then inoculating the seed culture into fresh LB medium until the bacterial concentration reaches OD0.05. 600nm When the concentration reaches 0.6-0.8, add an inducer, and after induction, separate and wash to obtain wet bacterial cells.

[0035] In some embodiments, the culture conditions for the recombinant bacteria are: temperature of 32-38℃, rotation speed of 150-250 rpm, and culture time of 10-16 h.

[0036] In some embodiments, the culture conditions for the above seed solution are: temperature of 32-38℃ and rotation speed of 150-250 rpm.

[0037] In some embodiments, the induction conditions are: 0.2-0.6 mM IPTG inducer, temperature of 25-30°C, and time of 10-16 h.

[0038] In some embodiments, the whole-cell transformation production system includes: acetaldehyde 1-70 g / L, ammonium formate 2-110 g / L, glucose 5-20 g / L, pyridoxal phosphate 0.01-0.05 g / L, and recombinant bacterial cells 1-10 g / L.

[0039] In some embodiments, the production system for whole-cell transformation has a pH of 6.0-9.0, a temperature of 15-40°C, a rotation speed of 100-250 rpm, and a reaction time of 3-10 h.

[0040] The above production method converts the substrates acetaldehyde and ammonium formate into L-alanine. The production cycle is significantly shortened compared to the existing technology of 16-35 hours, the atom utilization rate can reach 80%, and the production cost is low. Therefore, it has good prospects for industrial application.

[0041] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0042] 1. Selection of bacteria and plasmids

[0043] The following plasmids were purchased from Novagen: pET28a(+), pCDFDuet-1, Escherichia coli BL21, Escherichia coli DH5α, and Escherichia coli XL-Blue.

[0044] 2. Enzyme selection

[0045] (1) Selection of aldolase

[0046] The amino acid sequences of aldolases MmALD and MkALD were obtained from the NCBI database. Codon optimization was performed based on E. coli preferences, and two nucleotide sequences were synthesized using standard genetic engineering methods, as shown in SEQ ID NO.2 and SEQ ID NO.4, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO.1 and SEQ ID NO.3, respectively. EcoRI and HindIII restriction enzyme sites were added to both ends of the nucleotide sequences.

[0047] (2) Selection of lactate dehydrogenase

[0048] The amino acid sequences of lactate dehydrogenases BcLDH and MsLDH were obtained from the NCBI database. Codon optimization was performed based on E. coli preferences, and two nucleotide sequences were synthesized using standard genetic engineering methods, as shown in SEQ ID NO. 6 and SEQ ID NO. 8, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO. 5 and SEQ ID NO. 7, respectively. EcoRI and HindIII restriction enzyme sites were added to both ends of the nucleotide sequences.

[0049] (3) Selection of alanine dehydrogenase

[0050] The amino acid sequences of alanine dehydrogenases BfADH and MtADH were obtained from the NCBI database. Codon optimization was performed based on E. coli preferences, and two nucleotide sequences were synthesized using standard genetic engineering methods, as shown in SEQ ID NO. 10 and SEQ ID NO. 12, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO. 9 and SEQ ID NO. 11, respectively. Enzyme cleavage sites NdeI and XhoI were added to both ends of the nucleotide sequences.

[0051] 3. Construction of the three-enzyme co-expression system and cell culture

[0052] One enzyme from each of the selected aldolase, lactate dehydrogenase, and alanine dehydrogenase was chosen for co-expression. The encoding genes of all three enzymes were co-expressed using the pET28a(+) and pCDFDuet-1 double plasmids; pET28a(+) contained aldolase, and pCDFDuet-1 contained both lactate and alanine dehydrogenase. After obtaining the co-expression recombinant plasmids, both plasmids were simultaneously transformed into *Escherichia coli* BL21 competent cells. Positive transformants were obtained by screening with kanamycin and streptomycin plates, thus obtaining recombinant *E. coli*. The obtained recombinant bacteria were inoculated into fresh liquid culture medium, induced, centrifuged, and wet cells were obtained.

[0053] 4. Preparation of L-alanine from acetaldehyde and ammonium formate by whole-cell conversion

[0054] The conversion system consisted of acetaldehyde 1-70 g / L, ammonium formate 2-110 g / L, glucose 5-20 g / L, and pyridoxal phosphate 0.01-0.05 g / L. The pH was adjusted to 6.0-9.0, and the amount of fresh cells was 1-10 g / L. The conversion was then carried out at 15-40℃ and 100-250 rpm for 3-10 h. After the conversion, the L-alanine yield was determined by liquid chromatography.

[0055] 5. Sample detection and analysis

[0056] Take 800 μL of a sample diluted appropriately, add 200 μL of OPA derivatizing agent, mix well at 25℃ for 1 min, and inject immediately. Analyze the conversion solution using a Shimadzu 2030C high-performance liquid chromatograph (HPLC). The chromatographic conditions are: mobile phase methanol:water (V / V = 1:1), Inertsustain C18 column (4.6 × 250 mm, 5 μm), flow rate 1 mL / min, column temperature 30℃, injection volume 20 μL, and detection wavelength 333 nm.

[0057] Example 1

[0058] Construction of recombinant Escherichia coli

[0059] The fully synthesized ALD recombinant plasmid and pET28a(+) vector were double-digested with restriction endonucleases EcoRI and HindIII, respectively. ALD from different sources was then ligated into the pET28a(+) vector using T4 DNA ligase to obtain recombinant plasmid 1. The fully synthesized LDH recombinant plasmid and pCDFDuet-1 vector were double-digested with restriction endonucleases EcoRI and HindIII, respectively. The fully synthesized ADH recombinant plasmid and pCDFDuet-1 vector were double-digested with restriction endonucleases NdeI and XhoI, respectively. LDH and ADH from different sources were then ligated into the pCDFDuet vector in pairs using T4 DNA ligase to obtain recombinant plasmid 2. Recombinant plasmids 1 and 2 were then combined and transformed into E. coli BL21(DE3) competent cells to obtain recombinant Escherichia coli.

[0060] Example 2

[0061] Induction culture of recombinant Escherichia coli

[0062] 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 h to obtain a seed culture. The seed culture was then inoculated into fresh LB medium at a 2% inoculation rate and cultured at 37°C and 200 rpm until the bacterial concentration reached OD500.600nm When the concentration reaches 0.7, add 0.5 mM IPTG, induce at 28℃ for 15 h, centrifuge at 8000 rpm for 10 min, discard the supernatant, wash the wet bacterial cells twice with 0.9% physiological saline, centrifuge, and set aside.

[0063] Example 3

[0064] Comparison of transformation capabilities of various recombinant Escherichia coli strains

[0065] The collected recombinant E. coli were resuspended in a 50 mL system with a final cell concentration of 10 g / L, acetaldehyde 70 g / L, ammonium formate 110 g / L, glucose 20 g / L, pyridoxal phosphate 0.05 g / L, and pH 8.0. The reaction was carried out at 30 °C with a shaking speed of 200 rpm for 10 h. After transformation, the yield of L-alanine was determined by HPLC.

[0066] Table 1 Comparison of L-alanine production for various recombinant strains

[0067] E.coli BL21(DE3) / pET28a-MmALD+pCDFDuet-BcLDH-BfADH 85.7 E.coli BL21(DE3) / pET28a-MmALD+pCDFDuet-BcLDH-MtADH 102.3 E.coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-BfADH 97.6 E.coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH 139.4 E.coli BL21(DE3) / pET28a-MkALD+pCDFDuet-BcLDH-BfADH 79.9 E.coli BL21(DE3) / pET28a-MkALD+pCDFDuet-BcLDH-MtADH 115.3 E.coli BL21(DE3) / pET28a-MkALD+pCDFDuet-MsLDH-BfADH 72.5 E.coli BL21(DE3) / pET28a-MkALD+pCDFDuet-MsLDH-MtADH 83.8

[0068] Based on the comparison results of Example 3, it can be seen that the recombinant strain E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH has the highest L-alanine production. Therefore, this invention subsequently selected it as a whole-cell catalyst to study the L-alanine production efficiency under different reaction systems or conditions.

[0069] Example 4

[0070] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 1 g / L, acetaldehyde 1 g / L, ammonium formate 2 g / L, glucose 5 g / L, pyridoxal phosphate 0.01 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, and transformation time 10 h. HPLC analysis showed that the L-alanine yield was 2 g / L.

[0071] Example 5

[0072] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 1 g / L, acetaldehyde 8 g / L, ammonium formate 13 g / L, glucose 5 g / L, pyridoxal phosphate 0.01 g / L, pH 8.0, temperature 35℃, shaker speed 200 rpm, and transformation time 10 h. HPLC analysis showed that the L-alanine yield was 15.7 g / L.

[0073] Example 6

[0074] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 3 g / L, acetaldehyde 22 g / L, ammonium formate 33 g / L, glucose 7 g / L, pyridoxal phosphate 0.01 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, and transformation time 10 h. HPLC analysis showed that the L-alanine yield was 43.8 g / L.

[0075] Example 7

[0076] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 4 g / L, acetaldehyde 29 g / L, ammonium formate 42 g / L, glucose 10 g / L, pyridoxal phosphate 0.03 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, and transformation time 10 h. HPLC analysis showed that the L-alanine yield was 56.8 g / L.

[0077] Example 8

[0078] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 6 g / L, acetaldehyde 40 g / L, ammonium formate 59 g / L, glucose 12 g / L, pyridoxal phosphate 0.03 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, and transformation time 10 h. HPLC analysis showed that the L-alanine yield was 79.3 g / L.

[0079] Example 9

[0080] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 7 g / L, acetaldehyde 50 g / L, ammonium formate 75 g / L, glucose 14 g / L, pyridoxal phosphate 0.05 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, and transformation time 10 h. HPLC analysis showed that the L-alanine yield was 98.9 g / L.

[0081] Example 10

[0082] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 9 g / L, acetaldehyde 64 g / L, ammonium formate 94 g / L, glucose 18 g / L, pyridoxal phosphate 0.05 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, and transformation time 10 h. HPLC analysis showed that the L-alanine yield was 127.7 g / L.

[0083] Example 11

[0084] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 10 g / L, acetaldehyde 35 g / L, ammonium formate 52 g / L, glucose 20 g / L, pyridoxal phosphate 0.05 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, and transformation time 5 h. HPLC analysis showed that the L-alanine yield was 70 g / L.

[0085] Example 12

[0086] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 10 g / L, acetaldehyde 21 g / L, ammonium formate 34 g / L, glucose 20 g / L, pyridoxal phosphate 0.05 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, and transformation time 3 h. HPLC analysis showed that the L-alanine yield was 41.7 g / L.

[0087] Example 13

[0088] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 8 g / L, acetaldehyde 24 g / L, ammonium formate 37 g / L, glucose 16 g / L, pyridoxal phosphate 0.05 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, and transformation time 4 h. HPLC analysis showed that the L-alanine yield was 47.2 g / L.

[0089] Example 14

[0090] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 7 g / L, acetaldehyde 35 g / L, ammonium formate 53 g / L, glucose 15 g / L, pyridoxal phosphate 0.04 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, and transformation time 7 h. HPLC analysis showed that the L-alanine yield was 69.6 g / L.

[0091] Example 15

[0092] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 6 g / L, acetaldehyde 20 g / L, ammonium formate 31 g / L, glucose 11 g / L, pyridoxal phosphate 0.04 g / L, pH 6.0, temperature 35℃, shaker speed 200 rpm, and transformation time 5 h. HPLC analysis showed that the L-alanine yield was 39.3 g / L.

[0093] Example 16

[0094] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 5 g / L, acetaldehyde 17 g / L, ammonium formate 27 g / L, glucose 10 g / L, pyridoxal phosphate 0.03 g / L, pH 7.0, temperature 35℃, shaker speed 200 rpm, and transformation time 5 h. HPLC analysis showed that the L-alanine yield was 33.7 g / L.

[0095] Example 17

[0096] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 4 g / L, acetaldehyde 15 g / L, ammonium formate 24 g / L, glucose 8 g / L, pyridoxal phosphate 0.03 g / L, pH 7.5, temperature 35℃, shaking speed 200 rpm, and transformation time 5 h. HPLC analysis showed that the L-alanine yield was 30.1 g / L.

[0097] Example 18

[0098] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 2 g / L, acetaldehyde 8 g / L, ammonium formate 13 g / L, glucose 5 g / L, pyridoxal phosphate 0.02 g / L, pH 8.5, temperature 35℃, shaker speed 200 rpm, and transformation time 5 h. HPLC analysis showed that the L-alanine yield was 15.9 g / L.

[0099] Example 19

[0100] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 2 g / L, acetaldehyde 7 g / L, ammonium formate 12 g / L, glucose 5 g / L, pyridoxal phosphate 0.02 g / L, pH 9.0, temperature 35℃, shaker speed 200 rpm, and transformation time 5 h. HPLC analysis showed that the L-alanine yield was 14 g / L.

[0101] Example 20

[0102] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 6 g / L, acetaldehyde 42 g / L, ammonium formate 65 g / L, glucose 13 g / L, pyridoxal phosphate 0.03 g / L, pH 7.5, temperature 15℃, shaker speed 200 rpm, and transformation time 10 h. HPLC analysis showed that the L-alanine yield was 82.9 g / L.

[0103] Example 21

[0104] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 4 g / L, acetaldehyde 29 g / L, ammonium formate 45 g / L, glucose 9 g / L, pyridoxal phosphate 0.03 g / L, pH 7.5, temperature 25℃, shaker speed 200 rpm, and transformation time 10 h. HPLC analysis showed that the L-alanine yield was 56.9 g / L.

[0105] Example 22

[0106] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 5 g / L, acetaldehyde 50 g / L, ammonium formate 74 g / L, glucose 11 g / L, pyridoxal phosphate 0.03 g / L, pH 7.5, temperature 40℃, shaker speed 200 rpm, and transformation time 10 h. HPLC analysis showed that the L-alanine yield was 99.1 g / L.

[0107] Comparative Example 1

[0108] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 12 g / L, acetaldehyde 85 g / L, ammonium formate 125 g / L, glucose 25 g / L, pyridoxal phosphate 0.08 g / L, pH 7.5, temperature 35℃, shaker speed 200 rpm, and transformation time 15 h. HPLC analysis showed that the L-alanine yield was 25.8 g / L.

[0109] Comparative Example 2

[0110] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 5 g / L, acetaldehyde 35 g / L, ammonium formate 55 g / L, glucose 10 g / L, pyridoxal phosphate 0.03 g / L, pH 5.5, temperature 35℃, shaker speed 200 rpm, and transformation time 15 h. HPLC analysis showed that the L-alanine yield was 9.2 g / L.

[0111] Comparative Example 3

[0112] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 3 g / L, acetaldehyde 28 g / L, ammonium formate 46 g / L, glucose 5 g / L, pyridoxal phosphate 0.03 g / L, pH 9.5, temperature 35℃, shaker speed 200 rpm, and transformation time 15 h. HPLC analysis showed that the L-alanine yield was 9.6 g / L.

[0113] Comparative Example 4

[0114] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 4 g / L, acetaldehyde 30 g / L, ammonium formate 48 g / L, glucose 10 g / L, pyridoxal phosphate 0.03 g / L, pH 7.5, temperature 10℃, shaker speed 200 rpm, and transformation time 15 h. HPLC analysis showed that the L-alanine yield was 11.5 g / L.

[0115] Comparative Example 5

[0116] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 6 g / L, acetaldehyde 42 g / L, ammonium formate 66 g / L, glucose 15 g / L, pyridoxal phosphate 0.04 g / L, pH 8.0, temperature 45℃, shaker speed 200 rpm, and transformation time 15 h. HPLC analysis showed that the L-alanine yield was 11.9 g / L.

[0117] Comparative Example 6

[0118] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pET28a-MmALD+pCDFDuet-MsLDH-MtADH, bacterial cells were collected and placed in a 50 mL system with the following concentrations: cell wet weight 0.1 g / L, acetaldehyde 0.5 g / L, ammonium formate 1 g / L, glucose 1 g / L, pyridoxal phosphate 0.005 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, and transformation time 15 h. HPLC analysis showed that the L-alanine yield was 0.2 g / L.

[0119] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A recombinant bacterium for synthesizing L-alanine, characterized in that, The recombinant bacteria express at least the following amino acid sequences: MmALD (as shown in SEQ ID NO. 1), BcLDH (as shown in SEQ ID NO. 5), and BfADH (as shown in SEQ ID NO. 9); or Amino acid sequences as shown in SEQ ID NO.1 (MmALD), SEQ ID NO.5 (BcLDH), and SEQ ID NO.11 (MtADH); or Amino acid sequences such as MmALD (SEQ ID NO. 1), MsLDH (SEQ ID NO. 7), and BfADH (SEQ ID NO. 9); or Amino acid sequences as shown in SEQ ID NO.1 (MmALD), SEQ ID NO.7 (MsLDH), and SEQ ID NO.11 (MtADH); or Amino acid sequences such as MkALD (SEQ ID NO. 3), BcLDH (SEQ ID NO. 5), and BfADH (SEQ ID NO. 9); or Amino acid sequences such as MkALD (SEQ ID NO. 3), BcLDH (SEQ ID NO. 5), and MtADH (SEQ ID NO. 11); or Amino acid sequences such as MkALD (SEQ ID NO. 3), MsLDH (SEQ ID NO. 7), and BfADH (SEQ ID NO. 9); or The amino acid sequences are MkALD as shown in SEQ ID NO.3, MsLDH as shown in SEQ ID NO.7, and MtADH as shown in SEQ ID NO.

11.

2. The recombinant bacteria according to claim 1, characterized in that, The nucleotide sequence of MmALD is as shown in SEQ ID NO.2, and the nucleotide sequence of MkALD is as shown in SEQ ID NO.

4.

3. The recombinant bacteria according to claim 1, characterized in that, The nucleotide sequence of the BcLDH is as shown in SEQ ID NO.6, and the nucleotide sequence of the MsLDH is as shown in SEQ ID NO.

8.

4. The recombinant bacteria according to claim 1, characterized in that, The nucleotide sequence of BfADH is shown in SEQ ID NO.10, and the nucleotide sequence of MtADH is shown in SEQ ID NO.

12.

5. The method for preparing the recombinant bacteria according to any one of claims 1-4, characterized in that, include: The genes of MmALD or MkALD, BcLDH or MsLDH, and BfADH or MtADH are inserted into an expression vector to obtain a recombinant vector. The recombinant vector is then introduced into the starting strain to obtain the recombinant bacteria.

6. The preparation method according to claim 5, characterized in that, The expression vectors include pCDFDuet-1 and pACYCDuet-1.

7. The preparation method according to claim 6, characterized in that, The starting strain is Escherichia coli, which includes Escherichia coli BL21(DE3), Escherichia coli DH5α and Escherichia coli XL-Blue.

8. The use of the recombinant bacteria according to any one of claims 1-4 in the following aspects: (a) Catalytic synthesis of L-alanine; (b) Preparation of products for the catalytic synthesis of L-alanine.

9. A product for the catalytic synthesis of L-alanine, characterized in that, It contains the recombinant bacteria as described in any one of claims 1-4.

10. A method for synthesizing L-alanine, characterized in that, It includes adding the recombinant bacteria according to any one of claims 1-4 to a solution containing acetaldehyde and ammonium formate for whole-cell catalysis to obtain L-alanine.

11. The method according to claim 10, characterized in that, The whole-cell catalytic production system includes: acetaldehyde 1-70 g / L, ammonium formate 2-110 g / L, glucose 5-20 g / L, pyridoxal phosphate 0.01-0.05 g / L, and recombinant bacterial cells 1-10 g / L.

12. The method according to claim 10, characterized in that, The whole-cell catalytic production system has a pH of 6.0-9.0, a temperature of 15-40℃, a rotation speed of 100-250 rpm, and a reaction time of 3-10 h.

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