Method for producing L-alanine

Abstract

The invention discloses a method for producing L-alanine, according to the method, recombinant bacteria capable of expressing heterologous aldolase, lactic dehydrogenase and alanine dehydrogenase serve as a whole-cell catalyst, substrates acetaldehyde and ammonium formate are converted into L-alanine, and the method has the advantages of being high in production efficiency, high in atom utilization rate and low in cost. Meanwhile, the selected enzyme has the advantages of high activity, strong optical specificity and the like, so that the L-alanine is converted and produced by using the recombinant bacterium disclosed by the invention, the production efficiency is high, the method is green and environment-friendly, the cost is low, and the method has a good industrial application prospect.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and more particularly, to a method for producing L-alanine. Background Art

[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 the fields of food, medicine, and personal care.

[0003] The preparation methods of L-alanine mainly include extraction method, chemical synthesis method, and biotransformation method. The extraction method mainly hydrolyzes products with high protein content such as corn protein, with high production costs and unable to be industrially produced. The chemical synthesis method has the disadvantages of serious environmental pollution, low product purity, and high costs, and it is difficult to achieve industrial production. The biotransformation method has the advantages of high specificity, environmental friendliness, mild reaction conditions, and no need for multi-step separation and purification, and has currently received extensive attention.

[0004] At present, scholars at home and abroad have reported various biological preparation routes of L-alanine. For example, Chinese Patent CN102690762A uses L-aspartic acid as a substrate and catalyzes the preparation of L-alanine by Comamonas testosteroni that autonomously screens high-yield L-aspartate-β-decarboxylase, with a conversion ability of more than 390% (W / V) and an atom utilization rate of 66.9%. However, the above methods generally have the disadvantages of long conversion period, high substrate cost, and low atom utilization rate.

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

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

[0007] The present invention is implemented as follows:

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

[0009] In the second aspect, the present invention provides a preparation method of the above recombinant bacterium, 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 the recombinant bacterium.

[0010] In the third aspect, the present invention provides the application of the above recombinant bacterium in the following aspects:

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

[0012] (b) Preparation of products for catalytic synthesis of L-alanine.

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

[0014] Fifthly, the present invention provides a method for synthesizing L-alanine, which includes adding the above-mentioned recombinant bacterium into 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] By modifying the starting strain, the present invention changes the synthesis route of L-alanine, using acetaldehyde and ammonium formate as substrates, and generating L-alanine through the catalysis of the modified recombinant bacterium. The production method of the present invention can efficiently convert and produce L-alanine using low-cost substrates, and has the advantages of high yield, wide substrate sources, simple preparation process and low price; meanwhile, the enzyme selected in the present invention has advantages such as high activity and strong optical specificity. Therefore, using the recombinant bacterium of the present invention to convert and produce L-alanine has high production efficiency, is green and environmentally friendly, and has low cost, and has good industrial application prospects. Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can also be obtained based on these drawings without creative efforts.

[0018] Figure 1 It is the synthesis route of L-alanine in the present invention. Detailed Embodiments

[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. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified in the manufacturer are all conventional products that can be obtained through commercial purchase.

[0020] Such as Figure 1As shown in the figure, the synthetic route of L-alanine in the present invention is as follows: acetaldehyde and formic acid are converted into L-lactic acid by aldolase (ALD), pyruvic acid is obtained under the action of lactate dehydrogenase (LDH), and pyruvic acid is converted into L-alanine by alanine dehydrogenase (ADH). The coenzymes required in the reaction process are provided by the bacterial metabolism of glucose, and the coenzymes required for the reactions mediated by LDH and ADH can be complementary.

[0021] In order to be able to produce L-alanine by the above synthetic route, the present invention constructs a recombinant bacterium and uses it as a catalyst, which is added to a reaction system containing acetaldehyde and formic acid to achieve the production of L-alanine by the new route. For the above recombinant bacterium, it expresses at least ALD, LDH and ADH.

[0022] Furthermore, in order to further increase the yield of L-alanine on the basis of the above recombinant bacterium, the present invention compares and screens the sources of ALD, LDH and ADH.

[0023] Specifically, ALD includes MmALD (Genbank accession number: XMY63565.1) derived from Methanococcus maripaludis and MkALD (Genbank accession number: KEP43241.1) derived from Mycobacterium kansasii. The amino acid sequence of MmALD is as shown in SEQ ID NO.1, and the amino acid sequence of MkALD is as shown in SEQ ID NO.3.

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

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

[0026] In the experiments of the present invention, it was found that when the ALD, LDH, and ADH from the above sources were combined, the production efficiency of L-alanine was relatively high. Meanwhile, after obtaining the amino acid sequences of ALD, LDH, and ADH from the above sources, codon optimization was carried out according to the preference of the starting strain (such as Escherichia coli).

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

[0028] Correspondingly, the preparation method of the above 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 a starting strain to obtain the recombinant bacteria.

[0029] In some embodiments, the above expression vectors include pET28a(+) and pCDFDuet-1 plasmids.

[0030] In some embodiments, the starting strain of the above recombinant bacteria is Escherichia coli. In other embodiments, the starting strain of the recombinant bacteria can be other bacteria, and the present invention does not specifically limit it. Those skilled in the art can perform codon optimization according to different starting strains to adapt to a new reaction system.

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

[0032] The recombinant Escherichia coli of the present invention co-expresses the coding genes of 3 enzymes through dual plasmids. Optionally, pET28a(+) loads ALD, and pCDFDuet-1 loads 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, which is: adding the above recombinant bacteria into a solution containing acetaldehyde and ammonium formate for whole-cell catalysis to obtain L-alanine.

[0034] In some embodiments, the recombinant bacteria are induced and cultured before whole-cell transformation. The induction and culture process is as follows: The recombinant bacteria are inoculated into an LB medium containing 30 - 60 mg / L kanamycin and 30 - 60 mg / L streptomycin. After culture, a seed solution is obtained. Then, the seed solution is inoculated into a fresh LB medium until the bacterial concentration OD 600nm reaches 0.6 - 0.8. An inducer is added, and after induction, the wet bacterial cells are separated and washed to obtain the wet bacterial cells.

[0035] In some embodiments, the culture conditions for the above-mentioned recombinant bacteria are as follows: the temperature is 32 - 38 °C, the rotation speed is 150 - 250 rpm, and the culture time is 10 - 16 h.

[0036] In some embodiments, the culture conditions for the above-mentioned seed solution are as follows: the temperature is 32 - 38 °C, and the rotation speed is 150 - 250 rpm.

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

[0038] In some embodiments, the production system for whole-cell transformation 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 pH of the production system for whole-cell transformation is 6.0 - 9.0, the temperature is 15 - 40 °C, the rotation speed is 100 - 250 rpm, and the reaction time is 3 - 10 h.

[0040] By the above production method, the substrates acetaldehyde and ammonium formate are converted into L-alanine. Its production cycle is significantly shortened compared with 16 - 35 h of the prior art, the atom utilization rate can reach 80%, and the production cost is low. Therefore, it has good industrial application prospects.

[0041] The features and properties of the present invention are further described in detail below in conjunction with embodiments.

[0042] 1. Selection of bacteria and plasmids

[0043] pET28a(+) plasmid, pCDFDuet-1 plasmid, Escherichia coli BL21, Escherichia coli DH5α, and Escherichia coli XL-Blue purchased from Novagen.

[0044] 2. Selection of enzymes

[0045] (1) Selection of aldolase

[0046] The amino acid sequences of aldolases MmALD and MkALD were obtained from the NCBI database, codon-optimized according to the Escherichia coli preference, and two nucleotide sequences were synthesized by total synthesis through routine genetic engineering operations, 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. The restriction enzyme sites EcoRI and HindIII 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-optimized according to the Escherichia coli preference, and two nucleotide sequences were synthesized by total synthesis through routine genetic engineering operations, 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. The restriction enzyme sites EcoRI and HindIII 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-optimized according to the Escherichia coli preference, and two nucleotide sequences were synthesized by total synthesis through routine genetic engineering operations, 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. The restriction enzyme 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] For each type of the above-selected aldolase, lactate dehydrogenase, and alanine dehydrogenase, one enzyme was arbitrarily selected for three-enzyme combination co-expression. The pET28a(+) and pCDFDuet-1 dual plasmids were used to co-express the coding genes of the 3 enzymes; pET28a(+) carried the aldolase, and pCDFDuet-1 carried the lactate dehydrogenase and alanine dehydrogenase. After obtaining the co-expression recombinant plasmids, the two recombinant plasmids were simultaneously transferred into Escherichia coli BL21 competent cells, and positive transformants were screened using plates containing kanamycin and streptomycin, thus obtaining recombinant Escherichia coli. The obtained recombinant bacteria were inoculated into fresh liquid medium, induced to culture, and centrifuged to obtain wet cells.

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

[0054] Conversion system: acetaldehyde 1 - 70 g / L, ammonium formate 2 - 110 g / L, glucose 5 - 20 g / L, pyridoxal phosphate 0.01 - 0.05 g / L, adjust the pH between 6.0 - 9.0, the fresh cell mass is 1 - 10 g / L, then at 15 - 40 °C, 100 - 250 rpm, convert for 3 - 10 h. After the conversion is completed, the yield of L-alanine is determined by liquid chromatography.

[0055] 5. Detection and analysis of samples

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

[0057] Example 1

[0058] Construction of recombinant Escherichia coli

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

[0060] Example 2

[0061] Inductive culture of recombinant Escherichia coli

[0062] Inoculate the recombinant Escherichia coli into an LB medium containing 50 mg / L kanamycin and 50 mg / L streptomycin, culture at 37 °C, 200 rpm for 12 h to obtain a seed solution. Inoculate the seed solution into a fresh LB medium at an inoculation amount of 2%, and culture at 37 °C, 200 rpm until the cell density OD600nm Reached 0.7. Add 0.5 mM IPTG, induce for 15 h at 28°C, then centrifuge at 8000 rpm for 10 min. Discard the supernatant, wash the wet cells twice with 0.9% physiological saline, centrifuge, and reserve for use.

[0063] Example 3

[0064] Comparison of transformation abilities of various recombinant Escherichia coli

[0065] Resuspend the collected recombinant Escherichia coli 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, pH 8.0, react at 30°C, with a shaker speed of 200 rpm and a transformation time of 10 h. After the transformation, determine the yield of L-alanine by HPLC.

[0066] Table 1 Comparison of L-alanine yields corresponding to various recombinant bacteria

[0067] Recombinant bacterium L-alanine (g / L) 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 bacterium E. coli BL21(DE3) / pET28a-MmALD + pCDFDuet-MsLDH-MtADH has the highest yield of L-alanine. Therefore, in the follow-up of this invention, it is selected as the whole-cell catalyst to study the production efficiency of L-alanine under different reaction systems or reaction conditions.

[0069] Example 4

[0070] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-MmALD + pCDFDuet-MsLDH-MtADH is completed, collect the bacterial cells. In a 50 mL system, the wet cell weight is 1 g / L, acetaldehyde is 1 g / L, ammonium formate is 2 g / L, glucose is 5 g / L, pyridoxal phosphate is 0.01 g / L, pH 8.0, temperature is 30°C, shaker speed is 200 rpm, and transformation time is 10 h. The HPLC determination result shows that the yield of L-alanine is 2 g / 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-MmALD + pCDFDuet-MsLDH-MtADH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 1 g / L, acetaldehyde was 8 g / L, ammonium formate was 13 g / L, glucose was 5 g / L, pyridoxal phosphate was 0.01 g / L, pH was 8.0, the temperature was 35 °C, the shaker speed was 200 rpm, and the conversion time was 10 h. The HPLC measurement result showed that the L-alanine yield was 15.7 g / 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-MmALD + pCDFDuet-MsLDH-MtADH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 3 g / L, acetaldehyde was 22 g / L, ammonium formate was 33 g / L, glucose was 7 g / L, pyridoxal phosphate was 0.01 g / L, pH was 8.0, the temperature was 30 °C, the shaker speed was 200 rpm, and the conversion time was 10 h. The HPLC measurement result showed that the L-alanine yield was 43.8 g / 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-MmALD + pCDFDuet-MsLDH-MtADH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 4 g / L, acetaldehyde was 29 g / L, ammonium formate was 42 g / L, glucose was 10 g / L, pyridoxal phosphate was 0.03 g / L, pH was 8.0, the temperature was 30 °C, the shaker speed was 200 rpm, and the conversion time was 10 h. The HPLC measurement result showed that the L-alanine yield was 56.8 g / 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-MmALD + pCDFDuet-MsLDH-MtADH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 6 g / L, acetaldehyde was 40 g / L, ammonium formate was 59 g / L, glucose was 12 g / L, pyridoxal phosphate was 0.03 g / L, pH was 8.0, the temperature was 30 °C, the shaker speed was 200 rpm, and the conversion time was 10 h. The HPLC measurement result showed that the L-alanine yield was 79.3 g / 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-MmALD + pCDFDuet-MsLDH-MtADH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 7 g / L, acetaldehyde was 50 g / L, ammonium formate was 75 g / L, glucose was 14 g / L, pyridoxal phosphate was 0.05 g / L, pH was 8.0, the temperature was 30 °C, the shaker speed was 200 rpm, and the conversion time was 10 h. The HPLC measurement result showed that the L-alanine yield was 98.9 g / 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-MmALD + pCDFDuet-MsLDH-MtADH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 9 g / L, acetaldehyde was 64 g / L, ammonium formate was 94 g / L, glucose was 18 g / L, pyridoxal phosphate was 0.05 g / L, pH was 8.0, the temperature was 30 °C, the shaker speed was 200 rpm, and the conversion time was 10 h. The HPLC measurement result showed that the L-alanine yield was 127.7 g / L.

[0083] Example 11

[0084] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-MmALD + pCDFDuet-MsLDH-MtADH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 10 g / L, acetaldehyde was 35 g / L, ammonium formate was 52 g / L, glucose was 20 g / L, pyridoxal phosphate was 0.05 g / L, pH was 8.0, the temperature was 30 °C, the shaker speed was 200 rpm, and the conversion time was 5 h. The HPLC measurement result showed that the L-alanine yield was 70 g / 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-MmALD + pCDFDuet-MsLDH-MtADH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 10 g / L, acetaldehyde was 21 g / L, ammonium formate was 34 g / L, glucose was 20 g / L, pyridoxal phosphate was 0.05 g / L, pH was 8.0, the temperature was 30 °C, the shaker speed was 200 rpm, and the conversion time was 3 h. The HPLC measurement result showed that the L-alanine yield was 41.7 g / 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-MmALD + pCDFDuet-MsLDH-MtADH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 8 g / L, acetaldehyde was 24 g / L, ammonium formate was 37 g / L, glucose was 16 g / L, pyridoxal phosphate was 0.05 g / L, pH was 8.0, the temperature was 30 °C, the shaker speed was 200 rpm, and the conversion time was 4 h. The HPLC measurement result showed that the L-alanine yield was 47.2 g / L.

[0089] Example 14

[0090] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-MmALD + pCDFDuet-MsLDH-MtADH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 7 g / L, acetaldehyde was 35 g / L, ammonium formate was 53 g / L, glucose was 15 g / L, pyridoxal phosphate was 0.04 g / L, pH was 8.0, the temperature was 30 °C, the shaker speed was 200 rpm, and the conversion time was 7 h. The HPLC measurement result showed that the L-alanine yield was 69.6 g / L.

[0091] Example 15

[0092] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-MmALD + pCDFDuet-MsLDH-MtADH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 6 g / L, acetaldehyde was 20 g / L, ammonium formate was 31 g / L, glucose was 11 g / L, pyridoxal phosphate was 0.04 g / L, pH was 6.0, the temperature was 35 °C, the shaker speed was 200 rpm, and the conversion time was 5 h. The HPLC measurement result showed that the L-alanine yield was 39.3 g / 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-MmALD + pCDFDuet-MsLDH-MtADH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 5 g / L, acetaldehyde was 17 g / L, ammonium formate was 27 g / L, glucose was 10 g / L, pyridoxal phosphate was 0.03 g / L, pH was 7.0, the temperature was 35 °C, the shaker speed was 200 rpm, and the conversion time was 5 h. The HPLC measurement result showed that the L-alanine yield was 33.7 g / L.

[0095] Example 17

[0096] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-MmALD + pCDFDuet-MsLDH-MtADH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 4 g / L, acetaldehyde was 15 g / L, ammonium formate was 24 g / L, glucose was 8 g / L, pyridoxal phosphate was 0.03 g / L, pH was 7.5, the temperature was 35 °C, the shaking speed of the shaker was 200 rpm, and the conversion time was 5 h. The HPLC measurement result showed that the L-alanine yield was 30.1 g / L.

[0097] Example 18

[0098] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-MmALD + pCDFDuet-MsLDH-MtADH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 2 g / L, acetaldehyde was 8 g / L, ammonium formate was 13 g / L, glucose was 5 g / L, pyridoxal phosphate was 0.02 g / L, pH was 8.5, the temperature was 35 °C, the shaking speed of the shaker was 200 rpm, and the conversion time was 5 h. The HPLC measurement result showed that the L-alanine yield was 15.9 g / L.

[0099] Example 19

[0100] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-MmALD + pCDFDuet-MsLDH-MtADH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 2 g / L, acetaldehyde was 7 g / L, ammonium formate was 12 g / L, glucose was 5 g / L, pyridoxal phosphate was 0.02 g / L, pH was 9.0, the temperature was 35 °C, the shaking speed of the shaker was 200 rpm, and the conversion time was 5 h. The HPLC measurement result showed that the L-alanine yield was 14 g / L.

[0101] Example 20

[0102] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-MmALD + pCDFDuet-MsLDH-MtADH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 6 g / L, acetaldehyde was 42 g / L, ammonium formate was 65 g / L, glucose was 13 g / L, pyridoxal phosphate was 0.03 g / L, pH was 7.5, the temperature was 15 °C, the shaking speed of the shaker was 200 rpm, and the conversion time was 10 h. The HPLC measurement result showed that the L-alanine yield was 82.9 g / L.

[0103] Example 21

[0104] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-MmALD + pCDFDuet-MsLDH-MtADH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 4 g / L, acetaldehyde was 29 g / L, ammonium formate was 45 g / L, glucose was 9 g / L, pyridoxal phosphate was 0.03 g / L, pH was 7.5, the temperature was 25 °C, the shaking speed of the shaker was 200 rpm, and the conversion time was 10 h. The HPLC measurement result showed that the L-alanine yield was 56.9 g / L.

[0105] Example 22

[0106] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-MmALD + pCDFDuet-MsLDH-MtADH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 5 g / L, acetaldehyde was 50 g / L, ammonium formate was 74 g / L, glucose was 11 g / L, pyridoxal phosphate was 0.03 g / L, pH was 7.5, the temperature was 40 °C, the shaking speed of the shaker was 200 rpm, and the conversion time was 10 h. The HPLC measurement result showed that the L-alanine yield was 99.1 g / L.

[0107] Comparative Example 1

[0108] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-MmALD + pCDFDuet-MsLDH-MtADH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 12 g / L, acetaldehyde was 85 g / L, ammonium formate was 125 g / L, glucose was 25 g / L, pyridoxal phosphate was 0.08 g / L, pH was 7.5, the temperature was 35 °C, the shaking speed of the shaker was 200 rpm, and the conversion time was 15 h. The HPLC measurement result showed that the L-alanine yield was 25.8 g / L.

[0109] Comparative Example 2

[0110] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-MmALD + pCDFDuet-MsLDH-MtADH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 5 g / L, acetaldehyde was 35 g / L, ammonium formate was 55 g / L, glucose was 10 g / L, pyridoxal phosphate was 0.03 g / L, pH was 5.5, the temperature was 35 °C, the shaking speed of the shaker was 200 rpm, and the conversion time was 15 h. The HPLC measurement result showed that the L-alanine yield was 9.2 g / L.

[0111] Comparative Example 3

[0112] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-MmALD + pCDFDuet-MsLDH-MtADH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 3 g / L, acetaldehyde was 28 g / L, ammonium formate was 46 g / L, glucose was 5 g / L, pyridoxal phosphate was 0.03 g / L, pH was 9.5, the temperature was 35 °C, the shaker speed was 200 rpm, and the conversion time was 15 h. The HPLC measurement result showed that the L-alanine yield was 9.6 g / L.

[0113] Comparative Example 4

[0114] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-MmALD + pCDFDuet-MsLDH-MtADH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 4 g / L, acetaldehyde was 30 g / L, ammonium formate was 48 g / L, glucose was 10 g / L, pyridoxal phosphate was 0.03 g / L, pH was 7.5, the temperature was 10 °C, the shaker speed was 200 rpm, and the conversion time was 15 h. The HPLC measurement result showed that the L-alanine yield was 11.5 g / L.

[0115] Comparative Example 5

[0116] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-MmALD + pCDFDuet-MsLDH-MtADH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 6 g / L, acetaldehyde was 42 g / L, ammonium formate was 66 g / L, glucose was 15 g / L, pyridoxal phosphate was 0.04 g / L, pH was 8.0, the temperature was 45 °C, the shaker speed was 200 rpm, and the conversion time was 15 h. The HPLC measurement result showed that the L-alanine yield was 11.9 g / L.

[0117] Comparative Example 6

[0118] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-MmALD + pCDFDuet-MsLDH-MtADH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 0.1 g / L, acetaldehyde was 0.5 g / L, ammonium formate was 1 g / L, glucose was 1 g / L, pyridoxal phosphate was 0.005 g / L, pH was 8.0, the temperature was 30 °C, the shaker speed was 200 rpm, and the conversion time was 15 h. The HPLC measurement result showed that the L-alanine yield was 0.2 g / L.

[0119] The above are only the preferred embodiments of the present invention and are 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 within the protection scope of the present invention.

Claims

1. A recombinant bacterium for synthesizing L-alanine, characterized in that, The recombinant bacterium expresses at least aldolase, lactate dehydrogenase and alanine dehydrogenase.

2. The recombinant bacterium according to claim 1, characterized in that, The aldolase includes MmALD derived from Methanococcus maripaludis and MkALD derived from Mycobacterium kansasii; Preferably, the amino acid sequence of MmALD is as shown in SEQ ID NO.1, and the amino acid sequence of MkALD is as shown in SEQ ID NO.3; Preferably, 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 bacterium according to claim 1, characterized in that, The lactate dehydrogenase includes BcLDH derived from Bacillus cereus and MsLDH derived from Marinobacter subterrani; Preferably, the amino acid sequence of BcLDH is as shown in SEQ ID NO.5, and the amino acid sequence of MsLDH is as shown in SEQ ID NO.7; Preferably, the nucleotide sequence of BcLDH is as shown in SEQ ID NO.6, and the nucleotide sequence of MsLDH is as shown in SEQ ID NO.

8.

4. The recombinant bacterium according to claim 1, characterized in that The alanine dehydrogenase includes BfADH derived from Bacteroides fragilis and MtADH derived from Mycobacterium tuberculosis; Preferably, the amino acid sequence of BfADH is as shown in SEQ ID NO.9, and the amino acid sequence of MtADH is as shown in SEQ ID NO.11; Preferably, the nucleotide sequence of BfADH is as shown in SEQ ID NO.10, and the nucleotide sequence of MtADH is as shown in SEQ ID NO.

12.

5. The preparation method of the recombinant bacterium according to any one of claims 1-4, characterized in that It 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 the recombinant bacterium; Preferably, the expression vector includes pCDFDuet-1 and pACYCDuet-1; Preferably, the starting strain is Escherichia coli, including Escherichia coli BL21(DE3), Escherichia coli DH5α and Escherichia coli XL-Blue.

6. Use of the recombinant bacterium according to any one of claims 1-4 in the following aspects: (a) Catalyzing the synthesis of L-alanine and its downstream products; (b) Preparing a product for catalyzing the synthesis of L-alanine.

7. A product for catalytic synthesis of L-alanine, characterized in that: Containing the recombinant bacterium according to any one of claims 1-4.

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

9. The method according to claim 8, wherein The production system of the whole-cell catalysis 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 bacterium cells 1-10 g / L.

10. The method according to claim 8, wherein The pH of the whole-cell catalysis production system is 6.0 - 9.0, the temperature is 15 - 40 °C, the rotation speed is 100 - 250 rpm, and the reaction time is 3 - 10 h.

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