Recombinant bacteria, construction method and application thereof, and production method of 3-aminopropionic acid

Abstract

The invention discloses a recombinant bacterium, a construction method and application thereof and a production method of 3-aminopropionic acid. The production method comprises the following steps: co-expressing three enzymes by adopting double plasmids pET28a (+) and pCDFDuet-1; according to the method, pET28a (+) is loaded with a carboxylic acid reductase gene, pCDFDuet-1 is loaded with a transaminase gene and a glutamate dehydrogenase gene to obtain recombinant plasmids, the two recombinant plasmids are converted into corresponding strains to obtain recombinant bacteria, and the recombinant bacteria are used as a biocatalyst to convert a cheap substrate malonic acid into 3-aminopropionic acid. The production method of the 3-aminopropionic acid is high in production efficiency, environmentally friendly and low in cost, and the selected enzyme has the advantages of being high in activity, high in optical specificity and the like.

Description

[0001] The present invention claims priority. The application number of the prior application is: CN2024113048002, the name is: Recombinant bacteria and construction method and application and production method of 3-aminopropionic acid, and the application date of the prior application is: September 19, 2024. Technical Field

[0002] The invention belongs to the field of biotechnology and relates to the production of alanine, in particular to a recombinant bacterium and a construction method and application thereof, as well as a production method of 3-aminopropionic acid. Background Art

[0003] 3-Aminopropionic acid, also known as β-alanine, is the only β-type amino acid in nature. It is also a non-protein amino acid that exists in certain bacteria, fungi and plants. It is widely used in food, medicine, chemical industry and other fields.

[0004] The preparation methods of 3-aminopropionic acid mainly include chemical synthesis and biotransformation. The chemical synthesis method mainly uses acrylic acid or acrylonitrile as substrate, which consumes a lot of energy during the synthesis process and has certain toxic effects on the environment and organisms. 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.

[0005] For example, Chinese patent CN113943691A uses pyruvate as a substrate and converts 3-aminopropionic acid through the coupled conversion of L-alanine dehydrogenase and L-phenylalanine mutase. The yield of 3-aminopropionic acid is only 0.035 mM.

[0006] The 3-aminopropionic acid preparation routes in the prior art generally have disadvantages such as low atom utilization and low yield. Therefore, it is urgent to create a production method for 3-aminopropionic acid with high yield, easy industrial production and low cost. Summary of the invention

[0007] In order to solve the above problems, the first object of the present invention is to provide a recombinant bacterium, which co-expresses carboxylic acid reductase, transaminase and glutamate dehydrogenase.

[0008] The second object of the present invention is to provide a method for constructing the above-mentioned recombinant bacteria, using pET28a(+) and pCDFDuet-1 double plasmids to co-express three enzymes; pET28a(+) is loaded with the gene of carboxylic acid reductase, and pCDFDuet-1 is loaded with the gene of transaminase and the gene of glutamate dehydrogenase to obtain a recombinant vector, and then the two recombinant vectors are transformed into the corresponding strains to obtain recombinant bacteria.

[0009] The third object of the present invention is to provide the application of the above-mentioned recombinant bacteria;

[0010] Furthermore, provided is the use of the recombinant bacteria in producing 3-aminopropionic acid and its downstream products.

[0011] Furthermore, the above recombinant bacteria can be used as a biocatalyst to convert the substrate malonate into 3-aminopropionic acid.

[0012] The last objective of the present invention is to provide a novel method for producing 3-aminopropionic acid. The present invention adopts pET28a(+) and pCDFDuet-1 double plasmids to co-express three enzymes; pET28a(+) is loaded with the gene of carboxylic acid reductase, and pCDFDuet-1 is loaded with the gene of transaminase and the gene of glutamate dehydrogenase to obtain a recombinant vector, and then the two recombinant vectors are transformed into corresponding strains to obtain recombinant bacteria, and the recombinant bacteria are used as biocatalysts to convert the substrate malonate into 3-aminopropionic acid.

[0013] The method for producing 3-aminopropionic acid provided by the invention has high yield, high production efficiency, is green and environmentally friendly, and has low cost, and has good industrial application prospects.

[0014] In order to achieve the above object, the present invention adopts the following technical solutions:

[0015] The present invention first provides a recombinant bacterium, which contains genes encoding carboxylic acid reductase, transaminase and glutamate dehydrogenase.

[0016] As a preferred embodiment of the present invention, the carboxylic acid reductase is from Nocardia cyriacigeorgica (Genbank No. WP_014351049.1) and Rhodococcus sp. (Genbank No. WP_366238297.1), and is named NcCAR and RsCAR, respectively.

[0017] As a preferred embodiment of the present invention, the nucleotide sequence of the carboxylic acid reductase is shown as SEQ ID NO.1 and / or SEQ ID NO.3; the amino acid sequence of the carboxylic acid reductase is shown as SEQ ID NO.2 and / or SEQ ID NO.4.

[0018] As a preferred embodiment of the present invention, the transaminase is from Lactococcus cremoris (Genbank No. BBC74517.1) and Methanoculleus bourgensis (Genbank No. SAI87498.1), and is named LcTA and MbTA respectively.

[0019] As a preferred embodiment of the present invention, the transaminase nucleotide sequence is shown as SEQ ID NO.5 and / or SEQ ID NO.7; the transaminase amino acid sequence is shown as SEQ ID NO.6 and / or SEQ ID NO.8.

[0020] As a preferred embodiment of the present invention, the glutamate dehydrogenase is derived from Pyrobaculum calidifontis (Genbank No. ABO09023.1) and Thermococcus litoralis (Genbank No. AAA72393.1), and is named PcGluDH and TlGluDH, respectively.

[0021] As a preferred embodiment of the present invention, the glutamate dehydrogenase nucleotide sequence is SEQ ID NO.9 and / or SEQ ID NO.11; the glutamate dehydrogenase amino acid sequence is SEQ ID NO.10 and / or SEQ ID NO.12.

[0022] Secondly, the present invention provides a method for constructing the above-mentioned recombinant bacteria, which comprises: using a first vector and a second vector to co-express three enzymes; the first vector is loaded with a gene for carboxylic acid reductase, and the second vector is loaded with a gene for transaminase and a gene for glutamate dehydrogenase, and after obtaining the recombinant vectors, the two recombinant vectors are simultaneously transferred into competent Escherichia coli cells to obtain recombinant bacteria through screening.

[0023] As a preferred embodiment of the present invention, the first vector is a plasmid, and / or the second vector is a plasmid;

[0024] More preferably, the first vector is pET28a(+) or pCDFDuet-1, and the second vector is pCDFDuet-1 or pET28a(+).

[0025] As a preferred embodiment of the present invention, the competent cells are Escherichia coli competent cells.

[0026] More preferably, the Escherichia coli competent cells include Eschrichia coli BL21, Escherichia coli DH5α or Escherichia coli XL-Blue.

[0027] Most preferably, the E. coli competent cells are Eschrichia coli BL21.

[0028] Again, the present invention provides the use of the above-mentioned recombinant bacteria in the production of β-alanine and its downstream products.

[0029] Finally, the present invention provides a method for producing 3-aminopropionic acid, which comprises performing whole-cell conversion of malonic acid by the above-mentioned recombinant bacteria or the recombinant bacteria obtained by the above-mentioned construction method to obtain 3-aminopropionic acid; in the whole-cell conversion production system, the pH is 6.0-9.0, the temperature is 15-40°C, the shaking speed is 100-250rpm, and the conversion time is 8-24h.

[0030] Furthermore, the production method includes inoculating the above-mentioned recombinant bacteria or the recombinant bacteria obtained by the above-mentioned construction method into a liquid culture medium, inducing culture, centrifuging, and obtaining wet bacteria; resuspending the obtained wet bacteria in a whole-cell transformation production system, pH 6.0-9.0, reacting at 15-40°C, shaking speed 100-250rpm, transformation time 8-24h, to obtain 3-aminopropionic acid.

[0031] As a preferred embodiment of the present invention, the whole-cell transformation production system includes 1-5 g / L of recombinant bacteria, 1-100 g / L of malonic acid, 1-5 g / L of L-glutamic acid, 1-80 g / L of ammonium acetate, 10-100 g / L of glucose and 0.01-0.05 g / L of pyridoxal phosphate.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1) The present invention provides a recombinant bacterium and a method for producing 3-aminopropionic acid, wherein three enzymes are simultaneously expressed in Escherichia coli, and used as biocatalysts to convert the cheap substrate malonate into 3-aminopropionic acid. Malonic acid is converted into 3-oxopropionic acid by carboxylic acid reductase, 3-oxopropionic acid is converted into 3-aminopropionic acid by transaminase with L-glutamic acid as an amino donor, and 2-ketoglutaric acid is converted into L-glutamic acid by glutamate dehydrogenase, thereby realizing the recycling of L-glutamic acid, with high production efficiency, green environmental protection and low cost.

[0034] 2) The enzyme selected in the present invention has the advantages of high activity and strong optical specificity.

[0035] 3) The substrate malonic acid in the present invention has a wide source, a simple preparation process and a low price, and is an ideal substrate.

[0036] 4) The recombinant bacteria of the present invention are used to transform and produce 3-aminopropionic acid, which has high yield, high production efficiency, high conversion rate, is green and environmentally friendly, and has low cost, and has good prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0038] Figure 1 It is the synthesis route of 3-aminopropionic acid in the present invention. DETAILED DESCRIPTION

[0039] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0040] The present invention uses recombinant bacteria as a biocatalyst to prepare 3-aminopropionic acid with a cheap substrate malonate. The present invention simultaneously expresses three enzymes in Escherichia coli, namely carboxylic acid reductase (CAR), transaminase (TA) and glutamate dehydrogenase (GluDH).

[0041] See also Figure 1 Reaction principle: Malonic acid is converted into 3-oxopropionic acid by carboxylic acid reductase. Under the action of transaminase, 3-oxopropionic acid is converted into 3-aminopropionic acid with L-glutamate as the amino donor. 2-ketoglutarate is converted into L-glutamate by glutamate dehydrogenase to achieve the recycling of L-glutamate. The coenzymes required in the reaction are provided by the metabolism of glucose by the bacteria.

[0042] Bacteria and plasmids used in the present invention:

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

[0044] Selection of enzymes used in the present invention:

[0045] (1) Selection of carboxylic acid reductase

[0046] The amino acid sequences of carboxylic acid reductase NcCAR and RsCAR 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.1 and SEQ ID NO.3, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO.2 and SEQ ID NO.4, respectively. Restriction sites EcoRI and HindIII were added at both ends of the nucleotide sequence.

[0047] (2) Selection of transaminase

[0048] The amino acid sequences of transaminases LcTA and MbTA 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.5 and SEQ ID NO.7, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO.6 and SEQ ID NO.8, respectively. Restriction sites EcoRI and HindIII were added at both ends of the nucleotide sequence.

[0049] (3) Selection of glutamate dehydrogenase

[0050] The amino acid sequences of glutamate dehydrogenase PcGluDH and TlGluDH 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.9 and SEQ ID NO.11, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO.10 and SEQ ID NO.12, respectively. Restriction sites NdeI and XhoI were added at both ends of the nucleotide sequence.

[0051] In the present invention, the construction of the three-enzyme co-expression system and bacterial culture:

[0052] One enzyme from each of the above selected carboxylic acid reductase, transaminase and glutamate dehydrogenase is selected to co-express the three enzymes.

[0053] The pET28a(+) and pCDFDuet-1 double plasmids were used to co-express three enzymes; pET28a(+) was loaded with the gene of carboxylic acid reductase, and pCDFDuet-1 was loaded with the gene of transaminase and the gene of glutamate dehydrogenase. After obtaining the co-expression recombinant plasmid, the two recombinant plasmids were simultaneously transferred into Escherichia coli BL21 competent cells, and the positive transformants were screened using a plate containing kanamycin and streptomycin, that is, the recombinant bacteria were obtained. The obtained recombinant bacteria were inoculated into a fresh liquid culture medium, induced culture, centrifuged, and wet bacteria were obtained.

[0054] Whole cell conversion of malonate to produce 3-aminopropionic acid:

[0055] Conversion system: 1-100 g / L malonic acid, 1-5 g / L L-glutamic acid, 1-80 g / L ammonium acetate, 10-100 g / L glucose, 0.01-0.05 g / L pyridoxal phosphate, pH adjusted to 6.0-9.0, fresh cell volume 1-5 g / L, then at 15-40 ° C, 100-250 rpm, conversion for 8-24 hours. After the conversion is completed, the yield of 3-aminopropionic acid is determined by liquid chromatography.

[0056] 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 (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] This example provides the construction of recombinant Escherichia coli:

[0060] The fully synthetic CAR recombinant plasmid and the pET28a(+) vector were double-digested with restriction endonucleases EcoRI and HindIII, respectively, and CARs from different sources were connected to the pET28a(+) vector using T4 DNA ligase to obtain recombinant plasmid 1; the fully synthetic TA recombinant plasmid and the pCDFDuet-1 vector were double-digested with restriction endonucleases EcoRI and HindIII, respectively, and the fully synthetic GluDH recombinant plasmid and the pCDFDuet-1 vector were double-digested with restriction endonucleases NdeI and XhoI, respectively, and TA and GluDH from different sources were connected to the pCDFDuet vector in pairs using 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] This example provides the 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 solution. The seed solution was inoculated into fresh LB medium at a 2% inoculation amount, 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 solution was centrifuged at 8000 rpm for 10 minutes, and the supernatant was discarded to obtain the recombinant E. coli, and the recombinant E. coli was washed twice with 0.9% physiological saline, centrifuged, and set aside.

[0064] Example 3

[0065] This example provides a comparison of the transformation abilities of various recombinant E. coli

[0066] The collected recombinant E. coli was resuspended in a 50 mL system, with a final cell concentration of 5 g / L, 100 g / L malonic acid, 5 g / L L-glutamic acid, 80 g / L ammonium acetate, 100 g / L glucose, 0.05 g / L pyridoxal phosphate, pH 8.0, and reacted at 30°C, with a shaking speed of 200 rpm and a conversion time of 24 h. After the conversion, the yield of 3-aminopropionic acid was determined by HPLC, and the results are shown in Table 1.

[0067] Table 1. Comparison of 3-aminopropionic acid production of various recombinant bacteria

[0068] Recombinant bacteria β-Alanine(g / L) E.coliBL21(DE3) / pET28a-NcCAR+pCDFDuet-LcTA-PcGluDH 61.1 E.coliBL21(DE3) / pET28a-NcCAR+pCDFDuet-LcTA-TlGluDH 46.8 E.coliBL21(DE3) / pET28a-NcCAR+pCDFDuet-MbTA-PcGluDH 72.4 E.coliBL21(DE3) / pET28a-NcCAR+pCDFDuet-MbTA-TlGluDH 65.3 E.coliBL21(DE3) / pET28a-RsCAR+pCDFDuet-LcTA-PcGluDH 85.1 E.coliBL21(DE3) / pET28a-RsCAR+pCDFDuet-LcTA-TlGluDH 57.8 E.coliBL21(DE3) / pET28a-RsCAR+pCDFDuet-MbTA-PcGluDH 74.5 E.coliBL21(DE3) / pET28a-RsCAR+pCDFDuet-MbTA-TlGluDH 51.2

[0069] Example 4

[0070] This example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 1g / L, malonic acid is 1g / L, L-glutamic acid is 1g / L, ammonium acetate is 1g / L, glucose is 10g / L, pyridoxal phosphate is 0.01g / L, pH 8.0, temperature is 30 ° C, shaker speed is 200rpm, and conversion time is 24h. HPLC measurement results show that the yield of 3-aminopropionic acid is 0.83g / L.

[0071] Example 5

[0072] This example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 1g / L, malonic acid is 9g / L, L-glutamic acid is 1g / L, ammonium acetate is 7g / L, glucose is 10g / L, pyridoxal phosphate is 0.01g / L, pH 8.0, temperature is 35 ° C, shaker speed is 200rpm, and conversion time is 24h. HPLC measurement results show that the yield of 3-aminopropionic acid is 7.5g / L.

[0073] Example 6

[0074] This example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 1.5g / L, malonic acid is 28g / L, L-glutamic acid is 2g / L, ammonium acetate is 22g / L, glucose is 30g / L, pyridoxal phosphate is 0.01g / L, pH 8.0, temperature is 30 ° C, shaker speed is 200rpm, and conversion time is 24h. HPLC measurement results show that the yield of 3-aminopropionic acid is 23.7g / L.

[0075] Example 7

[0076] This example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 2g / L, malonic acid is 41g / L, L-glutamic acid is 3g / L, ammonium acetate is 33g / L, glucose is 40g / L, pyridoxal phosphate is 0.03g / L, pH 8.0, temperature is 30°C, and the shaking speed is 200rpm. The conversion time is 24h. HPLC measurement results show that the yield of 3-aminopropionic acid is 34.4g / L.

[0077] Example 8

[0078] This example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 3g / L, malonic acid is 59g / L, L-glutamic acid is 3g / L, ammonium acetate is 47g / L, glucose is 60g / L, pyridoxal phosphate is 0.03g / L, pH 8.0, temperature is 30°C, and the shaking speed is 200rpm. The conversion time is 24h. HPLC measurement results show that the yield of 3-aminopropionic acid is 49.7g / L.

[0079] Example 9

[0080] This example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 3.5g / L, malonic acid is 70g / L, L-glutamic acid is 4g / L, ammonium acetate is 56g / L, glucose is 70g / L, pyridoxal phosphate is 0.05g / L, pH 8.0, temperature is 30 ° C, shaker speed is 200rpm, and conversion time is 24h. HPLC measurement results show that the yield of 3-aminopropionic acid is 59.2g / L.

[0081] Example 10

[0082] This example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 4.5g / L, malonic acid is 88g / L, L-glutamic acid is 5g / L, ammonium acetate is 70g / L, glucose is 90g / L, pyridoxal phosphate is 0.05g / L, pH 8.0, temperature is 30 ° C, shaker speed is 200rpm, and conversion time is 24h. HPLC measurement results show that the yield of 3-aminopropionic acid is 73.8g / L.

[0083] Embodiment 11

[0084] This example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 5g / L, malonic acid is 49g / L, L-glutamic acid is 3g / L, ammonium acetate is 39g / L, glucose is 50g / L, pyridoxal phosphate is 0.05g / L, pH 8.0, temperature is 30 ° C, shaker speed is 200rpm, and conversion time is 12h. HPLC measurement results show that the yield of 3-aminopropionic acid is 41.5g / L.

[0085] Example 12

[0086] This example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 5g / L, malonic acid is 32g / L, L-glutamic acid is 3g / L, ammonium acetate is 26g / L, glucose is 30g / L, pyridoxal phosphate is 0.05g / L, pH 8.0, temperature is 30 ° C, shaker speed is 200rpm, and conversion time is 8h. HPLC measurement results show that the yield of 3-aminopropionic acid is 27.2g / L.

[0087] Example 13

[0088] This example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 4g / L, malonic acid is 33g / L, L-glutamic acid is 2g / L, ammonium acetate is 26g / L, glucose is 40g / L, pyridoxal phosphate is 0.05g / L, pH 8.0, temperature is 30°C, shaking speed is 200rpm, and conversion time is 10h. HPLC measurement results show that the yield of 3-aminopropionic acid is 27.6g / L.

[0089] Embodiment 14

[0090] This example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 3.5g / L, malonic acid is 50g / L, L-glutamic acid is 3g / L, ammonium acetate is 40g / L, glucose is 55g / L, pyridoxal phosphate is 0.04g / L, pH 8.0, temperature is 30 ° C, shaker speed is 200rpm, and conversion time is 17h. HPLC measurement results show that the yield of 3-aminopropionic acid is 41.7g / L.

[0091] Embodiment 15

[0092] This example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 3g / L, malonic acid is 29g / L, L-glutamic acid is 2g / L, ammonium acetate is 23g / L, glucose is 30g / L, pyridoxal phosphate is 0.04g / L, pH 6.0, temperature is 35 ° C, shaker speed is 200rpm, and conversion time is 12h. HPLC measurement results show that the yield of 3-aminopropionic acid is 24.4g / L.

[0093] Example 16

[0094] This example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 2.5g / L, malonic acid is 26g / L, L-glutamic acid is 2g / L, ammonium acetate is 21g / L, glucose is 30g / L, pyridoxal phosphate is 0.03g / L, pH 7.0, temperature is 35 ° C, shaker speed is 200rpm, and conversion time is 12h. HPLC measurement results show that the yield of 3-aminopropionic acid is 21.9g / L.

[0095] Embodiment 17

[0096] This example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 2g / L, malonic acid is 20g / L, L-glutamic acid is 2g / L, ammonium acetate is 16g / L, glucose is 25g / L, pyridoxal phosphate is 0.03g / L, pH 7.5, temperature is 35 ° C, shaker speed is 200rpm, and conversion time is 12h. HPLC measurement results show that the yield of 3-aminopropionic acid is 16.8g / L.

[0097] Embodiment 18

[0098] This example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 1g / L, malonic acid is 11g / L, L-glutamic acid is 1g / L, ammonium acetate is 9g / L, glucose is 20g / L, pyridoxal phosphate is 0.02g / L, pH 8.5, temperature is 35 ° C, shaker speed is 200rpm, and conversion time is 12h. HPLC measurement results show that the yield of 3-aminopropionic acid is 9.3g / L.

[0099] Embodiment 19

[0100] This example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 1g / L, malonic acid is 10g / L, L-glutamic acid is 1g / L, ammonium acetate is 8g / L, glucose is 20g / L, pyridoxal phosphate is 0.02g / L, pH 9.0, temperature is 35 ° C, shaker speed is 200rpm, and conversion time is 12h. HPLC measurement results show that the yield of 3-aminopropionic acid is 8.4g / L.

[0101] Embodiment 20

[0102] This example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 3g / L, malonic acid is 60g / L, L-glutamic acid is 4g / L, ammonium acetate is 48g / L, glucose is 60g / L, pyridoxal phosphate is 0.03g / L, pH 7.5, temperature is 15°C, shaker speed is 200rpm, and conversion time is 24h. HPLC measurement results show that the yield of 3-aminopropionic acid is 50.1g / L.

[0103] Embodiment 21

[0104] This example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 2g / L, malonic acid is 39g / L, L-glutamic acid is 3g / L, ammonium acetate is 31g / L, glucose is 50g / L, pyridoxal phosphate is 0.03g / L, pH 7.5, temperature is 25°C, shaking speed is 200rpm, and conversion time is 24h. HPLC measurement results show that the yield of 3-aminopropionic acid is 33.1g / L.

[0105] Embodiment 22

[0106] This example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 2.5g / L, malonic acid is 51g / L, L-glutamic acid is 3g / L, ammonium acetate is 41g / L, glucose is 50g / L, pyridoxal phosphate is 0.03g / L, pH 7.5, temperature is 40 ° C, shaker speed is 200rpm, and conversion time is 24h. HPLC measurement results show that the yield of 3-aminopropionic acid is 43.1g / L.

[0107] Comparative Example 1

[0108] This comparative example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 6g / L, malonic acid is 110g / L, L-glutamic acid is 7g / L, ammonium acetate is 90g / L, glucose is 110g / L, pyridoxal phosphate is 0.08g / L, pH7.5, temperature is 35 ° C, shaker speed is 200rpm, and conversion time is 36h. HPLC measurement results show that the yield of 3-aminopropionic acid is 16.1g / L.

[0109] Comparative Example 2

[0110] This comparative example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 2.5g / L, malonic acid is 50g / L, L-glutamic acid is 3g / L, ammonium acetate is 40g / L, glucose is 50g / L, pyridoxal phosphate is 0.03g / L, pH 5.5, temperature is 35 ° C, shaker speed is 200rpm, and conversion time is 36h. HPLC measurement results show that the yield of 3-aminopropionic acid is 5.9g / L.

[0111] Comparative Example 3

[0112] This comparative example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 1.5g / L, malonic acid is 27g / L, L-glutamic acid is 2g / L, ammonium acetate is 23g / L, glucose is 40g / L, pyridoxal phosphate is 0.03g / L, pH 9.5, temperature is 35 ° C, shaker speed is 200rpm, and conversion time is 36h. HPLC measurement results show that the yield of 3-aminopropionic acid is 2.7g / L.

[0113] Comparative Example 4

[0114] This comparative example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 2g / L, malonic acid is 36g / L, L-glutamic acid is 3g / L, ammonium acetate is 30g / L, glucose is 50g / L, pyridoxal phosphate is 0.03g / L, pH 7.5, temperature is 10°C, shaker speed is 200rpm, and conversion time is 36h. HPLC measurement results show that the yield of 3-aminopropionic acid is 5.8g / L.

[0115] Comparative Example 5

[0116] This comparative example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 3g / L, malonic acid is 57g / L, L-glutamic acid is 3g / L, ammonium acetate is 46g / L, glucose is 60g / L, pyridoxal phosphate is 0.04g / L, pH 8.0, temperature is 45 ° C, shaker speed is 200rpm, and conversion time is 36h. HPLC measurement results show that the yield of 3-aminopropionic acid is 7.3g / L.

[0117] Comparative Example 6

[0118] This comparative example is based on the induction expression method described in Example 2. After the induction expression of E.coli BL21 (DE3) / pET28a-RsCAR + pCDFDuet-LcTA-PcGluDH is completed, the recombinant E. coli is collected. In a 50mL system, the cell wet weight is 0.1g / L, malonic acid is 0.8g / L, L-glutamic acid is 0.5g / L, ammonium acetate is 0.7g / L, glucose is 5g / L, pyridoxal phosphate is 0.005g / L, pH 8.0, temperature is 30 ° C, shaker speed is 200rpm, and conversion time is 36h. HPLC measurement results show that the yield of 3-aminopropionic acid is 0.14g / L.

[0119] The present invention provides a recombinant bacterium and a method for producing 3-aminopropionic acid. Three enzymes are simultaneously expressed in Escherichia coli, which are used as biocatalysts to convert malonate, a cheap substrate, into 3-aminopropionic acid. Carboxylic acid reductase is used to convert malonate into 3-oxopropionic acid. Under the action of transaminase, 3-oxopropionic acid is converted into 3-aminopropionic acid using L-glutamic acid as an amino donor. Glutamate dehydrogenase is used to convert 2-ketoglutarate into L-glutamic acid, thereby realizing the recycling of L-glutamic acid. The production efficiency is high, the product is green and environmentally friendly, and the cost is low.

[0120] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A recombinant bacterium, characterized in that: The recombinant bacteria contain genes encoding carboxylic acid reductase, transaminase and glutamate dehydrogenase.

2. A recombinant bacterium according to claim 1, characterized in that: The nucleotide sequence of the carboxylic acid reductase is shown in SEQ ID NO.1 and / or SEQ ID NO.3; the amino acid sequence of the carboxylic acid reductase is shown in SEQ ID NO.2 and / or SEQ ID NO.

4.

3. A recombinant bacterium according to claim 1, characterized in that: The transaminase nucleotide sequence is shown in SEQ ID NO.5 and / or SEQ ID NO.7; the transaminase amino acid sequence is shown in SEQ ID NO.6 and / or SEQ ID NO.

8.

4. A recombinant bacterium according to claim 1, characterized in that: The glutamate dehydrogenase nucleotide sequence is SEQ ID NO.9 and / or SEQ ID NO.11; the glutamate dehydrogenase amino acid sequence is SEQ ID NO.10 and / or SEQ ID NO.

12.

5. A method for constructing a recombinant bacterium according to any one of claims 1 to 4, characterized in that: The construction method comprises: using a first vector to load the gene of carboxylic acid reductase, and a second vector to load the gene of transaminase and the gene of glutamate dehydrogenase, after obtaining the recombinant vector, the two recombinant vectors are simultaneously transferred into competent cells, and the recombinant bacteria are obtained by screening.

6. The method for constructing a recombinant bacterium according to claim 5, characterized in that: The first vector is a plasmid, and / or the second vector is a plasmid; Preferably, the first vector is pET28a(+) or pCDFDuet-1, and the second vector is pCDFDuet-1 or pET28a(+).

7. The method for constructing a recombinant bacterium according to claim 5, characterized in that: The competent cells are Escherichia coli competent cells, Preferably, the Escherichia coli competent cells include Eschrichia coli BL21, Escherichia coli DH5α or Escherichia coli XL-Blue.

8. A use of the recombinant bacterium according to any one of claims 1 to 4, characterized in that: The recombinant bacteria is used in producing 3-aminopropionic acid and its downstream products.

9. A method for producing 3-aminopropionic acid, characterized in that: The production method comprises performing whole-cell conversion of malonate using the recombinant bacteria described in any one of claims 1 to 4 or the recombinant bacteria obtained by the construction method described in any one of claims 5 to 7 to obtain 3-aminopropionic acid; in the whole-cell conversion production system, the pH is 6.0-9.0, the temperature is 15-40°C, the shaking speed is 100-250 rpm, and the conversion time is 8-24 hours.

10. The method for producing β-alanine according to claim 9, characterized in that: The whole cell transformation production system includes 1-5 g / L of recombinant bacteria, 1-100 g / L of malonic acid, 1-5 g / L of L-glutamic acid, 1-80 g / L of ammonium acetate, 10-100 g / L of glucose and 0.01-0.05 g / L of pyridoxal phosphate.

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