Method for producing R-3-aminobutyric acid

Abstract

The invention discloses a method for producing R-3-aminobutyric acid through whole-cell catalysis, which is characterized in that acetaldehyde and ammonium acetate are converted into R-3-aminobutyric acid by constructing recombinant bacteria for expressing aldolase, 3-hydracrylic acid dehydrogenase and diaminopimelate dehydrogenase. According to the production method disclosed by the invention, the R-3-aminobutyric acid can be efficiently converted and produced by utilizing low-price substrates (acetaldehyde and ammonium acetate), the yield is relatively high, the sources of the substrates are wide, the preparation process is simple, and the price is low; meanwhile, the selected enzyme has the advantages of high activity, strong optical specificity and the like, so that the recombinant bacterium disclosed by the invention is used for converting and producing the R-3-aminobutyric acid, 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 specifically, to a method for producing R-3-aminobutyric acid. Background Art

[0002] R-3-aminobutyric acid is a precursor substance of the pharmaceutical intermediate R-3-aminobutanol. R-3-aminobutanol is an important raw material for synthesizing the anti-AIDS drug dolutegravir. With the increasing influence of dolutegravir in the market, the market demand for R-3-aminobutyric acid is also continuously increasing.

[0003] The preparation methods of R-3-aminobutyric acid mainly include chemical synthesis method and biotransformation method. The chemical synthesis method has the disadvantages of serious environmental pollution, difficult operation and harsh reaction conditions, and it is difficult to realize industrial production. The biotransformation method has the advantages of high specificity, environmental friendliness and mild reaction conditions, and has currently received extensive attention.

[0004] At present, scholars at home and abroad have reported various biological preparation routes of R-3-aminobutyric acid. Andreas et al. used crotonic acid as a substrate and prepared R-3-aminobutyric acid by catalysis with the aspartase mutant BSASP-C6. After reacting for 100 h, the substrate conversion rate was only 60%. Chinese Patent CN113122563A used glucose or glycerol as a carbon source and prepared R-3-aminobutyric acid by microbial fermentation. After fermenting for 48 h, the yield of R-3-aminobutyric acid was only 1.96 g / L.

[0005] The above-reported R-3-aminobutyric acid preparation routes generally have the disadvantages of low yield, high substrate cost and low conversion efficiency. Therefore, there is an urgent need to provide a method for preparing R-3-aminobutyric acid with low cost and high efficiency. 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 R-3-aminobutyric acid. Using the recombinant bacteria of the present invention for transformation and production of R-3-aminobutyric acid has the advantages of high production efficiency, environmental friendliness and low cost.

[0007] The present invention provides a new route for synthesizing R-3-aminobutyric acid by whole-cell transformation method using acetaldehyde and ammonium acetate as substrates. First, acetaldehyde and acetic acid are converted into (S)-3-hydroxybutyric acid by aldolase (ALD). Under the action of 3-hydroxypropionate dehydrogenase (PDH), acetoacetic acid is obtained. R-3-aminobutyric acid is converted from acetoacetic acid by diaminopimelate dehydrogenase (DAPDH). The coenzymes required during the reaction process are provided by the bacteria metabolizing glucose. The coenzymes required for the reactions mediated by PDH and DAPDH can be complementary to achieve coenzyme recycling. The specific synthesis route is as Figure 1 shown.

[0008] The present invention is implemented as follows:

[0009] In a first aspect, the present invention provides a recombinant bacterium for synthesizing R-3-aminobutyric acid, which comprises ALD, PDH, and DAPDH;

[0010] Among them, ALD includes StALD and LsALD; the amino acid sequence of StALD is shown in SEQ ID NO.1, and the amino acid sequence of LsALD is shown in SEQ ID NO.2.

[0011] PDH includes CePDH and KpPDH; the amino acid sequence of CePDH is shown in SEQ ID NO.5, and the amino acid sequence of KpPDH is shown in SEQ ID NO.6.

[0012] DAPDH includes PmDAPDH and PdDAPDH; the amino acid sequence of PmDAPDH is shown in SEQ ID NO.9, and the amino acid sequence of PdDAPDH is shown in SEQ ID NO.10.

[0013] In some embodiments, the above-mentioned aldolase StALD is derived from Sphaerobacter thermophilus, Genbank number ACZ39383.1, its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.3.

[0014] In some embodiments, the above-mentioned aldolase LsALD is derived from Lachnellula suecica, Genbank number TVY82384.1, its amino acid sequence is shown in SEQ ID NO.2, and its nucleotide sequence is shown in SEQ ID NO.4.

[0015] In some embodiments, the above-mentioned 3-hydroxypropionate dehydrogenase CePDH is derived from Citrobacter europaeus, Genbank number CAD7564440.1, its amino acid sequence is shown in SEQ ID NO.5, and its nucleotide sequence is shown in SEQ ID NO.7.

[0016] In some embodiments, the above-mentioned 3-hydroxypropionate dehydrogenase KpPDH is derived from Klebsiella pneumoniae, Genbank number VED93542.1, its amino acid sequence is shown in SEQ ID NO.6, and its nucleotide sequence is shown in SEQ ID NO.8.

[0017] In some embodiments, the above-mentioned diaminopimelate dehydrogenase PmDAPDH is derived from Proteus mirabilis, with the Genbank number KXB99891.1. Its amino acid sequence is as shown in SEQ ID NO.9, and its nucleotide sequence is as shown in SEQ ID NO.11.

[0018] In some embodiments, the above-mentioned diaminopimelate dehydrogenase PdDAPDH is derived from Paenibacillus dendritiformis, with the Genbank number WP_407054926.1. Its amino acid sequence is as shown in SEQ ID NO.10, and its nucleotide sequence is as shown in SEQ ID NO.12.

[0019] After the inventors obtained the amino acid sequences of ALD, PDH, and DAPDH from the above sources, they performed codon optimization according to the Escherichia coli preference and synthesized the optimized nucleotide sequences by total synthesis.

[0020] In some embodiments, the method for constructing the above-mentioned recombinant bacterium is as follows: ligate the gene fragments of ALD, PDH, and DAPDH to an expression vector, and then introduce the obtained recombinant expression vector into a starting strain to obtain the above-mentioned recombinant bacterium.

[0021] In some embodiments, the above-mentioned expression vectors include pET28a(+) and pCDFDuet-1 plasmids.

[0022] In some embodiments, the starting strain of the above-mentioned recombinant bacterium is Escherichia coli. In other embodiments, the starting strain of the recombinant bacterium can be other bacteria, and the present invention does not specifically limit it.

[0023] In some embodiments, the above-mentioned Escherichia coli includes Escherichia coli BL21, Escherichia coli DH5α, and Escherichia coli XL-Blue.

[0024] The recombinant Escherichia coli of the present invention co-expresses the coding genes of 3 enzymes through two plasmids. Optionally, pET28a(+) loads ALD, and pCDFDuet-1 loads PDH and DAPDH. Then, the two recombinant plasmids are transformed into a starting strain to obtain a recombinant bacterium.

[0025] In a second aspect, the present invention provides a whole-cell catalyst containing the above-mentioned recombinant bacterium.

[0026] Using the above-mentioned recombinant bacterium as a whole-cell catalyst, the expressed ALD, PDH, and DAPDH can efficiently convert acetaldehyde and ammonium acetate into R-3-aminobutyric acid.

[0027] In a third aspect, the present invention provides a nucleic acid molecule encoding a protein having the function of converting acetaldehyde and ammonium acetate into R-3-aminobutyric acid, and the nucleic acid molecule includes genes encoding ALD, PDH, and DAPDH.

[0028] In some embodiments, ALD includes StALD and LsALD; the nucleotide sequence of StALD is shown in SEQ ID NO.3, and the nucleotide sequence of LsALD is shown in SEQ ID NO.4.

[0029] In some embodiments, PDH includes CePDH and KpPDH; the nucleotide sequence of CePDH is shown in SEQ ID NO.7, and the nucleotide sequence of KpPDH is shown in SEQ ID NO.8.

[0030] In some embodiments, DAPDH includes PmDAPDH and PdDAPDH; the nucleotide sequence of PmDAPDH is shown in SEQ ID NO.11, and the nucleotide sequence of PdDAPDH is shown in SEQ ID NO.12.

[0031] In a fourth aspect, the present invention provides an expression vector carrying the above nucleic acid molecule.

[0032] In a fifth aspect, the present invention provides a microbial cell containing the above nucleic acid molecule or expression vector.

[0033] In a sixth aspect, the present invention provides the use of the above recombinant bacterium, whole cell catalyst, nucleic acid molecule, expression vector or microorganism in the synthesis of R-3-aminobutyric acid and its downstream products.

[0034] In a seventh aspect, the present invention provides a method for synthesizing R-3-aminobutyric acid, which includes adding the above recombinant bacterium into a solution containing acetaldehyde and ammonium acetate for whole cell conversion to obtain R-3-aminobutyric acid.

[0035] In some embodiments, the recombinant bacterium is induced to culture before whole cell conversion, and the induction culture process is as follows: inoculating the recombinant bacterium into an LB medium containing 30-60 mg / L kanamycin and 30-60 mg / L streptomycin, culturing to obtain a seed solution, then inoculating the seed solution into a fresh LB medium until the cell density OD 600nm reaches 0.6-0.8, adding an inducer, and separating and washing after induction to obtain wet cells.

[0036] In some embodiments, the culture conditions of the above recombinant bacterium are: temperature is 32-38 °C, rotation speed is 150-250 rpm, and culture time is 10-16 h.

[0037] In some embodiments, the culture conditions of the above-mentioned seed liquid are as follows: the temperature is 32 - 38 °C, and the rotation speed is 150 - 250 rpm.

[0038] In some embodiments, the above-mentioned 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.

[0039] In some embodiments, the production system for whole-cell transformation includes: acetaldehyde 1 - 60 g / L, ammonium acetate 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.

[0040] 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 8 - 24 h.

[0041] The present invention has the following beneficial effects:

[0042] The present invention provides a method for producing R-3-aminobutyric acid by whole-cell catalysis. By constructing a recombinant bacterium expressing aldolase, 3-hydroxypropionate dehydrogenase, and diaminopimelate dehydrogenase, acetaldehyde and ammonium acetate are converted into R-3-aminobutyric acid. The production method of the present invention can efficiently convert and produce R-3-aminobutyric acid using low-cost substrates (acetaldehyde and ammonium acetate), with a high yield, wide substrate sources, simple preparation process, and low price. At the same time, the enzymes selected in the present invention have advantages such as high activity and strong optical specificity. Therefore, using the recombinant bacterium of the present invention to transform and produce R-3-aminobutyric acid has high production efficiency, is environmentally friendly and low-cost, and has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use 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 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.

[0044] Figure 1 It is the synthesis route of R-3-aminobutyric acid in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0045] 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 described clearly and completely below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0046] The features and performance of the present invention will be further described in detail below in conjunction with the embodiments.

[0047] 1. Selection of Bacteria and Plasmids

[0048] pET28a plasmid, pCDFDuet-1 plasmid, Escherichia coli BL21, Escherichia coli DH5α, and Escherichia coli XL-Blue purchased from Novagen.

[0049] 2. Selection of Enzymes

[0050] (1) Selection of Aldolase

[0051] The amino acid sequences of aldolases StALD and LsALD were obtained from the NCBI database, codon-optimized according to the Escherichia coli preference, and two nucleotide sequences were synthesized by total synthesis through conventional genetic engineering operations, as shown in SEQ ID NO.3 and SEQ ID NO.4 respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively. Restriction enzyme sites EcoRI and HindIII were added to both ends of the nucleotide sequences.

[0052] (2) Selection of 3-Hydroxypropionate Dehydrogenase

[0053] The amino acid sequences of 3-hydroxypropionate dehydrogenases CePDH and KpPDH were obtained from the NCBI database, codon-optimized according to the Escherichia coli preference, and two nucleotide sequences were synthesized by total synthesis through conventional genetic engineering operations, as shown in SEQ ID NO.7 and SEQ ID NO.8 respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO.5 and SEQ ID NO.6 respectively. Restriction enzyme sites EcoRI and HindIII were added to both ends of the nucleotide sequences.

[0054] (3) Selection of Diaminopimelate Dehydrogenase

[0055] The amino acid sequences of diaminopimelate dehydrogenase PmDAPDH and PdDAPDH were obtained from the NCBI database, codon-optimized according to the Escherichia coli preference, and two nucleotide sequences were synthesized by total synthesis through conventional genetic engineering operations, as shown in SEQ ID NO.11 and SEQ ID NO.12 respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO.9 and SEQ ID NO.10 respectively. Restriction enzyme sites NdeI and XhoI were added to both ends of the nucleotide sequences.

[0056] 3. Construction of a three-enzyme co-expression system and cell culture

[0057] For the co-expression of three enzymes, any one enzyme from each of the above-selected aldolase, 3-hydroxypropionate dehydrogenase, and diaminopimelate dehydrogenase was selected for three-enzyme combination co-expression. The pET28a(+) and pCDFDuet-1 dual plasmids were used to co-express the encoding genes of the 3 enzymes; pET28a(+) carried the aldolase, and pCDFDuet-1 carried the 3-hydroxypropionate dehydrogenase and diaminopimelate 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.

[0058] 4. Preparation of R-3-aminobutyric acid by whole-cell conversion of acetaldehyde and ammonium acetate

[0059] Conversion system: acetaldehyde 1 - 60 g / L, ammonium acetate 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, and then at 15 - 40 °C, 100 - 250 rpm, convert for 8 - 24 h. After the conversion, the yield of R-3-aminobutyric acid was measured by liquid chromatography.

[0060] 5. Detection and analysis of samples

[0061] 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. The conversion solution was analyzed by a Shimadzu 2030C high-performance liquid chromatograph (HPLC). The chromatographic conditions were as follows: the mobile phase was methanol: water (1:1), an Inertsustain C18 chromatographic column (4.6×250 mm, 5 μm) was used, the flow rate was 1 mL / min, the column temperature was 30 °C, the injection volume was 20 μL, and the detection wavelength was 333 nm.

[0062] Example 1

[0063] Construction of Recombinant Escherichia coli

[0064] 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 PDH recombinant plasmid and the pCDFDuet-1 vector were respectively double-digested with restriction enzymes EcoRI and HindIII, and the fully synthesized DAPDH recombinant plasmid and the pCDFDuet-1 vector were respectively double-digested with restriction enzymes NdeI and XhoI. PDH and DAPDH from different sources were ligated in pairs to the pCDFDuet vector by T4 DNA ligase to obtain recombinant plasmid 2; different combinations of recombinant plasmids 1 and 2 were transformed into E. coli BL21(DE3) competent cells to obtain recombinant Escherichia coli.

[0065] Example 2

[0066] Inductive Cultivation of Recombinant Escherichia coli

[0067] The recombinant Escherichia 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 solution. The seed solution was inoculated into fresh LB medium at an inoculation amount of 2%, and cultured at 37 °C and 200 rpm until the cell density OD 600nm reached 0.7. 0.5 mM IPTG was added, and after induction at 28 °C for 15 h, the cells were centrifuged at 8000 rpm for 10 min. The supernatant was discarded, and the wet cells were washed twice with 0.9% normal saline, and then centrifuged for standby.

[0068] Example 3

[0069] Comparison of Transformation Capabilities of Various Recombinant Escherichia coli

[0070] The collected recombinant Escherichia coli was resuspended in a 50 mL system with a final cell concentration of 10 g / L, acetaldehyde 60 g / L, ammonium acetate 110 g / L, glucose 20 g / L, pyridoxal phosphate 0.05 g / L, pH 8.0, and reacted at 30 °C with a shaker speed of 200 rpm for 24 h. After the transformation, the yield of R-3-aminobutyric acid was determined by HPLC.

[0071] Table 1 Comparison of R-3-aminobutyric Acid Yields of Various Recombinant Bacteria

[0072]

[0073]

[0074] Example 4

[0075] According to the inducible expression method described in Example 2, after the inducible expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 1 g / L, acetaldehyde was 7 g / L, ammonium acetate 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 24 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 16.1 g / L.

[0076] Example 5

[0077] According to the inducible expression method described in Example 2, after the inducible expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 3 g / L, acetaldehyde was 19 g / L, ammonium acetate was 34 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 24 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 43.9 g / L.

[0078] Example 6

[0079] According to the inducible expression method described in Example 2, after the inducible expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 4 g / L, acetaldehyde was 25 g / L, ammonium acetate was 44 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 24 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 57.5 g / L.

[0080] Example 7

[0081] According to the inducible expression method described in Example 2, after the inducible expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 6 g / L, acetaldehyde was 35 g / L, ammonium acetate was 62 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 24 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 81.4 g / L.

[0082] Example 8

[0083] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 7 g / L, acetaldehyde was 43 g / L, ammonium acetate was 76 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 24 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 99.7 g / L.

[0084] Example 9

[0085] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 9 g / L, acetaldehyde was 55 g / L, ammonium acetate was 100 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 24 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 126.1 g / L.

[0086] Example 10

[0087] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 10 g / L, acetaldehyde was 30 g / L, ammonium acetate was 55 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 12 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 69.2 g / L.

[0088] Example 11

[0089] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 10 g / L, acetaldehyde was 18 g / L, ammonium acetate was 33 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 8 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 41.6 g / L.

[0090] Example 12

[0091] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 8 g / L, acetaldehyde was 20 g / L, ammonium acetate was 36 g / L, glucose was 16 g / L, pyridoxal phosphate was 0.05 g / L, pH was 8.0, the temperature was 30 °C, the shaking speed of the shaker was 200 rpm, and the conversion time was 10 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 46.6 g / L.

[0092] Example 13

[0093] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 7 g / L, acetaldehyde was 30 g / L, ammonium acetate was 54 g / L, glucose was 15 g / L, pyridoxal phosphate was 0.04 g / L, pH was 8.0, the temperature was 30 °C, the shaking speed of the shaker was 200 rpm, and the conversion time was 17 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 68.9 g / L.

[0094] Example 14

[0095] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 6 g / L, acetaldehyde was 17 g / L, ammonium acetate 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 shaking speed of the shaker was 200 rpm, and the conversion time was 12 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 38.9 g / L.

[0096] Example 15

[0097] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 5 g / L, acetaldehyde was 14 g / L, ammonium acetate was 26 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 12 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 32.5 g / L.

[0098] Example 16

[0099] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 4 g / L, acetaldehyde was 13 g / L, ammonium acetate 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 shaker speed was 200 rpm, and the conversion time was 12 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 30.3 g / L.

[0100] Example 17

[0101] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 2 g / L, acetaldehyde was 7 g / L, ammonium acetate 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 shaker speed was 200 rpm, and the conversion time was 12 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 16.1 g / L.

[0102] Example 18

[0103] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 2 g / L, acetaldehyde was 6 g / L, ammonium acetate 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 shaker speed was 200 rpm, and the conversion time was 12 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 13.8 g / L.

[0104] Example 19

[0105] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 6 g / L, acetaldehyde was 36 g / L, ammonium acetate 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 shaker speed was 200 rpm, and the conversion time was 24 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 83.5 g / L.

[0106] Example 20

[0107] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 4 g / L, acetaldehyde was 25 g / L, ammonium acetate 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 shaker speed was 200 rpm, and the conversion time was 24 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 57.5 g / L.

[0108] Example 21

[0109] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 7 g / L, acetaldehyde was 43 g / L, ammonium acetate was 77 g / L, glucose was 11 g / L, pyridoxal phosphate was 0.03 g / L, pH was 7.5, the temperature was 40 °C, the shaker speed was 200 rpm, and the conversion time was 24 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 99.6 g / L.

[0110] Example 22

[0111] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 12 g / L, acetaldehyde was 70 g / L, ammonium acetate 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 shaker speed was 200 rpm, and the conversion time was 36 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 21.4 g / L.

[0112] Comparative Example 1

[0113] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 1 g / L, acetaldehyde was 1 g / L, ammonium acetate was 2 g / L, glucose was 5 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 24 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 2.3 g / L.

[0114] Comparative Example 2

[0115] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 5 g / L, acetaldehyde was 30 g / L, ammonium acetate was 60 g / L, glucose was 10 g / L, pyridoxal phosphate was 0.03 g / L, pH was 5.5, the temperature was 35 °C, the shaker speed was 200 rpm, and the conversion time was 36 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 12.1 g / L.

[0116] Comparative Example 3

[0117] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 3 g / L, acetaldehyde was 24 g / L, ammonium acetate was 45 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 36 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 10.7 g / L.

[0118] Comparative Example 4

[0119] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the bacterial cells were collected. In a 50 mL system, the wet cell weight was 4 g / L, acetaldehyde was 26 g / L, ammonium acetate was 50 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 24 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 7.3 g / L.

[0120] Comparative Example 5

[0121] According to the induced expression method described in Example 2, after the induced expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH was completed, the cells were collected. In a 50 mL system, the wet cell weight was 6 g / L, acetaldehyde was 36 g / L, ammonium acetate was 70 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 36 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 16.9 g / L.

[0122] Comparative Example 6

[0123] According to the induced expression method described in Example 2, after the induced expression of E. coli BL21(DE3) / pET28a-LsALD + pCDFDuet-CePDH-PmDAPDH 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 acetate 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 36 h. The HPLC measurement result showed that the yield of R-3-aminobutyric acid was 0.26 g / L.

[0124] 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 can have various modifications and changes. 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 R-3-aminobutyric acid, characterized in that: Contains genes encoding aldolase, 3-hydroxypropionate dehydrogenase, and diaminopimelate dehydrogenase.

2. The recombinant bacterium according to claim 1, characterized in that The aldolase includes StALD and LsALD; the amino acid sequence of StALD is shown in SEQ ID NO.1, and the amino acid sequence of LsALD is shown in SEQ ID NO.2; Preferably, the 3-hydroxypropionate dehydrogenase includes CePDH and KpPDH; the amino acid sequence of CePDH is shown in SEQ ID NO.5, and the amino acid sequence of KpPDH is shown in SEQ ID NO.6; Preferably, the diaminopimelate dehydrogenase includes PmDAPDH and PdDAPDH; the amino acid sequence of the PmDAPDH is shown in SEQ ID NO.9, and the amino acid sequence of the PdDAPDH is shown in SEQ ID NO.

10.

3. A whole cell catalyst, characterized in that Contains the recombinant bacteria according to claim 1 or 2.

4. A nucleic acid molecule encoding a protein having the function of converting acetaldehyde and ammonium acetate into R-3-aminobutyric acid, characterized in that: The nucleic acid molecule includes genes encoding aldolase, 3-hydroxypropionate dehydrogenase, and diaminopimelate dehydrogenase; Preferably, the aldolase comprises StALD and LsALD; the nucleotide sequence of StALD is shown in SEQ ID NO.3, and the nucleotide sequence of LsALD is shown in SEQ ID NO.4; Preferably, the 3-hydroxypropionate dehydrogenase includes CePDH and KpPDH; the nucleotide sequence of CePDH is shown in SEQ ID NO.7, and the nucleotide sequence of KpPDH is shown in SEQ ID NO.8; Preferably, the diaminopimelate dehydrogenase includes PmDAPDH and PdDAPDH; the nucleotide sequence of the PmDAPDH is shown in SEQ ID NO.11, and the nucleotide sequence of the PdDAPDH is shown in SEQ ID NO.

12.

5. An expression vector, characterized in that: The expression vector carries the nucleic acid molecule of claim 4.

6. A microbial cell, characterized in that Containing the nucleic acid molecule according to claim 4 or the expression vector according to claim 5.

7. Use of the recombinant bacteria according to claim 1 or 2, or the whole cell catalyst according to claim 3, or the nucleic acid molecule according to claim 4, or the expression vector according to claim 5, or the microbial cell according to claim 6 in synthesizing R-3-aminobutyric acid and its downstream products.

8. A method for synthesizing R-3-aminobutyric acid, characterized in that: The method comprises adding the recombinant bacteria according to claim 1 or 2 into a solution containing acetaldehyde and ammonium acetate for whole-cell catalysis to obtain R-3-aminobutyric acid.

9. The method according to claim 8, characterized in that The whole-cell catalytic production system comprises: 1-60 g / L of acetaldehyde, 2-110 g / L of ammonium acetate, 5-20 g / L of glucose, 0.01-0.05 g / L of pyridoxal phosphate, and 1-10 g / L of recombinant bacterial cells.

10. The method according to claim 9, characterized in that The pH of the whole-cell catalytic production system is 6.0-9.0, the temperature is 15-40° C., and the reaction time is 8-24 hours.

Images