A process for the production of (s)-2-amino-4-hydroxybutyric acid

Abstract

The application discloses a method for producing (S)-2-amino-4-hydroxybutyric acid and belongs to the technical field of biotechnology. The method comprises the following steps: constructing a gene engineering bacterium capable of expressing L-amino acid deaminase, 4-hydroxy-2-ketoglutarate aldolase, transaminase and glutamate dehydrogenase; and then converting a substrate L-alanine, formaldehyde and ammonia into (S)-2-amino-4-hydroxybutyric acid through whole cell transformation. The method has the advantages of high conversion efficiency and low production cost. Meanwhile, the selected enzyme has the advantages of high activity and strong optical specificity. Therefore, the enzyme is used for producing (S)-2-amino-4-hydroxybutyric acid, and the production efficiency is high, the production is green and environment-friendly, the cost is low, and the method has a good industrial application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a method for producing (S)-2-amino-4-hydroxybutyric acid. BACKGROUND

[0002] L-Homoserine, also known as (S)-2-amino-4-hydroxybutyric acid, is an important intermediate for biosynthesis of essential amino acids L-methionine and L-threonine, and is also a platform compound for synthesis of various C4 compounds and L-phosphinothricin, and is widely used in medicine, agriculture, cosmetics and perfume industry.

[0003] The preparation methods of (S)-2-amino-4-hydroxybutyric acid mainly include chemical synthesis method and biological transformation method. The chemical synthesis method has problems of harsh reaction conditions, long reaction steps, low production efficiency and low safety, and is gradually replaced. The biological transformation method has advantages of high specificity, green environmental protection, mild reaction conditions and no need for multi-step separation and purification, and has been widely concerned at present.

[0004] At present, some domestic and foreign scholars have reported some biological preparation routes of (S)-2-amino-4-hydroxybutyric acid, for example, Ning Li et al. constructed (S)-2-amino-4-hydroxybutyric acid production bacteria by modifying the metabolic pathway of corynebacterium glutamicum, and the yield of (S)-2-amino-4-hydroxybutyric acid reached 63.5g / L after 96h fermentation, and the space-time yield was 0.66g / L / h.

[0005] The above reported biological synthesis routes of (S)-2-amino-4-hydroxybutyric acid have disadvantages of low space-time yield, low production efficiency and high substrate cost, therefore, it is urgent to provide a (S)-2-amino-4-hydroxybutyric acid preparation method with high efficiency and low cost.

[0006] In view of this, the present application is proposed. SUMMARY

[0007] The purpose of the present application is to provide a method for producing (S)-2-amino-4-hydroxybutyric acid, which is constructed by expressing L-amino acid deaminase, 4-hydroxy-2-ketoglutaric acid aldolase, transaminase and glutamate dehydrogenase in a genetically engineered bacterium, and then converting the substrates L-alanine, formaldehyde and ammonia into (S)-2-amino-4-hydroxybutyric acid by whole cell transformation, which has the advantages of high conversion efficiency and low production cost.

[0008] The present application provides a new synthesis route in order to improve the defects of low space-time yield, low production efficiency and high substrate cost of (S)-2-amino-4-hydroxybutyric acid in the prior art.

[0009] For the synthesis of (S)-2-amino-4-hydroxybutyric acid, the present application converts L-alanine into pyruvic acid by L-amino acid deaminase (LAAD), converts pyruvic acid and formaldehyde into 4-hydroxy-2-oxobutyric acid by 4-hydroxy-2-ketovalerate aldolase (HOA), converts 4-hydroxy-2-oxobutyric acid into L-homoserine by transaminase (TA), converts L-homoserine into alpha-ketoglutarate by taking L-glutamic acid as an amino donor, converts alpha-ketoglutarate into L-glutamic acid by glutamate dehydrogenase (GluDH), constructs an L-glutamic acid recycling system, and provides coenzyme NADPH required in the reaction process by metabolizing glucose by the bacteria. Figure 1

[0010] Based on the above synthesis route, the present application provides a genetically engineered bacterium for producing (S)-2-amino-4-hydroxybutyric acid, which comprises genes encoding LAAD, HOA, TA and GluDH.

[0011] According to the above reaction principle, the inventors introduce genes encoding LAAD, HOA, TA and GluDH into a host bacterium to obtain a genetically engineered bacterium, which is used as a biological catalyst to convert substrates L-alanine, formaldehyde and ammonia into (S)-2-amino-4-hydroxybutyric acid.

[0012] In some embodiments, the LAAD comprises BpLAAD and PvLAAD, the amino acid sequence of BpLAAD is shown in SEQ ID NO. 1, and the amino acid sequence of PvLAAD is shown in SEQ ID NO. 2.

[0013] In the present application, BpLAAD is derived from Bordetella pertussis, Genbank number CAE41061.1, and PvLAAD is derived from Proteus vulgaris, Genbank number BAA90864.1. After obtaining the amino acid sequences of BpLAAD and PvLAAD, the inventors perform codon optimization according to the preference of Escherichia coli, and synthesize two optimized nucleotide sequences by a total synthesis method.

[0014] In some embodiments, the nucleotide sequence of BpLAAD is shown in SEQ ID NO. 9, and the nucleotide sequence of PvLAAD is shown in SEQ ID NO. 10.

[0015] In some embodiments, the HOA comprises PsHOA and EcHOA, the amino acid sequence of PsHOA is shown in SEQ ID NO. 3, and the amino acid sequence of EcHOA is shown in SEQ ID NO. 4.

[0016] ​In the present application, PsHOA is derived from Pseudomonas sp, Genbank No. CAA43227.1; EcHOA is derived from Escherichia coli, Genbank No. BAA13057.1. After obtaining the amino acid sequences of PsHOA and EcHOA, the inventors performed codon optimization according to the preference of Escherichia coli, and synthesized two optimized nucleotide sequences by total synthesis.

[0017] In some embodiments, the nucleotide sequence of PsHOA is as shown in SEQ ID NO. 11; the nucleotide sequence of EcHOA is as shown in SEQ ID NO. 12.

[0018] In some embodiments, the transaminase comprises BsTA and SsTA; the amino acid sequence of BsTA is as shown in SEQ ID NO. 5; the amino acid sequence of SsTA is as shown in SEQ ID NO. 6.

[0019] In the present application, BsTA is derived from Bacillus subtilis, Genbank No. QHF59561.1, Genbank No. SPC35538.1; SsTA is derived from Streptococcus salivarius, Genbank No. KXU58123.1. After obtaining the amino acid sequences of BsTA and SsTA, the inventors performed codon optimization according to the preference of Escherichia coli, and synthesized two optimized nucleotide sequences by total synthesis.

[0020] In some embodiments, the nucleotide sequence of BsTA is as shown in SEQ ID NO. 13; the nucleotide sequence of SsTA is as shown in SEQ ID NO. 14.

[0021] In some embodiments, the glutamate dehydrogenase comprises SsGluDH and PpGluDH; the amino acid sequence of SsGluDH is as shown in SEQ ID NO. 7; the amino acid sequence of PpGluDH is as shown in SEQ ID NO. 8.

[0022] In the present application, SsGluDH is derived from Streptomyces sp., Genbank No. WP_031092864.1; PpGluDH is derived from Palaeococcus pacificus, Genbank No. WP_048165779.1. After obtaining the amino acid sequences of SsGluDH and PpGluDH, the inventors performed codon optimization according to the preference of Escherichia coli, and synthesized two optimized nucleotide sequences by total synthesis.

[0023] In some embodiments, the nucleotide sequence of SsGluDH is shown as SEQ ID NO. 15; and the nucleotide sequence of PpGluDH is shown as SEQ ID NO. 16.

[0024] In some embodiments, the method for constructing the genetically engineered bacterium comprises: inserting the genes of LAAD, HOA, TA and GluDH into an expression vector to obtain a recombinant vector, and then introducing the recombinant vector into a host bacterium to obtain the genetically engineered bacterium.

[0025] In some embodiments, the expression vector comprises pCDFDuet-1 and pACYCDuet-1 plasmids.

[0026] In some embodiments, the host of the genetically engineered bacterium is Escherichia coli.

[0027] In some embodiments, the Escherichia coli is selected from any one of Escherichia coli BL21 (DE3), Escherichia coli DH5a and Escherichia coli XL-Blue.

[0028] In the present application, one enzyme is selected from each of the above-mentioned LAAD, HOA, TA and GluDH, and four-enzyme combination co-expression is performed. The way of introducing the plasmid carrying the coding genes of the above-mentioned enzymes can be that any two coding genes exist on the same plasmid, or that the four genes exist on different plasmids, or other introduction methods, which are not limited by the present application.

[0029] More preferably, the pCDFDuet-1 and pACYCDuet-1 double plasmids co-express the coding genes of the four enzymes; the pCDFDuet-1 loads LAAD and HOA, and the pACYCDuet-1 loads TA and GluDH.

[0030] The recombinant vector that can be constructed by using the above-mentioned vector can be pCDFDuet-BpLAAD-PsHOA, pCDFDuet-BpLAAD-EcHOA, pCDFDuet-PvLAAD-PsHOA, pCDFDuet-PvLAAD-EcHOA, pACYCDuet-BsTA-SsGluDH, pACYCDuet-BsTA-PpGluDH and pACYCDuet-SsTA-PpGluDH.

[0031] In some embodiments, the recombinant vector is pCDFDuet-BpLAAD-PsHOA and pACYCDuet-BsTA-PpGluDH.

[0032] In a second aspect, the present application further provides a whole cell catalyst containing the genetically engineered bacteria described above. The whole cell catalyst described above can be used to convert the substrates L-alanine, formaldehyde and ammonia into (S)-2-amino-4-hydroxybutyric acid.

[0033] In a third aspect, the present application provides a method for producing L-homoserine, which comprises adding the genetically engineered bacteria described above into a conversion system containing the substrates L-alanine, formaldehyde and ammonia for whole cell conversion to obtain (S)-2-amino-4-hydroxybutyric acid.

[0034] In some embodiments, the production system for whole cell conversion comprises: L-alanine 1-60 g / L, formaldehyde 1-20 g / L, ammonium acetate 5-60 g / L, L-glutamic acid 1-5 g / L, glucose 10-100 g / L, pyridoxal phosphate 0.02-0.1 g / L, and the amount of the engineered bacteria 1-20 g / L.

[0035] In some embodiments, the production system for whole cell conversion has a pH of 6.0-9.0.

[0036] In some embodiments, the production system for whole cell conversion has a temperature of 15-40℃.

[0037] In some embodiments, the production system for whole cell conversion has a reaction time of 6-24 h.

[0038] The present application has the following advantages:

[0039] (1) The present application provides a new synthetic route for (S)-2-amino-4-hydroxybutyric acid, which is synthesized from L-alanine, formaldehyde and ammonia as substrates, through L-amino acid deaminase, 4-hydroxy-2-ketopentanoic acid aldolase, transaminase and glutamate dehydrogenase to convert them into (S)-2-amino-4-hydroxybutyric acid.

[0040] (2) The present application constructs a genetically engineered bacteria capable of expressing L-amino acid deaminase, 4-hydroxy-2-ketopentanoic acid aldolase, transaminase and glutamate dehydrogenase, which can be used to produce (S)-2-amino-4-hydroxybutyric acid. This method has the advantages of high conversion efficiency, high space-time yield and low production cost.

[0041] (3) The present application screens L-amino acid deaminase, 4-hydroxy-2-ketopentanoic acid aldolase, transaminase and glutamate dehydrogenase from specific sources, which can improve the production efficiency of (S)-2-amino-4-hydroxybutyric acid. In addition, the selected enzymes also have the advantages of high activity and strong optical specificity. Therefore, the genetically engineered bacteria of the present application can be used to produce (S)-2-amino-4-hydroxybutyric acid with high production efficiency, green environmental protection and good industrial application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0042] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required to be used in the embodiments will be briefly introduced as follows. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0043] Figure 1 The synthesis route of (S)-2-amino-4-hydroxybutyric acid in the present application. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely as follows. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not mentioned by the manufacturers are all conventional products that can be purchased in the market.

[0045] The features and performances of the present application will be further described in detail below in combination with the embodiments.

[0046] 1. Selection of bacteria and plasmids

[0047] pCDFDuet-1 plasmid, pACYCDuet-1 plasmid, Escherichia coli BL21(DE3), Escherichia coli DH5α, Escherichia coli XL-Blue purchased from Novagen company.

[0048] 2. Selection of enzymes

[0049] (1) Selection of L-amino acid deaminase

[0050] The amino acid sequences of L-amino acid deaminases BpLAAD and PvLAAD were obtained from the NCBI database, and were codon-optimized according to the preference of Escherichia coli. Two nucleotide sequences were synthesized by a conventional operation of genetic engineering in a total synthesis method, and were shown as SEQ ID NO. 9 and SEQ ID NO. 10, respectively. The amino acid sequences encoding the enzymes were shown as SEQ ID NO. 1 and SEQ ID NO. 2, respectively. Enzymatic cleavage sites EcoRI and HindIII were added at both ends of the nucleotide sequences.

[0051] (2) Selection of 4-hydroxy-2-ketovalerate aldolase

[0052] The amino acid sequences of 4-hydroxy-2-ketovalerate aldolase PsHOA and EcHOA were obtained from the NCBI database, and codon optimization was performed according to the preference of Escherichia coli. Two nucleotide sequences were synthesized by conventional genetic engineering operations in a full synthesis method, as shown in SEQ ID NO. 11 and SEQ ID NO. 12. The amino acid sequences of the enzymes are shown in SEQ ID NO. 3 and SEQ ID NO. 4. Enzyme cleavage sites NdeI and XhoI were added at both ends of the nucleotide sequence.

[0053] (3) Selection of transaminase

[0054] The amino acid sequences of transaminases BsTA and SsTA were obtained from the NCBI database, and codon optimization was performed according to the preference of Escherichia coli. One nucleotide sequence was synthesized by conventional genetic engineering operations in a full synthesis method, as shown in SEQ ID NO. 13 and SEQ ID NO. 14. The amino acid sequences of the enzymes are shown in SEQ ID NO. 5 and SEQ ID NO. 6. Enzyme cleavage sites EcoRI and HindIII were added at both ends of the nucleotide sequence.

[0055] (4) Selection of glutamate dehydrogenase

[0056] The amino acid sequences of glutamate dehydrogenases SsGluDH and PpGluDH were obtained from the NCBI database, and codon optimization was performed according to the preference of Escherichia coli. One nucleotide sequence was synthesized by conventional genetic engineering operations in a full synthesis method, as shown in SEQ ID NO. 15 and SEQ ID NO. 16. The amino acid sequences of the enzymes are shown in SEQ ID NO. 7 and SEQ ID NO. 8. Enzyme cleavage sites NdeI and XhoI were added at both ends of the nucleotide sequence.

[0057] 3. Construction of four-enzyme co-expression system and cultivation of bacterial cells

[0058] Each of the selected L-amino acid deaminase, 4-hydroxy-2-ketovalerate aldolase, transaminase, and glutamate dehydrogenase was selected for four-enzyme combination co-expression. The coding genes of the four enzymes were co-expressed using pCDFDuet-1 and pACYCDuet-1 double plasmids; pCDFDuet-1 loaded L-amino acid deaminase and 4-hydroxy-2-ketovalerate aldolase, and pACYCDuet-1 loaded transaminase and glutamate dehydrogenase. After obtaining the co-expression recombinant plasmid, the two recombinant plasmids were simultaneously transformed into Escherichia coli BL21(DE3) competent cells, and positive transformants were selected using a plate containing streptomycin and chloramphenicol, thereby obtaining recombinant Escherichia coli. The obtained recombinant bacteria were inoculated into fresh liquid medium, induced and cultured, centrifuged, and wet bacterial cells were obtained.

[0059] 4. Whole cell transformation of L-alanine, formaldehyde, L-glutamic acid and ammonium acetate to produce L-homoserine

[0060] Transformation system: L-alanine concentration is 1-60 g / L, formaldehyde concentration is 1-20 g / L, ammonium acetate 5-60 g / L, L-glutamic acid 1-5 g / L, glucose 10-100 g / L, pyridoxal phosphate (PLP) 0.02-0.1 g / L, adjust pH to 6.0-9.0, fresh bacterial amount is 1-20 g / L, then at 15-40 °C, 200 rpm, transform for 6-24 h. After transformation, the product is identified as (S)-2-amino-4-hydroxybutyric acid. The yield of (S)-2-amino-4-hydroxybutyric acid is determined by liquid chromatography.

[0061] 5. Detection analysis of samples

[0062] Take 800 μL of the sample diluted by appropriate times, add 200 μL of OPA derivatization agent, mix well at 25 °C for 1 min, and immediately inject. The transformation liquid is analyzed by Shimadzu 2030C high performance liquid chromatograph (HPLC), and the chromatographic conditions are as follows: the mobile phase is methanol: water (V / V = 1:1), an Inertsustain C18 chromatographic column (4.6 x 250 mm, 5 μm) is used, the flow rate is 1 mL / min, the column temperature is 30 °C, the injection amount is 20 μL, and the detection wavelength is 333 nm.

[0063] Example 1

[0064] Construction of recombinant E. coli

[0065] The synthetic LAAD recombinant plasmid and pCDFDuet-1 vector were double digested by restriction enzymes EcoRI and HindIII, respectively, and the LAAD from different sources was ligated to the pCDFDuet-1 vector by T4 DNA ligase. The synthetic HOA recombinant plasmid and pCDFDuet-1 vector were double digested by restriction enzymes NdeI and XhoI, respectively, and the LAAD and HOA from different sources were combined two by two and ligated to the pCDFDuet-1 vector by T4 DNA ligase to obtain recombinant plasmid 1. The synthetic TA recombinant plasmid and pACYCDuet-1 vector were double digested by restriction enzymes EcoRI and HindIII, respectively, and the synthetic GluDH recombinant plasmid and pACYCDuet-1 vector were double digested by restriction enzymes NdeI and XhoI, respectively, and the TA and GluDH from different sources were combined two by two and ligated to the pACYCDuet-1 vector to obtain recombinant plasmid 2. The different recombinant plasmids 1 and recombinant plasmids 2 were combined and transformed into E. coli BL21 (DE3) competent cells to obtain recombinant E. coli.

[0066] Example 2

[0067] Induced culture of recombinant E. coli

[0068] The recombinant E. coli was inoculated into LB medium containing 50 mg / L streptomycin and 50 mg / L chloramphenicol, 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 a 2% inoculation amount, and cultured at 37°C and 200 rpm until the bacterial concentration reached OD600 of 0.7. Then, 0.5 mM IPTG was added, and the culture was induced at 28°C for 15 h. After centrifugation at 8000 rpm for 10 min, the supernatant was discarded, and the wet bacterial cells were washed twice with 0.9% physiological saline, centrifuged, and stored for use. 600nm

[0069] Example 3

[0070] Comparison of transformation abilities of various recombinant E. coli

[0071] The collected recombinant E. coli was resuspended in 50 mL of a system, and the final concentration of the cells was 20 g / L, the concentration of L-alanine was 60 g / L, the concentration of formaldehyde was 20 g / L, the concentration of ammonium acetate was 60 g / L, the concentration of L-glutamic acid was 5 g / L, the concentration of glucose was 100 g / L, the concentration of PLP was 0.1 g / L, and the pH was 8.0. The reaction was carried out at 30°C, the shaking speed was 200 rpm, and the transformation time was 24 h. After the transformation was completed, the yield of (S)-2-amino-4-hydroxybutyric acid was determined by HPLC, and the detection results of the yield and the space-time yield of various recombinant bacteria are shown in Table 1. ​

[0072] Table 1 Comparison of the production of (S)-2-amino-4-hydroxybutyric acid and the space-time yield of various recombinant bacteria

[0073]

[0074] As can be seen from the production data in Table 1, the abilities of the genetically engineered bacteria constructed from different sources of LAAD, HOA, TA and GluDH to produce (S)-2-amino-4-hydroxybutyric acid are different, in which E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH produces the highest amount of L-homoserine.

[0075] Example 4

[0076] According to the induction culture method described in Example 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of cells was 1 g / L, L-alanine was 1 g / L, formaldehyde was 1 g / L, ammonium acetate was 5 g / L, L-glutamic acid was 1 g / L, glucose was 10 g / L, PLP was 0.02 g / L, pH was 8.0, temperature was 30°C, the shaking speed was 200 rpm, and the conversion time was 24 h. The HPLC determination result was that the production of (S)-2-amino-4-hydroxybutyric acid was 1.3 g / L.

[0077] Example 5

[0078] According to the induction culture method described in Example 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of cells was 3 g / L, L-alanine was 8 g / L, formaldehyde was 3 g / L, ammonium acetate was 8 g / L, L-glutamic acid was 1 g / L, glucose was 30 g / L, PLP was 0.02 g / L, pH was 8.0, temperature was 35°C, the shaking speed was 200 rpm, and the conversion time was 24 h. The HPLC determination result was that the production of (S)-2-amino-4-hydroxybutyric acid was 10.4 g / L.

[0079] Example 6

[0080] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of cells was 5 g / L, L-alanine was 15 g / L, formaldehyde was 5 g / L, ammonium acetate was 15 g / L, L-glutamic acid was 2 g / L, glucose was 50 g / L, PLP was 0.02 g / L, pH was 8.0, the temperature was 30°C, the rotation speed of the shaker was 200 rpm, and the conversion time was 24 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 19.7 g / L.

[0081] Embodiment 7

[0082] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of cells was 5 g / L, L-alanine was 15 g / L, formaldehyde was 5 g / L, ammonium acetate was 15 g / L, L-glutamic acid was 2 g / L, glucose was 50 g / L, PLP was 0.02 g / L, pH was 8.0, the temperature was 30°C, the rotation speed of the shaker was 200 rpm, and the conversion time was 24 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 19.7 g / L.

[0083] Embodiment 8

[0084] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of cells was 5 g / L, L-alanine was 15 g / L, formaldehyde was 5 g / L, ammonium acetate was 15 g / L, L-glutamic acid was 2 g / L, glucose was 50 g / L, PLP was 0.02 g / L, pH was 8.0, the temperature was 30°C, the rotation speed of the shaker was 200 rpm, and the conversion time was 24 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 19.7 g / L.

[0085] Embodiment 9

[0086] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of the cells was 15 g / L, L-alanine was 43 g / L, formaldehyde was 15 g / L, ammonium acetate was 40 g / L, L-glutamic acid was 5 g / L, glucose was 80 g / L, PLP was 0.1 g / L, pH was 8.0, the temperature was 30°C, the shaking speed was 200 rpm, and the conversion time was 24 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 55.6 g / L.

[0087] Embodiment 10

[0088] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of the cells was 15 g / L, L-alanine was 43 g / L, formaldehyde was 15 g / L, ammonium acetate was 40 g / L, L-glutamic acid was 5 g / L, glucose was 80 g / L, PLP was 0.1 g / L, pH was 8.0, the temperature was 30°C, the shaking speed was 200 rpm, and the conversion time was 24 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 55.6 g / L.

[0089] Embodiment 11

[0090] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of the cells was 15 g / L, L-alanine was 43 g / L, formaldehyde was 15 g / L, ammonium acetate was 40 g / L, L-glutamic acid was 5 g / L, glucose was 80 g / L, PLP was 0.1 g / L, pH was 8.0, the temperature was 30°C, the shaking speed was 200 rpm, and the conversion time was 24 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 55.6 g / L.

[0091] Embodiment 12

[0092] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of cells was 20 g / L, L-alanine was 16 g / L, formaldehyde was 6 g / L, ammonium acetate was 15 g / L, L-glutamic acid was 2 g / L, glucose was 50 g / L, PLP was 0.1 g / L, pH was 8.0, the temperature was 30°C, the rotation speed of the shaker was 200 rpm, and the conversion time was 6 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 21.2 g / L.

[0093] Embodiment 13

[0094] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of cells was 20 g / L, L-alanine was 16 g / L, formaldehyde was 6 g / L, ammonium acetate was 15 g / L, L-glutamic acid was 2 g / L, glucose was 50 g / L, PLP was 0.1 g / L, pH was 8.0, the temperature was 30°C, the rotation speed of the shaker was 200 rpm, and the conversion time was 6 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 21.2 g / L.

[0095] Embodiment 14

[0096] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of cells was 20 g / L, L-alanine was 16 g / L, formaldehyde was 6 g / L, ammonium acetate was 15 g / L, L-glutamic acid was 2 g / L, glucose was 50 g / L, PLP was 0.1 g / L, pH was 8.0, the temperature was 30°C, the rotation speed of the shaker was 200 rpm, and the conversion time was 6 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 21.2 g / L.

[0097] Embodiment 15

[0098] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of cells was 10 g / L, L-alanine was 13 g / L, formaldehyde was 5 g / L, ammonium acetate was 12 g / L, L-glutamic acid was 2 g / L, glucose was 50 g / L, PLP was 0.05 g / L, pH was 6.0, the temperature was 35°C, the rotation speed of the shaker was 200 rpm, and the conversion time was 12 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 16.9 g / L.

[0099] Embodiment 16

[0100] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of cells was 10 g / L, L-alanine was 13 g / L, formaldehyde was 5 g / L, ammonium acetate was 12 g / L, L-glutamic acid was 2 g / L, glucose was 50 g / L, PLP was 0.05 g / L, pH was 6.0, the temperature was 35°C, the rotation speed of the shaker was 200 rpm, and the conversion time was 12 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 16.9 g / L.

[0101] Embodiment 17

[0102] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of cells was 10 g / L, L-alanine was 13 g / L, formaldehyde was 5 g / L, ammonium acetate was 12 g / L, L-glutamic acid was 2 g / L, glucose was 50 g / L, PLP was 0.05 g / L, pH was 6.0, the temperature was 35°C, the rotation speed of the shaker was 200 rpm, and the conversion time was 12 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 16.9 g / L.

[0103] Embodiment 18

[0104] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of cells was 7 g / L, L-alanine was 10 g / L, formaldehyde was 4 g / L, ammonium acetate was 9 g / L, L-glutamic acid was 2 g / L, glucose was 40 g / L, PLP was 0.05 g / L, pH was 8.5, the temperature was 35°C, the rotation speed of the shaker was 200 rpm, and the conversion time was 12 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 12.9 g / L.

[0105] Embodiment 19

[0106] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of cells was 7 g / L, L-alanine was 10 g / L, formaldehyde was 4 g / L, ammonium acetate was 9 g / L, L-glutamic acid was 2 g / L, glucose was 40 g / L, PLP was 0.05 g / L, pH was 8.5, the temperature was 35°C, the rotation speed of the shaker was 200 rpm, and the conversion time was 12 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 12.9 g / L.

[0107] Embodiment 20

[0108] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of cells was 7 g / L, L-alanine was 10 g / L, formaldehyde was 4 g / L, ammonium acetate was 9 g / L, L-glutamic acid was 2 g / L, glucose was 40 g / L, PLP was 0.05 g / L, pH was 8.5, the temperature was 35°C, the rotation speed of the shaker was 200 rpm, and the conversion time was 12 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 12.9 g / L.

[0109] Embodiment 21

[0110] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of the cells was 10 g / L, L-alanine was 28 g / L, formaldehyde was 10 g / L, ammonium acetate was 26 g / L, L-glutamic acid was 3 g / L, glucose was 70 g / L, PLP was 0.05 g / L, pH was 7.5, the temperature was 25°C, the rotation speed of the shaker was 200 rpm, and the conversion time was 24 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 36.5 g / L.

[0111] Example 22

[0112] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of the cells was 10 g / L, L-alanine was 28 g / L, formaldehyde was 10 g / L, ammonium acetate was 26 g / L, L-glutamic acid was 3 g / L, glucose was 70 g / L, PLP was 0.05 g / L, pH was 7.5, the temperature was 25°C, the rotation speed of the shaker was 200 rpm, and the conversion time was 24 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 36.5 g / L.

[0113] Comparative Example 1

[0114] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of the cells was 10 g / L, L-alanine was 28 g / L, formaldehyde was 10 g / L, ammonium acetate was 26 g / L, L-glutamic acid was 3 g / L, glucose was 70 g / L, PLP was 0.05 g / L, pH was 7.5, the temperature was 25°C, the rotation speed of the shaker was 200 rpm, and the conversion time was 24 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 36.5 g / L.

[0115] Comparative Example 2

[0116] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of cells was 25 g / L, L-alanine was 60 g / L, formaldehyde was 20 g / L, ammonium acetate was 55 g / L, L-glutamic acid was 5 g / L, glucose was 100 g / L, PLP was 0.1 g / L, pH was 5.5, the temperature was 35°C, the rotation speed of the shaker was 200 rpm, and the conversion time was 36 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 14.4 g / L.

[0117] Comparative Example 3

[0118] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of cells was 25 g / L, L-alanine was 60 g / L, formaldehyde was 20 g / L, ammonium acetate was 55 g / L, L-glutamic acid was 5 g / L, glucose was 100 g / L, PLP was 0.1 g / L, pH was 5.5, the temperature was 35°C, the rotation speed of the shaker was 200 rpm, and the conversion time was 36 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 14.4 g / L.

[0119] Comparative Example 4

[0120] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of cells was 25 g / L, L-alanine was 60 g / L, formaldehyde was 20 g / L, ammonium acetate was 55 g / L, L-glutamic acid was 5 g / L, glucose was 100 g / L, PLP was 0.1 g / L, pH was 5.5, the temperature was 35°C, the rotation speed of the shaker was 200 rpm, and the conversion time was 36 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 14.4 g / L.

[0121] Comparative Example 5

[0122] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and the cells were cultured in a 50 mL system with a cell wet weight of 30 g / L, L-alanine of 50 g / L, formaldehyde of 17 g / L, ammonium acetate of 45 g / L, L-glutamic acid of 5 g / L, glucose of 90 g / L, PLP of 0.15 g / L, pH 8.0, temperature 45°C, shaking speed 200 rpm, and conversion time 36 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 13.4 g / L.

[0123] Comparative Example 6

[0124] According to the induction culture method described in Embodiment 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-BpLAAD-PsHOA + pACYCDuet-BsTA-PpGluDH was completed, the bacterial cells were collected, and the cells were cultured in a 50 mL system with a cell wet weight of 30 g / L, L-alanine of 50 g / L, formaldehyde of 17 g / L, ammonium acetate of 45 g / L, L-glutamic acid of 5 g / L, glucose of 90 g / L, PLP of 0.15 g / L, pH 8.0, temperature 45°C, shaking speed 200 rpm, and conversion time 36 h. The HPLC determination result was that the yield of (S)-2-amino-4-hydroxybutyric acid was 13.4 g / L.

[0125] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A genetically engineered bacterium for producing (S)-2-amino-4-hydroxybutyric acid, characterized in that, The genome of the genetically engineered bacteria contains genes encoding L-amino acid deaminase, 4-hydroxy-2-ketovalerate aldolase, transaminase, and glutamate dehydrogenase. The L-amino acid deaminase is BpLAAD, and its amino acid sequence is shown in SEQ ID NO.1; The 4-hydroxy-2-ketovalerate aldolase is PsHOA, and its amino acid sequence is shown in SEQ ID NO.3; The transaminases are BsTA and SsTA; the amino acid sequence of BsTA is shown in SEQ ID NO.5; the amino acid sequence of SsTA is shown in SEQ ID NO.6; The glutamate dehydrogenase is PpGluDH, and its amino acid sequence is shown in SEQ ID NO.

8.

2. The genetically engineered bacteria according to claim 1, characterized in that, The nucleotide sequence of BpLAAD is shown in SEQ ID NO.

9.

3. The genetically engineered bacterium according to claim 1, characterized in that, The nucleotide sequence of the PsHOA is shown in SEQ ID NO.

11.

4. The genetically engineered bacterium according to claim 1, characterized in that, The nucleotide sequence of the BsTA is shown in SEQ ID NO. 13; the nucleotide sequence of the SsTA is shown in SEQ ID NO.

14.

5. The genetically engineered bacterium according to claim 1, characterized in that, The nucleotide sequence of the PpGluDH is shown in SEQ ID NO.

16.

6. The genetically engineered bacteria according to any one of claims 1-5, characterized in that, The method for constructing the genetically engineered bacteria includes: inserting the genes of L-amino acid deaminase, 4-hydroxy-2-ketovalerate aldolase, transaminase and glutamate dehydrogenase into an expression vector to obtain a recombinant vector, and then introducing the recombinant vector into a host bacterium to obtain the genetically engineered bacteria.

7. The genetically engineered bacterium according to claim 6, characterized in that, The expression vectors include pCDFDuet-1 and pACYCDuet-1 plasmids.

8. The genetically engineered bacteria according to claim 6, characterized in that, The host of the genetically engineered bacteria is Escherichia coli.

9. The genetically engineered bacterium according to claim 8, characterized in that, The Escherichia coli is selected from Escherichia coli BL21(DE3), Escherichia coli DH5α and Escherichia coli Any of the XL-Blue colors.

10. The genetically engineered bacterium according to claim 6, characterized in that, The genetically engineered bacteria were obtained by inserting BpLAAD, PsHOA, SsTA / BsTA and PpGluDH into the expression vector to obtain a recombinant vector, and then the recombinant vector was introduced into the host bacteria to cultivate and screen for genetically engineered bacteria that produce (S)-2-amino-4-hydroxybutyric acid. The recombinant expression vectors include: pCDFDuet-BpLAAD-PsHOA, pACYCDuet-BsTA-PpGluDH, and pACYCDuet-SsTA-PpGluDH.

11. A whole-cell catalyst, characterized in that, It contains the genetically engineered bacteria as described in any one of claims 1-10.

12. A method for producing (S)-2-amino-4-hydroxybutyric acid, characterized in that, The method includes adding the genetically engineered bacteria according to any one of claims 1-10 into a solution containing L-alanine, formaldehyde and ammonia for whole-cell transformation to obtain the (S)-2-amino-4-hydroxybutyric acid.

13. The method according to claim 12, characterized in that, The whole-cell transformation production system includes: L-alanine 1-60 g / L, formaldehyde 1-20 g / L, ammonium acetate 5-60 g / L, L-glutamic acid 1-5 g / L, glucose 10-100 g / L, pyridoxal phosphate 0.02-0.1 g / L, and engineered bacterial cell count 1-20 g / L.

14. The method according to claim 12, characterized in that, In the whole-cell transformation production system, the pH is 6.0-9.

0.

15. The method according to claim 12, characterized in that, The temperature of the whole-cell transformation production system is 15-40℃.

16. The method according to claim 12, characterized in that, The reaction time of the whole-cell transformation production system is 6-24 h.

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