CN113122563A - Method for constructing R-3-aminobutyric acid production strain - Google Patents

Method for constructing R-3-aminobutyric acid production strain Download PDF

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CN113122563A
CN113122563A CN202110435474.9A CN202110435474A CN113122563A CN 113122563 A CN113122563 A CN 113122563A CN 202110435474 A CN202110435474 A CN 202110435474A CN 113122563 A CN113122563 A CN 113122563A
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aminobutyric acid
plasmid
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phba
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范文超
高书良
丁鹏
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Luoyang Huarong Biotechnology Co ltd
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Abstract

The invention constructs an R-3-aminobutyric acid producing strain through genetic engineering, which is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 22076. The R-3-aminobutyric acid producing strain can directly produce R-3-aminobutyric acid through fermentation, and has a wide development and application prospect.

Description

Method for constructing R-3-aminobutyric acid production strain
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a genetic engineering producing strain capable of directly producing R-3-aminobutyric acid through fermentation and a construction method thereof.
Background
R-3-aminobutyric acid, CAS No. 3775-73-3, is an important intermediate for synthesizing new HIV resistant medicament Lutelvir, and the synthesis method of the compound mainly comprises a chemical synthesis method and an enzyme catalysis method at present. The chemical synthesis method has harsh reaction conditions, needs a large amount of chemical raw materials, has high cost and heavy metal pollution, and is not beneficial to industrial mass production. The enzyme catalysis method generally needs to use the chemical raw material butenoic acid as a substrate, and can prepare the R-3-aminobutyric acid through aspartase or mutants. Both processes are highly dependent on the production and supply of chemical raw materials.
The economic production of R-3-aminobutyric acid by microbial fermentation is a research direction worth exploring. To date, there has been no literature report on the production of R-3-aminobutyric acid by fermentation. Therefore, the construction of engineering bacteria capable of directly biosynthesizing R-3-aminobutyric acid is challenging, and can be a technical breakthrough going to industrial application.
Disclosure of Invention
In order to investigate the feasibility of fermentation for the production of R-3-aminobutyric acid, the inventors engineered the most commonly used genetically engineered bacterial host Escherichia coli by metabolic engineering, such that the Escherichia coli overexpresses the genes for the following enzymes: 3-ketothiolase (phbA) or acetoacetyl coenzyme synthetase (atoB), acetoacetyl coenzyme A reductase (phbB), 3-hydroxybutyryl coenzyme A dehydratase (3-hydroxybutyryl-CoA dehydratase), thioesterase (thiolesterase), or aminolyase or aspartate ammonia lyase (AspB) so as to realize biosynthesis of R-3-aminobutyric acid, and finally obtain an engineering bacterium capable of producing R-3-aminobutyric acid. Specifically, the present invention includes the following technical solutions.
A method for constructing R-3-aminobutyric acid producing bacteria comprises the following steps:
A. plasmid a, which was constructed for the synthesis of crotonic acid (also known as crotonic acid), contains the following genes for the enzymes:
3-ketothiolase (PhbA) or acetoacetyl-coenzyme synthetase (AtoB),
acetoacetyl-CoA reductase (PhbB),
crotonase (Crt, also known as enoyl hydratase, 3-hydroxybutyryl-CoA dehydratase), and
thioesterases (thioesterases, Ydii),
the plasmid A can construct a synthetic pathway of crotonic acid (crotonic acid) in microorganisms such as Escherichia coli;
B. constructing a plasmid B for expression of an amino lyase, preferably aspartate ammonia lyase (AspB);
C. co-transferring the plasmid A constructed in the step A and the plasmid B constructed in the step B into escherichia coli to obtain a positive clone which simultaneously expresses genes of 3-ketothiolase (phbA) or acetoacetyl-coenzyme synthetase (atoB), acetoacetyl-coenzyme A reductase (phbB), crotonase (Crt), thioesterase (Ydii) and amino lyase (preferably aspartate ammonia lyase AspB);
D. and D, screening out a strain for producing the R-3-aminobutyric acid from the positive clone constructed in the step C.
Wherein, the construction of the plasmid A can comprise the following steps:
a-1, connecting 3-ketothiolase gene phbA, acetoacetyl coenzyme A reductase gene phbB, crotonase gene crt and thioesterase gene ydiI in series by Ribosome Binding Site (RBS) sequences RBS to obtain an expression element phbA-phbB-crt-ydi for expressing the 4 genes;
and A-2, loading the expression element phbA-phbB-crt-ydiI constructed in the step A-1 between BamH1/HindIII sites of a vector pTrcHis2B and at the downstream of a Trc promoter to obtain a pTrcHis2B-ABcy plasmid, namely plasmid A.
The plasmid transformation in step C may be calcium chloride transformation or electric transformation, preferably electric transformation.
Preferably, the above-mentioned 3-ketothiolase (PhbA) gene is NCBI: J04987;
the acetoacetyl coenzyme A reductase (PhbB) gene is NCBI: L01112;
the crotonase is derived from Clostridium beijerinckii (Clostridium beijerinckii), and the gene number is GenBank: AGF54251.1(KEGG, csr: Cspa _ c 04330K 01715);
the Gene of the thioesterase (Ydii) is Gene ID: 946190.
Preferably, the nucleotide sequence of the expression element phbA-phbB-crt-ydiI is SEQ ID NO. 1.
In one embodiment, the construction of the plasmid B may comprise the following steps:
the aspartate ammonia lyase gene AspB was cloned between the NcoI/NotI sites of the vector pCDFDuet-1, downstream of the T7 promoter, to obtain the pCDF-AspB plasmid.
Preferably, the gene of the above-mentioned aspartate ammonia lyase AspB is SEQ ID NO:2, which is a (N142V, H188A) mutant gene reported in patent document CN110791493A, SEQ ID NO:4, also referred to as AspB-11-D4 in the examples herein.
Correspondingly, the wild-type aspartate ammonia lyase is also referred to as AspB1 in the examples herein, and the coding gene is SEQ ID NO 3, which is the codon optimized gene SEQ ID NO 2 reported in patent document CN 110791493A.
The second object of the present invention is to provide an R-3-aminobutyric acid producing bacterium constructed and screened by the above method.
The producing strain can be Escherichia coli, which is the most widely used host in genetic engineering, preferably Escherichia coli BL21(DE3) or Escherichia coli MG 1655.
The preferred R-3-aminobutyric acid producing bacteria are deposited in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and are Escherichia coli (Escherichia coli), and the preservation number is CGMCC No. 22076.
The third object of the present invention is to provide the use of the above-mentioned R-3-aminobutyric acid-producing bacterium such as CGMCC No.22076 for producing R-3-aminobutyric acid.
Specifically, R-3-aminobutyric acid can be produced by fermentation of the above-mentioned R-3-aminobutyric acid producing bacterium such as CGMCC No. 22076.
The medium used in the fermentation may be any medium suitable for growth and fermentation of Escherichia coli. For example, the fermentation medium composition is as follows: 125mM MOPS (pH7.4), 20g/L glycerol or glucose, 10g/L peptone, 5g/L yeast powder, 5mM calcium pantothenate, 2.78mM Na2HPO4，5mM(NH4)2SO4，30mM NH4Cl，5g/L CaCO3。
Preferably, the fermentation medium comprises the addition of 10 μm IPTG.
The fermentation temperature is preferably about 37 ℃.
The gene engineering bacteria constructed by the invention can produce R-3-aminobutyric acid through fermentation, are worthy of further development and utilization, and realize industrial production together.
The Latin chemical name of the R-3-aminobutyric acid high-yield genetic engineering bacteria constructed by the invention is Escherichia coli, the Chinese name is Escherichia coli, namely, Escherichia coli, and the Escherichia coli has been preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation date is 26 months at 3 years at 26 days at 2021, the preservation address is the institute of microbiology of China academy of sciences No. 3 of the North West Lu 1 of the Korean district in Beijing, and the preservation number is CGMCC No. 22076.
Drawings
FIG. 1 is a metabolic scheme of biosynthesis of R-3-aminobutyric acid constructed in the present invention.
FIG. 2 is a schematic structural diagram of crotonic acid expression element phbA-phbB-crt-ydiI.
FIG. 3 is a schematic diagram of the structure of a plasmid containing a crotonic acid synthesis pathway constructed in the present invention.
Detailed Description
The present invention has altered the intrinsic metabolic pathway of Escherichia coli, as shown in FIG. 1, Escherichia coli produces acetyl-CoA with glucose, acetyl-CoA is catalyzed by 3-ketothiolase (PhbA) or acetoacetyl-CoA synthetase (AtoB) to produce acetoacetyl-CoA, further catalyzed by acetoacetyl-CoA reductase (PhbB) to produce 3-hydroxybutyryl-CoA, further catalyzed by 3-hydroxybutyryl-CoA dehydratase, crotonyl-CoA catalyzed by thioesterase (Ydii) to produce crotonic acid, crotonic acid is catalyzed by amino lyase such as aspartic acid ammonia lyase (AspB) to produce 3-aminobutyric acid.
The genes of these overexpressed enzymes PhbA or AtoB, PhbB, Crt, Ydii, AspB can be cloned individually on a plasmid and then transformed into the same E.coli competent cell individually or simultaneously; it is also possible to clone more than two enzyme genes on a single plasmid and then to transfer them separately or simultaneously into the same E.coli competent cell.
Among them, useful plasmid vectors include pTrcHis2B, pCDFDuet-1, etc., but are not limited thereto.
In a preferred embodiment, genes of 4 enzymes, namely PhbA or AtoB, PhbB, Crt and Ydii, can be cloned on a plasmid to construct a crotonic acid synthesis pathway; and the amino lyase, such as AspB, was cloned separately on one plasmid, and then both plasmids were co-transformed into E.coli competent cells.
For example, genes of 4 enzymes, PhbA, PhbB, Crt and Ydii, can be connected in series by a ribosome binding site sequence rbs to obtain an expression element or expression cassette phbA-phbB-Crt-YdiI as shown in FIG. 2. For convenience of description, it may be abbreviated as ABcy.
It is to be understood that, when constructing expression elements expressing these 4 enzyme genes, the order among them is not fixed, and they may be crossed or reversed, so long as sufficient expression of each enzyme is successfully achieved.
For the sake of simplicity of description herein, an enzyme such as PhbA is sometimes used in combination with its gene (DNA) name encoding it, and one skilled in the art will understand that they represent different substances in different instances of description. Their meaning will be readily understood by those skilled in the art based on the context and context. For example, for PhbA, when used to describe the function or class of 3-ketothiolase, refer to proteins; when described as a gene, refers to the gene encoding the enzyme.
It has also been found that the enzymatic activity of amino-lyases such as the aspartate ammonia lyase AspB must be sufficiently strong to catalyze the conversion of crotonic acid to 3-aminobutyric acid, opening up a complete metabolic pathway. For example, the wild-type aspartate ammonia lyase AspB1 (coding gene SEQ ID NO:3) has insufficient enzymatic activity, and cannot realize the conversion of crotonic acid into 3-aminobutyric acid in Escherichia coli; however, the enzyme activity of the mutant AspB-11-D4 (encoding gene is SEQ ID NO:2) of the mutant (N142V and H188A) is obviously higher than that of the wild enzyme, so that the biosynthesis of the 3-aminobutyric acid is realized.
The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
The addition amount, content and concentration of various substances are referred to herein, wherein the percentage refers to the mass percentage unless otherwise specified.
Examples
Materials and methods
The whole gene synthesis, primer synthesis and sequencing in the examples were performed by Jinzhi Biotechnology, Inc., Suzhou.
The molecular biological experiments in the examples include plasmid construction, digestion, ligation, competent cell preparation, transformation, culture medium preparation, and the like, and are mainly performed with reference to "molecular cloning experimental manual" (third edition), sambrook, d.w. rasel (american), translation of huang peitang et al, scientific press, beijing, 2002). The specific experimental conditions can be determined by simple experiments if necessary.
PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the supplier of the plasmid or DNA template. If necessary, it can be adjusted by simple experiments.
The plasmid template pET24a-AspB1 comprising the AspB1 gene fragment and the plasmid template pET24a-AspB-11-D4 comprising the AspB-11-D4-R gene fragment were offered by Zhejiang Rui Biotechnology Ltd.
Culture medium:
LB culture medium: 5g/L yeast extract, 10g/L tryptone, 10g/L sodium chloride. (20 g/L agar powder was additionally added to LB solid medium.)
The shake flask fermentation method comprises the following steps:
fermentation medium: 125mM MOPS (pH7.4), 20g/L glycerol or glucose, 10g/L peptone, 5g/L yeast powder, 5mM calcium pantothenate, 2.78mM Na2HPO4，5mM(NH4)2SO4，30mM NH4Cl，5g/L CaCO3。
A fermentation step:
fermenting in a small shake flask:
1. single colony LB test tube culture overnight, 1% inoculation 15ml medium (25ml triangle bottle);
2. 10 μm IPTG, corresponding antibiotic, was added at 37 ℃ and 200rpm and cultured for 48 h.
HPLC detection method:
the crotonic acid detection method comprises the following steps: agilent 1260 high performance liquid chromatograph; an HPX-87H chromatographic column is adopted, a column incubator is 35 ℃, 5mM sulfuric acid is used as a mobile phase, the flow rate is 0.4ml/min, the detection wavelength is 210nm, the sample injection amount is 5 mu l, and the operation is carried out for 32 minutes.
The detection method of the R-3-aminobutyric acid comprises the following steps: agilent 1260 high performance liquid chromatograph; a chromatographic column: SB-Aq 4.6 x 250 x 5; flow rate: 1 mL/min; wavelength: 205 nm; column temperature: 30 ℃; sample introduction amount: 5 mu l of the solution; mobile phase: NaH2PO4(0.75%): acetonitrile 85: 15; diluting liquid: water; operating time: for 10 min.
Example 1: construction of crotonic acid synthetic pathway in Escherichia coli
According to the path design of FIG. 1, the 3-ketothiolase (PhbA) gene is selected as NCBI: J04987; the gene of acetoacetyl coenzyme A reductase (PhbB) is NCBI: L01112; the gene of crotonase (Crt) is GenBank: AGF 54251.1; the Gene for thioesterase (Ydii) is Gene ID: 946190.
The rbs sequence and the gene sequence are connected in series according to elements required by gene expression to form an expression element phbA-phbB-crt-ydiI, the structure of which is shown in figure 2, the nucleotide sequence of which is SEQ ID NO 1, sent to Jinzhi corporation of Suzhou for full-sequence DNA synthesis.
After DNA sequence synthesis, the vector pTrcHis2B was loaded between BamH1/HindIII sites and downstream of the Trc promoter to obtain pTrcHis2B-ABCy plasmid, as shown in FIG. 3. The four genes can be transcriptionally expressed under the control of the trc promoter.
Example 2: fermentation verification of crotonic acid engineering bacteria
The pTrcHis2B-ABcy plasmid constructed in example 1 was transferred into competent cells of E.coli host BL21(DE3) (Invitrogen corporation) using an electrotransformation method to obtain a pTrcHis2B-ABcy/BL21(DE3) engineered bacterium. Plate scribing, selecting single clone, carrying out shake flask fermentation for 24h, sampling and detecting, and determining the yield level of crotonic acid. Referring to Table 1, the expression level of the shake flask fermentation can reach more than 3 g/L.
Example 3: construction of R-aminobutyric acid engineering bacteria
According to the inventor's patent document CN110791493A, the wild-type aspartate ammonia lyase AspB1 gene sequence (SEQ ID NO:3 in this case) and its (N142V, H188A) mutant AspB-11-D4 gene sequence (SEQ ID NO:2 in this case) were selected, respectively, cloned between the NcoI/NotI sites of the vector pCDFDuet-1, and transcription expression was controlled using the T7 promoter on the vector. The plasmid pCDF-AspB1 and the plasmid pCDF-AspB-11-D4 for expressing aspartate ammonia lyase AspB were obtained, respectively. Comprises the following steps.
3.1 the following primer pair AspB1-F/AspB1-R was designed:
AspB1-F：aaaccatggATGAACACCGACGTTCGTATCG；
AspB1-R：cccgcggccgcTTTACGACCAGCGATACCCG。
the AspB1 gene fragment was amplified using the primers AspB1-F and AspB1-R, and the PCR reaction system included: 10ng of plasmid template pET24a-AspB1, complimentary to HuaRui Biotechnology Limited, Zhejiang, 50pmol of a pair of primers AspB1-F and AspB1-R, 1 XTaq buffer, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 7mM MgCl2，(0mM、0.05mM、0.1mM、0.15mM、0.2mM)MnCl22.5 units of Taq enzyme (fermentas).
The PCR reaction conditions are as follows: 5min at 95 ℃; 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 2 min/kbp; 10min at 72 ℃.
And (3) recovering gel (Axygen DNA gel recovery kit AP-GX-50) PCR amplified fragments, carrying out NcoI/NotI double digestion, and then connecting the fragments with the pCDFDuet-1 vector subjected to the same double digestion treatment to complete the construction of the pCDF-AspB1 plasmid.
3.2 the following primer pair AspB-11-D4-F/AspB-11-D4-R was designed:
AspB-11-D4-F：aaaccatggATGAACACCGACGTTCGTAT；
AspB-11-D4-R:cccgcggccgcTTATTTACGACCAGCGATAC。
the AspB-11-D4-R gene fragment was amplified using the primer pair AspB-11-D4-F/AspB-11-D4-R, and the PCR reaction system included: 10ng Zhejiang HuaRui biotechnology limited company of complimentary plasmid template pET24a-AspB-11-D4, 50pmol of a pair of primers AspB-11-D4-F/AspB-11-D4-R, 1 XTaq buffer, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 7mM MgCl2，(0mM、0.05mM、0.1mM、0.15mM、0.2mM)MnCl22.5 units of Taq enzyme (fermentas).
The PCR reaction conditions are as follows: 5min at 95 ℃; 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 2 min/kbp; 10min at 72 ℃.
And (3) recovering gel (Axygen DNA gel recovery kit AP-GX-50) PCR amplified fragments, carrying out NcoI/NotI double digestion, and then connecting the fragments with the pCDFDuet-1 vector subjected to the same double digestion treatment to complete the construction of the pCDF-AspB-11-D4 plasmid.
3.3 Using the electrotransformation method, the plasmid pCDF-AspB1 was co-transferred with the plasmid pTrcHis2B-ABcy obtained in example 1 into competent cells of E.coli host BL21(DE3) (Invitrogen Co.) to obtain the engineered strain pTrcHis2B-ABcy & pCDF-AspB1/BL21(DE 3).
The plasmid pCDF-AspB-11-D4 was co-transferred with the plasmid pTrcHis2B-ABcy obtained in example 1 into E.coli host BL21(DE3) (Invitrogen) competent cells using an electrotransformation method to obtain the engineered strain pTrcHis2B-ABcy & pCDF-AspB-11-D4/BL21(DE 3).
Example 4: fermentation verification of R-aminobutyric acid engineering bacteria
In a blank plasmid pTrcHis2B control strain pTrcHis2B/BL21(DE3) which does not express ABCy, a strain pTrcHis2B-ABcy/BL21(DE3) which expresses ABCy but does not express AspB, and a blank plasmid control strain pTrcHis2B which does not express ABCy and AspB&pCDFDuet-1/BL21(DE3), strain pTrcHis2B-ABcy expressing ABcy and wild type AspB1&pCDF-AspB1/BL21(DE3), strain pTrcHis2B-ABCy expressing ABCy and the mutant enzyme AspB-11-D4&A single colony was picked from an LB plate of pCDF-AspB-11-D4/BL21(DE3), inoculated into 5mL of LB liquid medium containing 50. mu.g/mL of kanamycin sulfate, and cultured at 37 ℃ and 250rpm overnight. Then 1 v/v% inoculated into a 25ml Erlenmeyer flask containing 15ml of medium, cultured at 37 ℃ and 250rpm for 2-3h to OD600At 0.6-0.8, 10 μm IPTG and 50 μ g/mL kanamycin sulfate were added and cultured at 37 ℃ and 200rpm for 48 hours. Then centrifuging at 4 deg.C and 10000rpm for 10mAnd in, detecting the yield levels of crotonic acid and R-3-aminobutyric acid in the supernatant of the fermentation liquor. The results are shown in Table 1.
TABLE 1 content of crotonic acid and R-3-aminobutyric acid in fermentation broths of different strains
Figure BDA0003032816910000081
The experimental results show that the metabolic engineering approach of the invention is feasible, and the overexpression of the four enzymes PhbA, PhbB, Crt and Ydii can realize the production of crotonic acid (crotonic acid) by an escherichia coli fermentation method; the over-expression of five enzymes PhbA, PhbB, Crt, Ydii and AspB can realize the 3-aminobutyric acid production by an escherichia coli fermentation method, but the precondition is that the enzyme activity of the aspartate ammonia lyase AspB must be large enough to get through the biosynthesis step of converting crotonic acid into 3-aminobutyric acid in escherichia coli, otherwise, the enzyme activity only stays at the biosynthesis stage of crotonic acid.
The strain pTrcHis2B-ABcy & pCDF-AspB-11-D4/BL21(DE3) constructed by the invention has the production capacity of R-3-aminobutyric acid, can realize the accumulation of R-3-aminobutyric acid in fermentation liquor, has an industrial development prospect, is preserved by strains, and has a preservation number of CGMCC No. 22076.
Sequence listing
<110> Luoyang Huarong Biotechnology Co., Ltd
<120> method for constructing R-3-aminobutyric acid producing bacteria
<130> SHPI2110068
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3201
<212> DNA
<213> Artificial sequence ()
<400> 1
gagctcagga ggtaaaaaaa catgaccgac gttgttattg tgagtgcggc ccgtacggcc 60
gtgggcaaat tcggtggcag tctggcgaaa attccggccc cagaactggg tgcggttgtt 120
atcaaggcgg cgctggaacg tgcgggtgtg aaaccggagc aagttagtga ggtgatcatg 180
ggccaagttc tgacggccgg tagtggtcag aatccagcgc gccaagccgc gatcaaagcg 240
ggtctcccag cgatggtgcc agcgatgacg atcaacaagg tttgtggcag cggcctcaaa 300
gccgttatgc tggcggcgaa cgcgattatg gccggtgatg ccgagatcgt tgttgccggc 360
ggccaagaaa acatgagtgc ggcgccgcat gttctgccgg gcagccgtga tggcttccgc 420
atgggcgacg cgaaactggt tgacaccatg attgtggacg gtctgtggga cgtgtacaac 480
cagtatcaca tgggtattac ggccgagaac gtggcgaaag agtatggcat cacccgcgaa 540
gcccaagatg agttcgccgt tggtagccag aacaaagccg aggcggccca gaaagccggc 600
aagttcgatg aggagatcgt gccagtgctg atcccacagc gcaagggcga tccggtggcg 660
ttcaaaacgg acgagtttgt tcgccaaggc gcgacgctgg atagtatgag cggtctgaaa 720
ccagccttcg acaaagccgg taccgtgacc gcggcgaatg ccagtggtct gaatgatggt 780
gccgcggcgg ttgttgttat gagcgccgcc aaggccaaag aactgggtct gaccccactg 840
gcgacgatca agagctacgc caatgccggc gttgatccga aagttatggg catgggcccg 900
gttccagcga gtaaacgtgc gctgagtcgt gcggaatgga ccccgcaaga tttggatctg 960
atggagatca atgaggcgtt tgcggcccaa gcgctcgccg ttcaccagca gatgggctgg 1020
gacaccagca aggtgaatgt gaatggtggc gccattgcga tcggtcaccc gattggtgcg 1080
agcggttgtc gtattctggt gacgctgctg cacgagatga agcgtcgcga tgcgaaaaaa 1140
ggtctcgcga gtctgtgcat tggtggcggc atgggtgttg cgctggccgt tgaacgtaaa 1200
taaaggaggt aaaaaaacat ggcccgcatt gcgctggtta ccggtggtat cggcggtatc 1260
ggcaccagca tctgcacccg tctggcgaaa gatggctgca cggtggttgc gaattgccac 1320
ccaagtgaag cggccgccgc ggaagagtgg aaacaagcgc gcgccgccga aggtttcgat 1380
attgcggtgt tcaccgcgga cgttagtagc ttcgatgata gcgcccgcat ggttcgcgaa 1440
atcacggaac aagttggccc gatcgatatt ctggtgaatt gcgcgggcat cacccgtgac 1500
aagaccttca agaagatgga gcaagcccat tgggaggccg tgatcaatgt gaacctcaac 1560
agcgtgttca acgtgacccg tcaagtttgg gatggcatgc tggaacgtgg ctttggccgc 1620
attatcaaca tcagcagcgt gaacggccag cgcggtcagt ttggtcaagc caactacagc 1680
gcggccaaag cgggcatgca cggtttcacg atggcgctgg cgcaagaagg tgccagcaaa 1740
ggtgtgaccg tgaacaccat cagtccgggc tacgttgaaa cggcgatgac gctggccatg 1800
aacgacgatg tgcgcaacag catcatcagt ggtatcccaa tgcgccgcat ggcccagccg 1860
aatgagatcg cggcggcgat tgcctttctg gccggtgacg aaagtggcta catgaccggt 1920
gcgaatctgc cggtgaacgg tggtctgttc atgcattaac tgcataaagg aggtaaaaaa 1980
acatggaatt aaagaatgtg attcttgaaa aagaaggaca tttagctatt gttacaatta 2040
atagaccaaa agcactaaat gcattgaact ctgaaacatt aaaggatatt gatgcagttt 2100
tagatgattt agaaaatgat actaatgtat atgccgttat agtaactggt gcaggagaaa 2160
aatcttttgt tgctggagca gatatttcag aaatgaaaga tcttaacgaa gaacaaggta 2220
aagaatttgg aattttagga aacaaagttt tcagaagatt agaaaaattg gataagccag 2280
ttattgcagc agtatcagga tttgctcttg gtggcggatg cgaacttgct atgtcatgtg 2340
atattagaat agcttcagtg aaagctaaat ttggtcaacc agaagctggt cttggaataa 2400
ctccaggatt tggtggaact caaagattag ctagaattgt agggcctgga aaggctaaag 2460
aattaatata tacttgtgac ttaataaatg ctgaagaagc atatagaata ggtttagtaa 2520
acaaaatagt tccattagaa agtttaatgg atgaagctaa agctatggct aataaaattg 2580
cagctaatgc tccaaaggca gttgcttact gtaaagatgc aattgacaga ggaatgcaag 2640
tagatataga tgcagctata ttaatagaag cagaagactt tggaaagtgt tttgcaacag 2700
aggatcaaac tgaaggaatg actgcatttt tagaaagaag agcagaaaag aatttccaaa 2760
ataaataata actttaataa ggagatatac atgatatgga aacgaaaaat caccctggaa 2820
gcactgaatg ctatggggga aggaaacatg gtgggattgc tggatattcg ctttgaacat 2880
attggtgatg acacccttga agcgacaatg ccagtagact cacggacaaa gcagcctttc 2940
gggttgctgc atggaggtgc atctgtggta ctggccgaaa gtatcggttc cgttgccggt 3000
tatttatgta ccgaaggtga gcaaaaagtg gttggtctgg aaatcaatgc taaccacgtc 3060
cgctcggcac gagaagggcg ggtgcgcggc gtatgcaaac cgttgcatct cggttcgcgt 3120
caccaggtct ggcagattga aatcttcgat gagaaagggc gtttgtgctg ttcgtcacga 3180
ttgacgaccg ctattttgtg a 3201
<210> 2
<211> 1407
<212> DNA
<213> Artificial sequence ()
<400> 2
atgaacaccg acgttcgtat cgaaaaagac ttcctgggtg aaaaagaaat cccgaaagac 60
gcttactacg gtgttcagac catccgtgct accgaaaact tcccgatcac cggttaccgt 120
atccacccgg aactgatcaa atctctgggt atcgttaaaa aatctgctgc tctggctaac 180
atggaagttg gtctgctgga caaagaagtt ggtcagtaca tcgttaaagc tgctgacgaa 240
gttatcgaag gtaaatggaa cgaccagttc atcgttgacc cgatccaggg tggtgctggt 300
acctctatca acatgaacgc taacgaagtt atcgctaacc gtgctctgga actgatgggt 360
gaagaaaaag gtaactactc taaaatctct ccgaactctc acgttaacat gtctcagtct 420
accgttgacg ctttcccgac cgctacccac atcgctgttc tgtctctgct gaaccagctg 480
atcgaaacca ccaaatacat gcagcaggaa ttcatgaaaa aagctgacga attcgctggt 540
gttatcaaaa tgggtcgttg cgccctgcag gacgctgttc cgatcctgct gggtcaggaa 600
ttcgaagctt acgctcgtgt tatcgctcgt gacatcgaac gtatcgctaa cacccgtaac 660
aacctgtacg acatcaacat gggtgctacc gctgttggta ccggtctgaa cgctgacccg 720
gaatacatct ctatcgttac cgaacacctg gctaaattct ctggtcaccc gctgcgttct 780
gctcagcacc tggttgacgc tacccagaac accgactgct acaccgaagt ttcttctgct 840
ctgaaagttt gcatgatcaa catgtctaaa atcgctaacg acctgcgtct gatggcttct 900
ggtccgcgtg ctggtctgtc tgaaatcgtt ctgccggctc gtcagccggg ttcttctatc 960
atcccgggtc tggttgctcc ggttatgccg gaagttatga accaggttgc tttccaggtt 1020
ttcggtaacg acctgaccat cacctctgct tctgaagctg gtcagttcga actgaacgtt 1080
atggaaccgg ttctgttctt caacctgatc cagtctatct ctatcatgac caacgttttc 1140
aaatctttca ccgaaaactg cctgaaaggt atcaaagcta acgaagaacg tatgaaagaa 1200
tacgttgaaa aatctatcgg tatcatcacc gctatcaacc cgcacgttgg ttacgaaacc 1260
gctgctaaac tggctcgtga agcttacctg accggtgaat ctatccgtga actgtgcatc 1320
aaatacggtg ttctgaccga agaacagctg aacgaaatcc tgaacccgta cgaaatgacc 1380
cacccgggta tcgctggtcg taaataa 1407
<210> 3
<211> 1404
<212> DNA
<213> Artificial sequence ()
<400> 3
atgaacaccg acgttcgtat cgaaaaagac ttcctgggtg aaaaagaaat cccgaaagac 60
gcttactacg gtgttcagac catccgtgct accgaaaact tcccgatcac cggttaccgt 120
atccacccgg aactgatcaa atctctgggt atcgttaaaa aatctgctgc tctggctaac 180
atggaagttg gtctgctgga caaagaagtt ggtcagtaca tcgttaaagc tgctgacgaa 240
gttatcgaag gtaaatggaa cgaccagttc atcgttgacc cgatccaggg tggtgctggt 300
acctctatca acatgaacgc taacgaagtt atcgctaacc gtgctctgga actgatgggt 360
gaagaaaaag gtaactactc taaaatctct ccgaactctc acgttaacat gtctcagtct 420
accaacgacg ctttcccgac cgctacccac atcgctgttc tgtctctgct gaaccagctg 480
atcgaaacca ccaaatacat gcagcaggaa ttcatgaaaa aagctgacga attcgctggt 540
gttatcaaaa tgggtcgttg ccacctgcag gacgctgttc cgatcctgct gggtcaggaa 600
ttcgaagctt acgctcgtgt tatcgctcgt gacatcgaac gtatcgctaa cacccgtaac 660
aacctgtacg acatcaacat gggtgctacc gctgttggta ccggtctgaa cgctgacccg 720
gaatacatct ctatcgttac cgaacacctg gctaaattct ctggtcaccc gctgcgttct 780
gctcagcacc tggttgacgc tacccagaac accgactgct acaccgaagt ttcttctgct 840
ctgaaagttt gcatgatcaa catgtctaaa atcgctaacg acctgcgtct gatggcttct 900
ggtccgcgtg ctggtctgtc tgaaatcgtt ctgccggctc gtcagccggg ttcttctatc 960
atcccgggtc tggttgctcc ggttatgccg gaagttatga accaggttgc tttccaggtt 1020
ttcggtaacg acctgaccat cacctctgct tctgaagctg gtcagttcga actgaacgtt 1080
atggaaccgg ttctgttctt caacctgatc cagtctatct ctatcatgac caacgttttc 1140
aaatctttca ccgaaaactg cctgaaaggt atcaaagcta acgaagaacg tatgaaagaa 1200
tacgttgaaa aatctatcgg tatcatcacc gctatcaacc cgcacgttgg ttacgaaacc 1260
gctgctaaac tggctcgtga agcttacctg accggtgaat ctatccgtga actgtgcatc 1320
aaatacggtg ttctgaccga agaacagctg aacgaaatcc tgaacccgta cgaaatgacc 1380
cacccgggta tcgctggtcg taaa 1404

Claims (10)

1. A method for constructing R-3-aminobutyric acid producing bacteria comprises the following steps:
A. constructing a plasmid a for effecting crotonic acid synthesis comprising genes of 3-ketothiolase (PhbA) or acetoacetyl-coa synthetase (AtoB), acetoacetyl-coa reductase (PhbB), crotonase (Crt) and thioesterase (YdiI);
B. constructing a plasmid B for expressing aspartate ammonia lyase (AspB, or amino lyase);
C. co-transferring the plasmid A constructed in the step A and the plasmid B constructed in the step B into escherichia coli to obtain positive clone;
D. and D, screening out a strain for producing the R-3-aminobutyric acid from the positive clone constructed in the step C.
2. The method of claim 1, wherein the construction of plasmid a comprises the steps of:
a-1, connecting 3-ketothiolase gene phbA, acetoacetyl coenzyme A reductase gene phbB, crotonase gene crt and thioesterase gene ydiI in series by Ribosome Binding Site (RBS) sequences RBS to obtain an expression element phbA-phbB-crt-ydi for expressing the 4 genes;
and A-2, loading the expression element phbA-phbB-crt-ydiI constructed in the step A-1 between BamH1/HindIII sites of a vector pTrcHis2B and at the downstream of a Trc promoter to obtain a pTrcHis2B-ABcy plasmid, namely plasmid A.
3. The method of claim 1, wherein the nucleotide sequence of the expression element phbA-phbB-crt-ydiI is SEQ ID NO 1.
4. The method of claim 1, wherein the construction of plasmid B comprises the steps of:
the aspartate ammonia lyase gene AspB was cloned between the NcoI/NotI sites of the vector pCDFDuet-1, downstream of the T7 promoter, to obtain the pCDF-AspB plasmid.
5. The method according to claim 4, wherein the amino acid sequence of the aspartate ammonia lyase AspB is SEQ ID NO:2 (SEQ ID NO:3 reported in patent document CN 110791493A), and the coding gene thereof is SEQ ID NO:3 (SEQ ID NO:4 reported in patent document CN 110791493A).
6. An R-3-aminobutyric acid producing bacterium constructed by the method according to any one of claims 1 to 5.
7. The R-3-aminobutyric acid production bacteria as set forth in claim 6, wherein said R-3-aminobutyric acid production bacteria is Escherichia coli (Escherichia coli) deposited in China general microbiological culture Collection center with a collection number of CGMCC No. 22076.
8. Use of the R-3-aminobutyric acid producing bacteria of claim 7 for producing R-3-aminobutyric acid.
9. The use according to claim 8, wherein R-3-aminobutyric acid is produced by fermentation of the R-3-aminobutyric acid producing bacterium CGMCC No.22076 according to claim 7.
10. Use according to claim 6, wherein the fermentation medium consists of: 125mM MOPS (pH7.4), 20g/L glycerol or glucose, 10g/L peptone, 5g/L yeast powder, 5mM calcium pantothenate, 2.78mM Na2HPO4，5mM(NH4)2SO4，30mM NH4Cl，5g/L CaCO3。
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