CN113122563B - Method for constructing R-3-aminobutyric acid producing bacteria - Google Patents

Method for constructing R-3-aminobutyric acid producing bacteria Download PDF

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CN113122563B
CN113122563B CN202110435474.9A CN202110435474A CN113122563B CN 113122563 B CN113122563 B CN 113122563B CN 202110435474 A CN202110435474 A CN 202110435474A CN 113122563 B CN113122563 B CN 113122563B
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aminobutyric acid
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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 China general microbiological culture Collection center (CGMCC) No.22076. The R-3-aminobutyric acid producing strain can directly produce R-3-aminobutyric acid through fermentation, and has wide development and application prospects.

Description

Method for constructing R-3-aminobutyric acid producing bacteria
Technical Field
The invention belongs to the field of genetic engineering, and in particular relates to a genetic engineering production strain capable of directly producing R-3-aminobutyric acid through fermentation and a construction method thereof.
Background
R-3-aminobutyric acid, CAS number 3775-73-3, is an important intermediate for synthesizing anti-HIV drug dolutegravir, and the synthesis method of the compound at present mainly comprises a chemical synthesis method and an enzyme catalysis method. The chemical synthesis method has harsh reaction conditions, needs to use a large amount of chemical raw materials, has higher cost and heavy metal pollution, and is not beneficial to industrialized mass production. In the enzyme catalysis method, the chemical raw material butenoic acid is generally required to be used as a substrate, and R-3-aminobutyric acid can be prepared by aspartase or mutants. Both processes have a high degree of dependence on the production and supply of chemical raw materials.
The production of R-3-aminobutyric acid by an economical microbial fermentation method is a research direction worthy of exploration. To date, there has been no report on the production of R-3-aminobutyric acid by fermentation. Therefore, the construction of engineering bacteria capable of directly biosynthesis of R-3-aminobutyric acid is challenging, and the engineering bacteria are a technological breakthrough going to industrial application.
Disclosure of Invention
To investigate the feasibility of fermentation to produce R-3-aminobutyric acid, the inventors modified the most industrially used genetically engineered host E.coli by metabolic engineering such that E.coli overexpressed the following enzyme genes: 3-ketosulfur lyase (phbA) or acetoacetyl-CoA synthetase (atoB), acetoacetyl-CoA reductase (PhbB), 3-hydroxybutyryl-CoA dehydratase (3-hydroxybutyryl-CoA dehydrase), thioesterase (thioase), amino lyase or aspartate ammonia lyase (AspB) to effect biosynthesis of R-3-aminobutyric acid, and finally obtaining an engineering bacterium capable of producing R-3-aminobutyric acid. Specifically, the invention comprises the following technical scheme.
A method of constructing an R-3-aminobutyric acid producing bacterium comprising the steps of:
A. plasmid a was constructed for achieving crotonic acid (also known as butenoic acid) synthesis, comprising the genes for the following enzymes:
3-ketosulfur lyase (PhbA) or acetoacetyl-coenzyme synthetase (AtoB),
acetoacetyl-CoA reductase (PhbB),
crotonase (Crt, also known as enoyl hydratase, 3-hydroxybutyryl-CoA dehydratase, 3-hydroxybutyryl-CoA dehydrase), and
thioesterases (YdiI),
the plasmid A can construct a crotonic acid (butenoic acid) synthesis pathway in microorganisms such as E.coli;
B. constructing a plasmid B for expressing an amino lyase, preferably an 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 positive clones simultaneously expressing genes of 3-ketosulfur lyase (phbA) or acetoacetyl-coenzyme synthase (atoB), acetoacetyl-coenzyme A reductase (PhbB), crotonase (Crt), thioesterase (Ydi) and amino-lyase (preferably aspartate ammonia lyase AspB);
D. from the positive clones constructed in step C, strains producing R-3-aminobutyric acid were selected.
Wherein, the construction of the plasmid A can comprise the following steps:
a-1. Connecting 3-ketosulfur lyase gene phbA, acetoacetyl-CoA reductase gene phbB, crotonase gene crt and thioesterase gene ydiI in series by ribosome binding site (ribosomebinding site, RBS) sequence RBS to obtain expression element phbA-phbB-crt-ydiI for expressing these 4 genes;
a-2. The expression element phbA-phbB-crt-ydiI constructed in step A-1 was loaded between BamH1/HindIII sites of vector pTrcHis2B, downstream of Trc promoter, to obtain pTrcHis2B-ABcy plasmid, plasmid A.
The plasmid transfer in step C above may be a calcium chloride transformation or electrotransformation, preferably electrotransformation.
Preferably, the 3-ketosulfur lyase (PhbA) gene is NCBI J04987;
the acetoacetyl-CoA reductase (PhbB) gene is NCBI:L01112;
the crotonase is derived from Clostridium bellianum (Clostridium beijerinckii) with the gene number of GenBank: AGF54251.1 (KEGG, csr: cpa_c 04330K 01715);
the Gene of 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 steps of:
the aspartic acid ammonia lyase gene AspB was cloned between the NcoI/NotI sites of vector pcdfdurt-1, downstream of the T7 promoter, to obtain pCDF-AspB plasmid.
Preferably, the gene of the above aspartic acid ammonia lyase AspB is SEQ ID NO:2, which is the mutant gene SEQ ID NO:4 reported in patent document CN110791493A (N142V, H188A), also referred to herein as AspB-11-D4.
Correspondingly, the wild-type aspartate ammonia lyase, also referred to as AspB1 in the examples herein, has its coding gene of SEQ ID No. 3 and the codon optimized gene of SEQ ID No.2 reported in patent document CN110791493 a.
The second object of the present invention is to provide an R-3-aminobutyric acid producing bacterium constructed and selected by the above method.
The producing strain can be the most widely used host in genetic engineering, namely escherichia coli, preferably escherichia coli BL21 (DE 3) or escherichia coli MG1655.
The preferred R-3-aminobutyric acid producing bacteria are preserved in China general microbiological culture Collection center, which is Escherichia coli (CGMCC No. 22076).
A third object of the present invention is to provide the use of the above-mentioned R-3-aminobutyric acid producing bacteria 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 bacteria such as CGMCC No.22076.
The medium used in the fermentation may be any medium suitable for growth and fermentation of E.coli. For example, the fermentation medium composition is as follows: 125mM MOPS (pH 7.4), 20g/L glycerol or glucose, 10g/LPeptone, 5g/L yeast powder, 5mM calcium pantothenate, 2.78mM Na 2 HPO 4 ，5mM(NH 4 ) 2 SO 4 ，30mM NH 4 Cl，5g/L CaCO 3 。
Preferably, the fermentation medium contains 10 μm IPTG.
The fermentation temperature is preferably about 37 ℃.
The genetically engineered bacterium constructed by the invention can be used for producing R-3-aminobutyric acid through fermentation, and is worthy of further development and utilization, and industrial production is realized together.
The Latin chemical name of the high-yield R-3-aminobutyric acid genetic engineering bacteria constructed by the invention is Escherichia coli, the Chinese name is Escherichia coli, namely Escherichia coli, which is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, the preservation date is 2021, 3 months and 26 days, and the preservation address is China center for microorganisms with the preservation number of CGMCC No.22076, national institute of sciences No. 3, north Chen West road No. 1 in the Korean region of Beijing city.
Drawings
FIG. 1 is a metabolic scheme of the biosynthesis of R-3-aminobutyric acid constructed according to the present invention.
FIG. 2 is a schematic diagram of the structure of the crotonic acid expression element phbA-phbB-crt-ydiI.
FIG. 3 is a schematic diagram of the structure of a plasmid comprising the crotonic acid synthesis pathway constructed in accordance with the present invention.
Detailed Description
The invention changes the internal metabolic pathway of the escherichia coli, as shown in figure 1, the escherichia coli can generate acetyl coenzyme A by glucose, the acetyl coenzyme A is catalyzed by 3-ketosulfur lyase (PhbA) or acetoacetyl coenzyme synthetase (AtoB) to generate acetoacetyl coenzyme A, the acetoacetyl coenzyme A is further catalyzed by acetoacetyl coenzyme A reductase (PhbB) to generate 3-hydroxybutyryl coenzyme A, the crotonyl coenzyme A is further catalyzed by 3-hydroxybutyryl coenzyme A dehydratase (Crt), the crotonyl coenzyme A is catalyzed by thioesterase (Ydi) to generate crotonic acid, and the crotonic acid is catalyzed by amino lyase (AspB) to generate 3-aminobutyric acid.
Genes of these overexpressed enzymes PhbA or AtoB, phbB, crt, ydiI, aspB can be cloned separately on a plasmid and then transferred separately, or simultaneously, into the same competent cell of e.coli; it is also possible to clone genes for more than two enzymes on one plasmid and then to transfer them separately or simultaneously into the same E.coli competent cell.
Among them, available plasmid vectors include pTrcHis2B, pCDFDuet-1 and the like, but are not limited thereto.
In a preferred embodiment, the genes for the 4 enzymes PhbA or AtoB, phbB, crt, ydiI can be cloned on a plasmid to construct the crotonic acid synthesis pathway; and an amino lyase such as AspB is cloned separately on one plasmid, and then both plasmids are co-transferred into competent cells of e.coli.
For example, the genes for the 4 enzymes PhbA, phbB, crt, ydiI may be concatenated with the ribosome binding site sequence rbs to provide an expression element or cassette phbA-phbB-crt-ydiI as shown in FIG. 2. For convenience of description, it may be abbreviated as ABcy.
It will be appreciated that in constructing expression elements for expressing these 4 enzyme genes, the ordering between them is not constant, and they may be crossed over and reversed, so long as adequate expression of each enzyme is achieved successfully.
In this context, for ease of description, certain enzymes such as PhbA are sometimes used in combination with their coding gene (DNA) names, and those skilled in the art will appreciate that they represent different substances in different descriptive contexts. Those skilled in the art will readily understand their meaning depending on the context and context. For example, for PhbA, when used to describe the function or class of 3-ketosulfur lyase, refers to a protein; when described as a gene, it refers to the gene encoding the enzyme.
It has also been found in research that the enzyme activity of amino-lyase, such as asparate ammonia lyase AspB, must be strong enough to catalyze the conversion of crotonic acid to 3-aminobutyric acid, opening up a complete metabolic pathway. For example, the wild-type aspartic acid ammonia lyase AspB1 (encoding gene SEQ ID NO: 3) has insufficient enzyme activity to effect the conversion of crotonic acid to 3-aminobutyric acid in E.coli; however, the enzyme activity of the (N142V, H188A) mutant AspB-11-D4 (the coding gene is SEQ ID NO: 2) is obviously higher than that of the wild enzyme, so that the biosynthesis of 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 of the present invention and are not intended to limit the scope of the present invention.
The amounts, amounts and concentrations of various substances are referred to herein, wherein the percentages refer to percentages by mass unless otherwise specified.
Examples
Materials and methods
The whole gene synthesis, primer synthesis and sequencing in the examples were all performed by su Jin Weizhi biotechnology, inc.
Examples of molecular biology experiments include plasmid construction, digestion, ligation, competent cell preparation, transformation, medium preparation, etc., and are mainly described in "molecular cloning Experimental guidelines (third edition), J.Sam Broker, D.W. Lassel (America) code, huang Peitang, et al, scientific Press, beijing, 2002). The specific experimental conditions can be determined by simple experiments, if necessary.
The PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the plasmid or DNA template suppliers. Can be adjusted if necessary by simple tests.
Plasmid templates pET24a-AspB1 comprising the AspB1 gene fragment and plasmid template pET24a-AspB-11-D4 comprising the AspB-11-D4-R gene fragment were offered by Zhejiang Huai Biotechnology Co.
Culture medium:
LB medium: 5g/L yeast extract, 10g/L tryptone, 10g/L sodium chloride. (LB solid Medium additionally 20g/L agar powder.)
The shake flask fermentation method comprises the following steps:
fermentation medium: 125mM MOPS (pH 7.4), 20g/L glycerol or glucose, 10g/L peptone, 5g/L yeast powder, 5mM calcium pantothenate, 2.78mM Na 2 HPO 4 ，5mM(NH 4 ) 2 SO 4 ，30mM NH 4 Cl，5g/L CaCO 3 。
Fermentation:
and (3) fermenting in a small shaking bottle:
1. single colony LB test tube culture overnight, 1% inoculation in 15ml medium (25 ml triangle bottle);
2. 10 μm IPTG was added and the corresponding antibiotic was incubated at 37℃and 200rpm for 48h.
HPLC detection method:
the crotonic acid detection method comprises the following steps: agilent 1260 high performance liquid chromatograph; the sample is run for 32 minutes by adopting an HPX-87H chromatographic column, a column temperature box at 35 ℃ and 5mM sulfuric acid as a mobile phase, the flow rate is 0.4ml/min, the detection wavelength is 210nm, the sample injection amount is 5 μl.
The detection method of the R-3-aminobutyric acid comprises the following steps: agilent 1260 high performance liquid chromatograph; chromatographic column: SB-Aq 4.6x250 x 5; flow rate: 1mL/min; wavelength: 205nm; column temperature: 30 ℃; sample injection amount: 5 μl; mobile phase: naH (NaH) 2 PO 4 (0.75%): acetonitrile=85: 15; dilution liquid: water; run time: and 10min.
Example 1: construction of crotonic acid synthetic pathway in E.coli
According to the pathway design of FIG. 1, the 3-ketosulfur lyase (PhbA) gene was selected as NCBI J04987; the acetoacetyl-CoA reductase (PhbB) gene is NCBI: L01112; the crotonase (Crt) gene is GenBank AGF54251.1; the thioesterase (YdiI) Gene is Gene ID 946190.
According to the elements required for gene expression, the rbs sequence and the gene sequence are connected in series 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, and the complete sequence DNA synthesis is carried out by the company Jin Weizhi of Suzhou.
After DNA sequence synthesis, the vector pTrcHis2B was loaded between BamH1/HindIII sites of pTrcHis2B, downstream of the Trc promoter, to obtain pTrcHis2B-ABcy plasmid, as shown in FIG. 3. The four genes can be expressed transcriptionally 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 (DE 3) (Invitrogen) using an electrotransformation method to obtain pTrcHis2B-ABcy/BL21 (DE 3) engineering bacteria. And (3) streaking a flat plate, picking a monoclonal, carrying out shake flask fermentation for 24 hours, sampling and detecting, and determining the crotonic acid yield level. Referring to Table 1, shake flask fermentation expression levels can be above 3 g/L.
Example 3: construction of R-aminobutyric acid engineering bacteria
According to the inventors' patent document CN110791493A, the wild-type aspartic acid ammonia lyase gene sequence (SEQ ID NO:3 herein) and its (N142V, H188A) mutant AspB-11-D4 gene sequence (SEQ ID NO:2 herein) were selected, cloned between NcoI/NotI sites of vector pCDFDuet-1, respectively, and transcriptional expression was controlled using the T7 promoter on the vector. Plasmids pCDF-AspB1 and pCDF-AspB-11-D4 for expressing aspartate ammonia lyase were obtained, respectively. Comprises the following steps.
3.1 the following primer pairs AspB1-F/AspB1-R were designed:
AspB1-F：aaaccatggATGAACACCGACGTTCGTATCG；
AspB1-R：cccgcggccgcTTTACGACCAGCGATACCCG。
the AspB1 gene fragment was amplified using primers AspB1-F and AspB1-R, and the PCR reaction system included: 10ng Zhejiang HuaRui Biotechnology Co., ltd. Benefit plasmid template pET24a-AspB1, 50pmol a pair of primers AspB1-F and AspB1-R,1×Taq buffer,0.2mM dGTP,0.2mM dATP,1mM dCTP,1mM dTTP,7mM MgCl 2 ，(0mM、0.05mM、0.1mM、0.15mM、0.2mM)MnCl 2 2.5 units of Taq enzyme (ferrons).
The PCR reaction conditions were: 95 ℃ for 5min;94℃for 30s,55℃for 30s,72℃for 2min/kbp,30 cycles; and at 72℃for 10min.
And (3) carrying out PCR amplification fragments by using a gel recovery (Axygen DNA gel recovery kit AP-GX-50), carrying out NcoI/NotI double digestion, and connecting the fragments with the pCDFDuet-1 vector subjected to the same double digestion treatment to complete construction of the pCDF-AspB1 plasmid.
3.2 the following primer pairs AspB-11-D4-F/AspB-11-D4-R were designed:
AspB-11-D4-F：aaaccatggATGAACACCGACGTTCGTAT；
AspB-11-D4-R:cccgcggccgcTTATTTACGACCAGCGATAC。
the gene fragment of AspB-11-D4-R was amplified using the primer pair AspB-11-D4-F/AspB-11-D4-R, and the PCR reaction system comprised: 10ng Zhejiang HuaRui biotechnology Co., ltd. Benefit plasmid template pET24a-AspB-11-D4, 50pmol a pair of primers AspB-11-D4-F/AspB-11-D4-R,1×Taq buffer,0.2mM dGTP,0.2mM dATP,1mM dCTP,1mM dTTP,7mM MgCl 2 ，(0mM、0.05mM、0.1mM、0.15mM、0.2mM)MnCl 2 2.5 units of Taq enzyme (ferrons).
The PCR reaction conditions were: 95 ℃ for 5min;94℃for 30s,55℃for 30s,72℃for 2min/kbp,30 cycles; and at 72℃for 10min.
And (3) carrying out PCR amplification fragments by using a gel recovery (Axygen DNA gel recovery kit AP-GX-50), carrying out NcoI/NotI double digestion, and connecting the fragments with the pCDFDuet-1 vector subjected to the same double digestion treatment to complete construction of the pCDF-AspB-11-D4 plasmid.
3.3 Using the electrotransformation method, the plasmid pCDF-AspB1 was co-transformed with the plasmid pTrcHis2B-ABcy obtained in example 1 into competent cells of E.coli host BL21 (DE 3) (Invitrogen Co.) to obtain the engineering strain pTrcHis2B-ABcy & pCDF-AspB1/BL21 (DE 3).
Using the electrotransformation method, the plasmid pCDF-AspB-11-D4 was co-transformed into competent cells of E.coli host BL21 (DE 3) (Invitrogen) together with the plasmid pTrcHis2B-ABcy obtained in example 1 to obtain the engineering strain pTrcHis2B-ABcy & pCDF-AspB-11-D4/BL21 (DE 3).
Example 4: fermentation verification of R-aminobutyric acid engineering bacteria
The control strain pTrcHis2B was obtained from the blank plasmid pTrcHis2B control strain pTrcHis2B/BL21 (DE 3) which did not express ABcy, the strain pTrcHis2B-ABcy/BL21 (DE 3) which expressed ABcy but did not express aspB, and the blank plasmid control strain pTrcHis2B which did not express ABcy and aspB&pCDFDuet-1/BL21 (DE 3), strain pTrcHis2B-ABcy expressing ABcy and wild-type AspB1&pCDF-AspB1/BL21 (DE 3), the strain pTrcHis2B-ABcy expressing ABcy and the mutant enzyme AspB-11-D4&Single colonies were picked on LB plates of pCDF-AspB-11-D4/BL21 (DE 3), inoculated to 5mL of a culture medium containing 50. Mu.g/mL of kana sulfateThe culture was carried out overnight at 37℃and 250rpm in LB liquid medium for the mycin. Then 1v/v% inoculated in a 25ml triangular flask containing 15ml of medium, cultured at 37℃and 250rpm for 2-3 hours to OD 600 At 0.6-0.8, 10 μm IPTG was added, 50. Mu.g/mL kanamycin sulfate, and the mixture was incubated at 37℃and 200rpm for 48 hours. Then, the mixture was centrifuged at 10000rpm at 4℃for 10 minutes to measure the production levels of crotonic acid and R-3-aminobutyric acid in the supernatant of the fermentation broth. The results are shown in Table 1.
TABLE 1 content of Babbic acid and R-3-aminobutyric acid in fermentation broths of different strains
The experimental result shows that the metabolic engineering approach is feasible, and the overexpression of four enzymes PhbA, phbB, crt, ydiI can realize the production of crotonic acid (butenoic acid) by an escherichia coli fermentation method; the over-expression of five enzymes PhbA, phbB, crt, ydiI, aspB can realize the production of 3-aminobutyric acid by an escherichia coli fermentation method, but the premise is that the enzyme activity of aspartic acid ammonia lyase AspB is large enough to open the biosynthesis step of converting crotonic acid into 3-aminobutyric acid in escherichia coli, otherwise, the process can only stay in the biosynthesis stage of crotonic acid.
The strain pTrcHis2B-ABcy & pCDF-AspB-11-D4/BL21 (DE 3) 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 industrial development prospect, and has been subjected to strain preservation with the preservation number of CGMCC No.22076.
Sequence listing
<110> Luoyang Hua Rong Biotechnology Co., ltd
<120> method for constructing R-3-aminobutyric acid producing bacteria
<130> SHPI2110068
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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
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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
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<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
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atgaacaccg acgttcgtat cgaaaaagac ttcctgggtg aaaaagaaat cccgaaagac 60
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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 (7)

1. A method of constructing an R-3-aminobutyric acid producing bacterium comprising the steps of:
A. constructing a plasmid A for realizing crotonic acid synthesis, which comprises genes of 3-ketosulfur lyase PhbA, acetoacetyl-CoA reductase PhbB, crotonase Crt and thioesterase Ydi;
B. constructing a plasmid B for expressing an aspartate ammonia lyase AspB, wherein the nucleotide sequence of a coding gene of the aspartate ammonia lyase AspB is SEQ ID NO. 2;
C. 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 clones;
D. screening the positive clone constructed in the step C for a strain producing R-3-aminobutyric acid, wherein
The construction of the plasmid A comprises the following steps:
a-1, connecting 3-ketosulfur lyase gene phbA, acetoacetyl-CoA reductase gene phbB, crotonase gene crt and thioesterase gene ydiI in series by using ribosome binding site sequence rbs to obtain an expression element phbA-phbB-crt-ydiI for expressing the 4 genes, wherein the nucleotide sequence of the expression element phbA-phbB-crt-ydiI is SEQ ID NO 1;
a-2. The expression element phbA-phbB-crt-ydiI constructed in step A-1 was loaded between BamH1/HindIII sites of vector pTrcHis2B, downstream of Trc promoter, to obtain pTrcHis2B-ABcy plasmid, plasmid A.
2. The method of claim 1, wherein the construction of plasmid B comprises the steps of:
the aspartic acid ammonia lyase gene AspB was cloned between the NcoI/NotI sites of vector pcdfdurt-1, downstream of the T7 promoter, to obtain pCDF-AspB plasmid.
3. An R-3-aminobutyric acid producing bacterium constructed by the method according to claim 1 or 2.
4. The bacterium for producing R-3-aminobutyric acid according to claim 3, which is Escherichia coliEscherichia coli) The strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.22076.
5. The use of the bacterium for producing R-3-aminobutyric acid according to claim 4.
6. The use according to claim 5, wherein the production of R-3-aminobutyric acid is carried out by fermentation of the R-3-aminobutyric acid producing bacterium CGMCC No.22076 according to claim 4.
7. The 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 Na 2 HPO 4 ，5mM(NH 4 ) 2 SO 4 ，30mMNH 4 Cl，5g/L CaCO 3 。
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Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1357629A (en) * 2001-06-08 2002-07-10 清华大学 Chiral beta-(R)-oxybatic acid producing process with recombinant colon bacillus
CN103562398A (en) * 2011-03-22 2014-02-05 Opx生物工艺学公司 Microbial production of chemical products and related compositions, methods and systems
TW201410865A (en) * 2012-06-04 2014-03-16 Genomatica Inc Microorganisms and methods for production of 4-hydroxybutyrate, 1,4-butanediol and related compounds
CN103890185A (en) * 2011-08-19 2014-06-25 基因组股份公司 Microorganisms and methods for producing 2,4-pentadienoate, butadiene, propylene, 1,3-butanediol and related alcohols
CN104039973A (en) * 2012-01-06 2014-09-10 弗门尼舍有限公司 Genetically engineered yeast cells
WO2014152434A2 (en) * 2013-03-15 2014-09-25 Genomatica, Inc. Microorganisms and methods for producing butadiene and related compounds by formate assimilation
CN104312966A (en) * 2008-03-03 2015-01-28 焦耳无限科技公司 CO2 fixing engineered microorganisms producing carbon-based target products
CN107922957A (en) * 2015-06-23 2018-04-17 基因组股份公司 For producing microorganism and the method for the target product with the biosynthesis for dropping low-level accessory substance
CN108866028A (en) * 2017-05-09 2018-11-23 中国科学院微生物研究所 A kind of amino lyases mutant protein and its encoding gene and application
CN109576317A (en) * 2017-09-29 2019-04-05 上海弈柯莱生物医药科技有限公司 The method that enzyme process prepares R-3- aminobutyric acid
CN110438056A (en) * 2019-08-12 2019-11-12 江南大学 The building and application of the colibacillus engineering of one plant of production n-butyric acie
CN110724657A (en) * 2019-10-29 2020-01-24 江南大学 Strategy for regulating cell morphology and improving polylactic acid production performance
CN110791494A (en) * 2019-11-28 2020-02-14 江南大学 Aspartic enzyme mutant, recombinant expression vector and recombinant bacterium containing aspartic enzyme mutant and application
CN110791493A (en) * 2019-11-27 2020-02-14 浙江华睿生物技术有限公司 Aspartate ammonia lyase mutant and application thereof
CN112094793A (en) * 2020-09-23 2020-12-18 江南大学 Method for co-production of D-1,2, 4-butanetriol and PHB by metabolically modifying escherichia coli and application
WO2021058691A1 (en) * 2019-09-26 2021-04-01 Basf Se Method for the production of beta-alanine or salts thereof

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7456002B2 (en) * 2002-05-28 2008-11-25 Riken Mutant poly(3-hydroxyalkanoic acid) synthases
US9644005B2 (en) * 2011-09-21 2017-05-09 King Abdullah University Of Science And Technology Didemnin biosynthetic gene cluster in Tistrella mobilis
WO2016004334A1 (en) * 2014-07-03 2016-01-07 Genomatica, Inc. Microorganisms for producing 4c-5c compounds with unsaturation and methods related thereto
US20190218531A1 (en) * 2018-01-18 2019-07-18 Board Of Trustees Of Michigan State University Two phase sustainable photoproduction via co-cultivation of encapsulated, carbohydrate-producing cyanobacteria
US20200308613A1 (en) * 2019-04-01 2020-10-01 Cargill, Incorporated Methods and genetically modified cells for production of fatty acid esters

Patent Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1357629A (en) * 2001-06-08 2002-07-10 清华大学 Chiral beta-(R)-oxybatic acid producing process with recombinant colon bacillus
CN104312966A (en) * 2008-03-03 2015-01-28 焦耳无限科技公司 CO2 fixing engineered microorganisms producing carbon-based target products
CN103562398A (en) * 2011-03-22 2014-02-05 Opx生物工艺学公司 Microbial production of chemical products and related compositions, methods and systems
CN103890185A (en) * 2011-08-19 2014-06-25 基因组股份公司 Microorganisms and methods for producing 2,4-pentadienoate, butadiene, propylene, 1,3-butanediol and related alcohols
CN104039973A (en) * 2012-01-06 2014-09-10 弗门尼舍有限公司 Genetically engineered yeast cells
TW201410865A (en) * 2012-06-04 2014-03-16 Genomatica Inc Microorganisms and methods for production of 4-hydroxybutyrate, 1,4-butanediol and related compounds
WO2014152434A2 (en) * 2013-03-15 2014-09-25 Genomatica, Inc. Microorganisms and methods for producing butadiene and related compounds by formate assimilation
CN107922957A (en) * 2015-06-23 2018-04-17 基因组股份公司 For producing microorganism and the method for the target product with the biosynthesis for dropping low-level accessory substance
CN108866028A (en) * 2017-05-09 2018-11-23 中国科学院微生物研究所 A kind of amino lyases mutant protein and its encoding gene and application
CN109576317A (en) * 2017-09-29 2019-04-05 上海弈柯莱生物医药科技有限公司 The method that enzyme process prepares R-3- aminobutyric acid
CN110438056A (en) * 2019-08-12 2019-11-12 江南大学 The building and application of the colibacillus engineering of one plant of production n-butyric acie
WO2021058691A1 (en) * 2019-09-26 2021-04-01 Basf Se Method for the production of beta-alanine or salts thereof
CN110724657A (en) * 2019-10-29 2020-01-24 江南大学 Strategy for regulating cell morphology and improving polylactic acid production performance
CN110791493A (en) * 2019-11-27 2020-02-14 浙江华睿生物技术有限公司 Aspartate ammonia lyase mutant and application thereof
CN110791494A (en) * 2019-11-28 2020-02-14 江南大学 Aspartic enzyme mutant, recombinant expression vector and recombinant bacterium containing aspartic enzyme mutant and application
US10865404B1 (en) * 2019-11-28 2020-12-15 Jiangnan University Aspartase mutant, recombinant expression vector and recombinant bacterium containing aspartase mutant, and use thereof
CN112094793A (en) * 2020-09-23 2020-12-18 江南大学 Method for co-production of D-1,2, 4-butanetriol and PHB by metabolically modifying escherichia coli and application

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Microbial production of R-3-hydroxybutyric acid by recombinant E. coli harboring genes of phbA, phbB, and tesB;Qian Liu 等;《Appl Microbiol Biotechnol》;全文 *
合成生物学在微生物细胞工厂构建中的应用;戴住波;朱欣娜;张学礼;;生命科学(10);全文 *
聚-β-羟基丁酸(PHB)在细菌建立感受态中的作用;李文化, 谢志雄, 陈向东, 沈萍;微生物学杂志(06);全文 *
聚β羟基丁酸(PHB)的研究进展;刘宝全, 蒋本国;大连民族学院学报(04);全文 *

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