CN108060114A - A kind of Escherichia coli of fermenting and producing l-Alanine and its application - Google Patents
A kind of Escherichia coli of fermenting and producing l-Alanine and its application Download PDFInfo
- Publication number
- CN108060114A CN108060114A CN201810033876.4A CN201810033876A CN108060114A CN 108060114 A CN108060114 A CN 108060114A CN 201810033876 A CN201810033876 A CN 201810033876A CN 108060114 A CN108060114 A CN 108060114A
- Authority
- CN
- China
- Prior art keywords
- escherichia coli
- alanine
- recombinant strain
- asd
- coli recombinant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01012—Aspartate 4-decarboxylase (4.1.1.12)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y403/00—Carbon-nitrogen lyases (4.3)
- C12Y403/01—Ammonia-lyases (4.3.1)
- C12Y403/01001—Aspartate ammonia-lyase (4.3.1.1), i.e. aspartase
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Escherichia coli and its application the invention discloses a kind of fermenting and producing L alanine, belong to technical field of bioengineering.The present invention can introduce Aspartase AspA and L aspartic acid β decarboxylases AsD in the Escherichia coli of fermenting and producing fumaric acid, the turn-over capacity of L alanine in further overexpression YgaW gene and strengthenings recombinant bacterium, the new mushroom-seed culturing of glucose fermentation production L alanine can be utilized by constructing, using the recombinant bacterial strain in fermentation cylinder for fermentation 45h, L alanine yield reaches 147g/L, saccharic acid conversion ratio 79.0%.
Description
Technical field
Escherichia coli and its application the present invention relates to a kind of fermenting and producing l-Alanine belong to biotechnology neck
Domain.
Background technology
L-Alanine is the highest amino acid of content in a kind of nonessential amino acid and blood of human body, in l-Alanine
It is widely used in fields such as industry, daily use chemicals, food.In daily chemicals field, l-Alanine is the weight of synthesizing amino acid surfactant
Raw material is wanted, in field of food, l-Alanine can be as natural sweetener to improving the sense of taste of flavoring agent and the acid of organic acid.
Field of medicaments, l-Alanine are also to synthesize the primary raw material of vitamin B6 and aminopropanol.
The production technology of l-Alanine includes extraction method, chemical synthesis, enzyme catalysis method and fermentation method.Industrial production at present
It is upper mainly to use enzyme catalysis method, that is, pass through and cultivate micro- life rich in L-Aspartic acid-β-decarboxylase AsD (EC4.1.1.12) vigor
Object cell, catalysis L-Aspartic acid obtain l-Alanine.The characteristics of enzymatic conversion method technique is enzyme activity height, and equipment requirement is simple,
Extraction process is simple, but wild-type microorganisms culture production AsD is needed using fumaric acid or sodium glutamate as carbon source, growth cycle
Long and toxigenic capacity is high, while main raw material(s) L-Aspartic acid price is high, causes its production cost high.Xu You is strong etc. to be passed through
Clone Origin has L- asparagus fern ammonia in the asD genes of Comamonas testosteroni (Comonas testosteroni) at one plant
Heterogenous expression in the escherichia coli CICC11022S of sour enzyme (AspA) production capacity, structure conversion fumaric acid production the third ammonia of L-
The recombination engineering of acid, recombinant bacterium 9h conversion fumaric acid generate the l-Alanine of 112.7g/L, conversion ratio 93.8%.This method with
Big Escherichia coli are bacteria enzyme source strain and fumaric acid is substrate, and the cost of material of production significantly reduces, but fumaric acid
It is to be produced through petroleum refining, price locks into crude oil price, while petroleum resources are increasingly short and non-renewable.Zhang Xueli
Deng 5 competition approach of key using genetic engineering means deletion l-Alanine synthesis, overexpression rate-limiting step enzyme the third ammonia of L-
Acidohydrogenase strengthens production path of the pyruvic acid to l-Alanine, builds Escherichia coli recombinant strain, using glucose as raw material, from
Water prepares culture medium, and fermentation 48h can produce 114g/L l-Alanine.Fermentation raw material glucose is cheap and easy to get, culture medium into
Divide simply, anaerobic fermentation power consumption is low, and product yield is high.The construction method of structure l-Alanine production bacterial strain is single at present,
It is related to a plurality of contended path, genetic engineering is complicated for operation.
The content of the invention
To solve the above-mentioned problems, the present invention produces fumaric acid by Escherichia coli fermentation, utilizes Aspartase AspA
Aspartic acid is converted into, further utilizes L-Aspartic acid-β-decarboxylase AsD conversion aspartic acid production l-Alanine, structure
The new mushroom-seed culturing of efficient production l-Alanine.
First purpose of the present invention is to provide a kind of Escherichia coli recombinant strain, the Escherichia coli recombinant strain be by
In the Escherichia coli that fumaric acid can be produced, Aspartase AspA and L-Aspartic acid-β-decarboxylase AsD is overexpressed.
In one embodiment of the invention, the Aspartase AspA and L-Aspartic acid-β-decarboxylase AsD leads to
RBS1 of the nucleotide sequence as shown in SEQ ID NO.4 is crossed to be connected.
In one embodiment of the invention, the nucleotide sequence of the Aspartase AspA encoding genes aspA is such as
Shown in SEQ ID NO.1, the nucleotide sequence such as SEQ ID of the L-Aspartic acid-β-decarboxylase AsD encoding genes asD
Shown in NO.2.
In one embodiment of the invention, the Escherichia coli of the production fumaric acid are Escherichia coli CICC.23846.
In one embodiment of the invention, Aspartase AspA and L- described in pEtac plasmid expressing in series are passed through
Aspartic acid-beta-decarboxylase AsD.
In one embodiment of the invention, the Escherichia coli recombinant strain is encoded in l-Alanine transport protein AlaE
Gene YgaW upstream integrations constitutive promoter J23119.
In one embodiment of the invention, the nucleotide of the l-Alanine transport protein AlaE encoding genes YgaW
Sequence is as shown in SEQ ID NO.3.
Second object of the present invention is to provide the construction method of the Escherichia coli recombinant strain, and the method is specifically：
(1) using pEtac plasmids, using RBS1 of the nucleotide sequence as shown in SEQ ID NO.4 as connection peptide, table of connecting
Up to Aspartase AspA of the nucleotide sequence as shown in SEQ ID NO.1 and nucleotide sequence as shown in SEQ ID NO.2
L-Aspartic acid-β-decarboxylase AsD, construction recombination plasmid pETac-aspA-asD；
(2) recombinant plasmid pETac-aspA-asD is imported in Escherichia coli CICC.23846；
(3) in l-Alanine transport protein AlaE upstream region of gene of the nucleotide sequence as shown in SEQ ID NO.3 from integration
In constitutive promoter J23119, then recombinant bacterium described in steps for importing (2), recombination bacillus coli E.coli P-119 are obtained.
3rd purpose of the invention is to provide a kind of utilization recombination bacillus coli E.coli P-119 fermenting and producings L-
The method of alanine, the method are specifically：3~5% inoculum concentration of shake-flask seed is inoculated in fermentation medium, dilute sulfuric acid and ammonia
Water management pH is 6.8~7.2,0.1~1vvm of throughput, 34~36 DEG C of temperature, 80~120rpm is stirred, when OD600 reaches 10
When~15, the expression of 2~3g/L lactose induction Aspartase and L-Aspartic acid-β-decarboxylase is added in, fermentation process works as Portugal
When grape sugar concentration is less than 10~15g/L, 500~700g/L glucose solutions are added, every liter of zymotic fluid adds 20~40g grapes
Sugar is added 2~6 times altogether, terminates fermentation after glucose consumption is most.
In one embodiment of the present invention, fermentation medium components are：60~80g/L of initial glucose, dusty yeast 4~
6g/L, tryptone 1~3g/L, (NH4)2SO41~3g/L, K2HPO4·12H2O 3~5g/L, KH2PO43~5g/L,
MgSO4·7H2O0.4~0.6g/L, monohydrate potassium 0.2~0.4g/L, MnCl2·4H20.06~0.1g/L of O.
Fourth object of the present invention is to provide application of the Escherichia coli recombinant strain in l-Alanine is produced.
Usefulness of the present invention：
For Escherichia coli as most common cell factory, genetic background is clear, is being capable of the large intestine of fermenting and producing fumaric acid
Aspartase AspA and L-Aspartic acid-β-decarboxylase AsD is introduced in bacillus, the fumaric acid of fermenting and producing is converted into
L-Alanine, further overexpression AlaE enhance the turn-over capacity of l-Alanine in recombinant bacterium, construct can utilize it is honest and clean
Valency glucose fermentation produces the superior strain of l-Alanine, and the production path there is no relevant report at present at present.
Description of the drawings
Fig. 1：The structure of pEtac-aspA-asD co-expression plasmids.
Specific embodiment
All it is using routine experiment method, implementing material can obtain from commercial channels in following embodiments.
In following embodiments, escherichia coli (Escherichia coli) is starting strain, purchased from CICC, number
23846, the Escherichia coli of fumaric acid can be produced by being also applied for other.
Amino acid detection is using conventional high performance liquid chromatography detection.
Aspartase AspA Enzyme activity assays：Fumaric acid ammonium hydroxide (pH=8.5) solution for being about 180g/L by 27mL concentration
37 DEG C of preheatings, weigh the wet thallus of 0.5g or so in 250mL conical flasks respectively, add in 0.09gCTAB powder and 2.5mL ions
Water is added in the conversion fluid of preheating, and preservative film sealing is placed on 37 DEG C, reacts 15min in the shaking table of 200rpm.Reaction is completed
Afterwards, appropriate reaction solution is taken quickly to centrifuge dilution and carries out liquid phase detection.
L-Aspartic acid-β-decarboxylase AsD Enzyme activity assays：The aspartic acid solution that 15mL concentration is 30g/L (is used into hydrogen-oxygen
Change sodium solution and adjust pH 7.0) 37 DEG C of preheatings, the wet thallus of 1g or so inductions 12h is weighed respectively in 250mL conical flasks, is added in
0.09g CTAB powder and 14mL deionized waters, add in the conversion fluid of preheating, and preservative film sealing is placed on 37 DEG C, 200rpm
15min is reacted in shaking table.After the completion of reaction, appropriate reaction solution is taken quickly to centrifuge dilution and carries out liquid phase detection.
Enzyme activity unit (U)：1 μm of required enzyme amount of oL products of production per minute is defined as 1 enzyme-activity unit.
Glucose assays method：Divided using SBA-40 bio-sensings analyzer (Shandong Province academy sciences Biology Research Institute)
Analysis.
Produce the calculating of intensity：Produce intensity (g/L/h)=l-Alanine yield (g/L)/fermentation time (h).
Embodiment 1：The structure of double enzyme coexpression bacterial strains
(1) using bacterial genomes extracts kit extraction e. coli k12 and the genome of Comamonas testosteroni
DNA；
(2) cloned using primer AspA-up and AspA-down in table 1 from genome of E.coli DNA and obtain aspA bases
Cause is connected to expression vector pETac using SacI and NotI double digestions, obtains pETac-aspA；
(3) according to RBS intensity and the linear relationship of institute's modulin expression quantity statistically, design RBS1, RBS2,
RBS3, RBS4 adjust the expression intensity of two enzymes, use primer AsD-up1 and AsD-down, AsD-up2 and AsD-down, AsD-
Up3 and AsD-down, AsD-up4 and AsD-down are cloned from the genomic DNA of Comamonas testosteroni and are obtained asD bases
Cause；
(4) after 4 sections of asD gene sequencing obtaining above-mentioned clone are correct, HindIII and NotI double digestions are used, respectively
It is connected to pETac-aspA plasmids and obtains pETac-aspA-asD (such as Fig. 1), plasmid thermal shock is converted in escherichia coli
CICC23846 obtains recombinant bacterial strain E.coli PAD；
(6) above-mentioned 4 plants of engineered strains use LB culture mediums (100mg/L kanamycin sulfates), and 35 DEG C are cultivated 10h, according to
4% inoculum concentration is inoculated in Medium of shaking flask fermentation, and 2g/L lactose is added in during OD600=2.0, continues to cultivate, when glucose disappears
End fermentation is exhausted, centrifugation detects l-Alanine yield in supernatant, collects bacterium mud detection enzyme activity；
Fermentation medium components：Initial glucose 60g/L, dusty yeast 3g/L, tryptone 1g/L, (NH4)2SO42g/L,
K2HPO4·12H2O 4g/L, KH2PO44g/L, MgSO4·7H2O 0.5g/L, monohydrate potassium 0.3g/L, MnCl2·
4H2O0.08g/L。
(7) l-Alanine yield and Enzyme activity assay such as result such as table 2 are RBS1 under same culture conditions, between two genes
L-Alanine yield is up to 37.6g/L during sequence, and AspA and AsD enzyme activity are respectively 301U/g and 478U/g during fermentation ends.
1 co-expression plasmid of table structure uses primer
2 recombinant bacterium fermenting and producing l-Alanine of table
Embodiment 2：The structure of L-alanine with high yield Escherichia coli
Using the principle of Red homologous recombinations constitutive promoter J23119 is added in YgaW genomic upstreams
(BBa23119), strengthen AlaE protein expressions, l-Alanine is promoted to be converted by intracellular to extracellular.
(1) homologous recombination primer (such as table 3) is designed using software Primer Premier 5, using Kan-S, Kan-A to draw
Object, pKD4 are template, expand kan genes, and using YgaW-S, YgaW-A as primer, e. coli k12 genome is template, is expanded
YgaW genes obtain Kan+YgaW segments using fusion DNA vaccine, are connected to PMD19 and carry out gene sequencing；
(2) using 119-S and YgaW-A as primer, the PMD19 plasmids of correct Kan+YgaW segments are sequenced as template in connection,
PCR is obtained with homology arm, J23119 promoters, the knockout frame of Kan genes；
(3) pKD46 plasmids are imported in Escherichia coli CICC 23846, prepares electrocompetent cell, frame piece will be knocked out
Section is electroporated to carry out homologous recombination in competent cell, and screening obtains the positive colony bacterial strain with kan resistances；
(4) after 43 DEG C of the positive colony bacterial strain culture with kan resistances its is made to lose pKD46 plasmids, prepare electric-shock feeling by
State cell, electroporated plasmid pCP20.Conversion fluid is coated on the LB tablets containing ampicillin, 30 DEG C of culture 12h, screening
Positive transformant.
(5) transformant is transferred to the restructuring of 43 DEG C of culture 12h screening ampicillins and kalamycin resistance while missing again
Bacterium, J23119 promoters successful integration, imports co-expression plasmid pETac-aspA-asD (RBS1) is built in embodiment 1 at this time
Recombinant bacterium obtains l-Alanine production bacterial strain E.coli P-119
(6) above-mentioned engineered strain E.coli PAD RBS1 and E.coli P-119 are trained in the way of in embodiment 1
It supports, l-Alanine content in fermentation ends detection zymotic fluid, ferment 48h, E.coli P-119 production l-Alanine 49.8g/L,
37.6g/L compared to E.coli PAD RBS1 improves 32.4%.
The gene constructed J23119 promoters of table 3YgaW use primer
Embodiment 3：E.coli P-119 fermenting and producing l-Alanine
Seed culture based component：Dusty yeast 5g/L, peptone 10g/L, NaCl 5.0g/L, MgSO4·7H2O 1.0g/L。
Fermentation medium components：Initial glucose 70g/L, dusty yeast 5g/L, tryptone 2g/L, (NH4)2SO42g/L,
K2HPO4·12H2O 4g/L, KH2PO44g/L, MgSO4·7H2O 0.5g/L, monohydrate potassium 0.3g/L, MnCl2·
4H2O0.08g/L。
It is inoculated in from the appropriate E.coli P-119 of inclined-plane picking in seed culture medium, 35 DEG C, 200rpm shaken cultivations 10~
12h, shake-flask seed are inoculated according to 10% inoculum concentration in fermentation tank, in 5L fermentation tanks culture medium liquid amount be 3L, dilute sulfuric acid and
It is 6.8~7.2 that ammonium hydroxide, which controls fermentation pH, and throughput 0.5vvm, 35 DEG C of temperature stirs 100rpm, when OD600 reaches 25~30
When, 2g/L lactose induction Aspartase and aspartic acid-beta-decarboxylation expression of enzymes are added in, when concentration of glucose is less than 10~15g/
During L, 600g/L glucose solutions are added, every liter of zymotic fluid adds 60g glucose, adds altogether 2 times, is tied after glucose consumption is most
Beam ferments.
As a result：Ferment 45h, and l-Alanine yield reaches 122.0g/L, saccharic acid conversion ratio 64.2%.
Embodiment 4：Recombinant bacterial strain E.coli Δ 5D2 fermenting and producing l-Alanine
Seed culture based component：Dusty yeast 5g/L, peptone 10g/L, NaCl 5.0g/L, MgSO4·7H2O 1.0g/L。
Fermentation medium components：Initial glucose 70g/L, dusty yeast 5g/L, tryptone 2g/L, (NH4)2SO42g/L,
K2HPO4·12H2O 4g/L, KH2PO44g/L, MgSO4·7H2O 0.5g/L, monohydrate potassium 0.3g/L, MnCl2·
4H2O0.08g/L。
It is inoculated in from the appropriate E.coli P-119 of inclined-plane picking in seed culture medium, 35 DEG C, 200rpm shaken cultivations 10~
12h, shake-flask seed are inoculated according to 4% inoculum concentration in fermentation tank, and culture medium liquid amount is 3L, dilute sulfuric acid and ammonia in 5L fermentation tanks
Water management fermentation pH is 6.8~7.2, and throughput 0.5vvm, 35 DEG C of temperature stirs 100rpm, when OD600 reaches 10~15,
2g/L lactose induction Aspartase and aspartic acid-beta-decarboxylation expression of enzymes are added in, when concentration of glucose is less than 10~15g/L
When, 600g/L glucose solutions are added, every liter of zymotic fluid adds 30g glucose, adds altogether 4 times, is tied after glucose consumption is most
Beam ferments.
As a result：Ferment 45h, and l-Alanine yield reaches 147g/L, saccharic acid conversion ratio 79.0%, realizes the height of l-Alanine
Effect production.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art without departing from the spirit and scope of the present invention, can do various change and modification, therefore the protection model of the present invention
Enclosing be subject to what claims were defined.
Sequence table
<110>Southern Yangtze University
<120>A kind of Escherichia coli of fermenting and producing l-Alanine and its application
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 1437
<212> DNA
<213>Artificial sequence
<400> 1
atgtcaaaca acattcgtat cgaagaagat ctgttgggta ccagggaagt tccagctgat 60
gcctactatg gtgttcacac tctgagagcg attgaaaact tctatatcag caacaacaaa 120
atcagtgata ttcctgaatt tgttcgcggt atggtaatgg ttaaaaaagc cgcagctatg 180
gcaaacaaag agctgcaaac cattcctaaa agtgtagcga atgccatcat tgccgcatgt 240
gatgaagtcc tgaacaacgg aaaatgcatg gatcagttcc cggtagacgt ctaccagggc 300
ggcgcaggta cttccgtaaa catgaacacc aacgaagtgc tggccaatat cggtctggaa 360
ctgatgggtc accaaaaagg tgaatatcag tacctgaacc cgaacgacca tgttaacaaa 420
tgtcagtcca ctaacgacgc ctacccgacc ggtttccgta tcgcagttta ctcttccctg 480
attaagctgg tagatgcgat taaccaactg cgtgaaggct ttgaacgtaa agctgtcgaa 540
ttccaggaca tcctgaaaat gggtcgtacc cagctgcagg acgcagtacc gatgaccctc 600
ggtcaggaat tccgcgcttt cagcatcctg ctgaaagaag aagtgaaaaa catccaacgt 660
accgctgaac tgctgctgga agttaacctt ggtgcaacag caatcggtac tggtctgaac 720
acgccgaaag agtactctcc gctggcagtg aaaaaactgg ctgaagttac tggcttccca 780
tgcgtaccgg ctgaagacct gatcgaagcg acctctgact gcggcgctta tgttatggtt 840
cacggcgcgc tgaaacgcct ggctgtgaag atgtccaaaa tctgtaacga cctgcgcttg 900
ctctcttcag gcccacgtgc cggcctgaac gagatcaacc tgccggaact gcaggcgggc 960
tcttccatca tgccagctaa agtaaacccg gttgttccgg aagtggttaa ccaggtatgc 1020
ttcaaagtca tcggtaacga caccactgtt accatggcag cagaagcagg tcagctgcag 1080
ttgaacgtta tggagccggt cattggccag gccatgttcg aatccgttca cattctgacc 1140
aacgcttgct acaacctgct ggaaaaatgc attaacggca tcactgctaa caaagaagtg 1200
tgcgaaggtt acgtttacaa ctctatcggt atcgttactt acctgaaccc gttcatcggt 1260
caccacaacg gtgacatcgt gggtaaaatc tgtgccgaaa ccggtaagag tgtacgtgaa 1320
gtcgttctgg aacgcggtct gttgactgaa gcggaacttg acgatatttt ctccgtacag 1380
aatctgatgc acccggctta caaagcaaaa cgctatactg atgaaagcga acagtaa 1437
<210> 2
<211> 1002
<212> DNA
<213>Artificial sequence
<400> 2
atgagcaagg attatcagag tctggcgaaa ttgagcccgt tcgagctcaa ggatgagttg 60
atcaagatcg cctcgagcga cggaaaccgc ctcatgctca atgcggggcg gggcaatccc 120
aactttctgg ccacaacgcc gagaagagca ttttttcgcc tgggcttgtt cgctgcagcc 180
gaatcggagc tttcctactc ttatatgaca acggtaggtg ttggcggact ggcgaagatc 240
gacgggattg agggtcgctt tgaacgttat atcgccgaga accgggatca ggagggcgtg 300
cgctttctcg gtaaatcact gagctatgtc cgtgaccagc tgggcttgga cccggccgcc 360
ttcctgcacg aaatggttga cggtattctg ggctgcaatt accccgttcc cccgcggatg 420
ctgaacatca gtgaaaaaat cgtacgccag tacatcatcc gggaaatggg ggccgatgca 480
atacccagtg agtccgtgaa cctgtttgca gtcgaagggg gaacggctgc catggcttac 540
attttcgaga gcctgaagct caacggtttg ctcaaggccg gcgacaaggt tgccattggg 600
atgccggttt tcacgccgta tatagaaatt ccggagctgg cccagtacgc actggaggaa 660
gtcgccatca acgcggatcc gagcctcaat tggcagtatc cggactctga gctggacaag 720
ctcaaggacc cggccatcaa gatctttttc tgcgtcaacc ccagcaatcc gccttcggtg 780
aagatggatc agcgaagtct ggaacgcgtg cgcaacatcg ttgcagaaca tcgtccggat 840
ctgatgatcc tgaccgacga tgtctatgga acttttgcag atgacttcca gtctctcttc 900
gcgatttgcc cggaaaatac gcttctggtc tactcattct ccaaatactt tggcgccact 960
ggctggcgtt tgggggtcgt ggccgctcat caacaaaatt aa 1002
<210> 3
<211> 450
<212> DNA
<213>Artificial sequence
<400> 3
atgttctcac cgcagtcacg cttgcgtcat gcagttgcag atacgttcgc gatggttgtt 60
tactgttctg tcgtgaacat gtgtattgaa gttttcctct ccggaatgag cttcgaacag 120
tctttttatt ccagattggt agcgattccg gtgaacatct taattgcatg gccatacggt 180
atgtaccgtg atctgtttat gcgcgcggca cgcaaagtta gcccgtcggg ctggataaaa 240
aatctggcgg atatcctggc ttatgtgacg ttccagtcac cggtgtatgt ggcgatcttg 300
ttagtggtgg gcgcagactg gcatcagatt atggcggcgg tcagttcaaa catcgttgtt 360
tcgatgttga tgggggcggt ttatggctac ttcctcgatt attgccgccg actgtttaaa 420
gtcagccgtt accagcaggt aaaagcctga 450
<210> 4
<211> 37
<212> DNA
<213>Artificial sequence
<400> 4
ttaaggagga attcttatga gcaaggatta tcagagt 37
<210> 5
<211> 35
<212> DNA
<213>Artificial sequence
<400> 5
acgcgtcgac atgtcaaaca acattcgtat cgaag 35
<210> 6
<211> 47
<212> DNA
<213>Artificial sequence
<400> 6
aaatatgcgg ccgcgaattc aagcttttac tgttcgcttt catcagt 47
<210> 7
<211> 46
<212> DNA
<213>Artificial sequence
<400> 7
cccaagcttt taaggaggaa ttcttatgag caaggattat cagagt 46
<210> 8
<211> 52
<212> DNA
<213>Artificial sequence
<400> 8
cccaagcttg attaaagagg agaaatacca tatgagcaag gattatcaga gt 52
<210> 9
<211> 51
<212> DNA
<213>Artificial sequence
<400> 9
cccaagcttg tcacacagga aacctaccat atgagcaagg attatcagag t 51
<210> 10
<211> 71
<212> DNA
<213>Artificial sequence
<400> 10
cccaagcttc gcttaaggag gatctacatt cttatgagca cgtcagacga atgagcaagg 60
attatcagag t 71
<210> 11
<211> 35
<212> DNA
<213>Artificial sequence
<400> 11
aaatatgcgg ccgcttaatt ttgttgatga gcggc 35
<210> 12
<211> 65
<212> DNA
<213>Artificial sequence
<400> 12
catctccatt aacatcccat tacgctttta ttaaggagca ttagcgtgta ggctggagct 60
gcttc 65
<210> 13
<211> 55
<212> DNA
<213>Artificial sequence
<400> 13
gctagcatta tacctaggac tgagctagct gtcaacatat gaatatcctc cttag 55
<210> 14
<211> 80
<212> DNA
<213>Artificial sequence
<400> 14
ttgacagcta gctcagtcct aggtataatg ctagcgaatt cattaaagag gagaaaggta 60
ccatgttctc accgcagtca 80
<210> 15
<211> 19
<212> DNA
<213>Artificial sequence
<400> 15
tcaggctttt acctgctgg 19
<210> 16
<211> 24
<212> DNA
<213>Artificial sequence
<400> 16
catctccatt aacatcccat tacg 24
Claims (10)
1. a kind of Escherichia coli recombinant strain, which is characterized in that the Escherichia coli recombinant strain is by that can produce fumaric acid
In Escherichia coli, Aspartase AspA and L-Aspartic acid-β-decarboxylase AsD is overexpressed.
2. Escherichia coli recombinant strain according to claim 1, which is characterized in that the Aspartase AspA and L- asparagus ferns
Propylhomoserin-β-decarboxylase AsD is connected by RBS1 of the nucleotide sequence as shown in SEQ ID NO.4.
3. Escherichia coli recombinant strain according to claim 1, which is characterized in that the Aspartase AspA encoding genes
The nucleotide sequence of aspA is as shown in SEQ ID NO.1, the nucleosides of the L-Aspartic acid-β-decarboxylase AsD encoding genes asD
Acid sequence is as shown in SEQ ID NO.2.
4. Escherichia coli recombinant strain according to claim 1, which is characterized in that the Escherichia coli of the production fumaric acid are big
Enterobacteria CICC.23846.
5. Escherichia coli recombinant strain according to claim 1, which is characterized in that by described in pEtac plasmid expressing in series
Aspartase AspA and L-Aspartic acid-β-decarboxylase AsD.
6. Escherichia coli recombinant strain according to claim 1, which is characterized in that the Escherichia coli recombinant strain is in the third ammonia of L-
Acid transporter albumin A laE encoding gene YgaW upstream integrations constitutive promoter J23119.
7. Escherichia coli recombinant strain according to claim 1, which is characterized in that the l-Alanine transport protein AlaE is compiled
The nucleotide sequence of code gene YgaW is as shown in SEQ ID NO.3.
A kind of 8. method using Escherichia coli recombinant strain fermenting and producing l-Alanine described in claim 1, which is characterized in that
The method is specifically：3~5% inoculum concentration of shake-flask seed is inoculated in fermentation medium, dilute sulfuric acid and ammonium hydroxide control pH 6.8
~7.2,0.1~1vvm of throughput, 34~36 DEG C of temperature stir 80~120rpm, when OD600 reaches 10~15, addition 2~
3g/L lactose induces the expression of Aspartase and L-Aspartic acid-β-decarboxylase, and fermentation process is less than 10 when concentration of glucose
During~15g/L, 500~700g/L glucose solutions are added, every liter of zymotic fluid adds 20~40g glucose, adds altogether 2~6 times,
Terminate fermentation after glucose consumption is most.
9. according to the method described in claim 8, it is characterized in that, fermentation medium components are：60~80g/ of initial glucose
L, 4~6g/L of dusty yeast, tryptone 1~3g/L, (NH4)2SO41~3g/L, K2HPO4·12H2O 3~5g/L, KH2PO4 3
~5g/L, MgSO4·7H20.4~0.6g/L of O, monohydrate potassium 0.2~0.4g/L, MnCl2·4H20.06~0.1g/ of O
L。
10. application of the Escherichia coli recombinant strain described in claim 1 in l-Alanine is produced.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810033876.4A CN108060114B (en) | 2018-01-15 | 2018-01-15 | A kind of Escherichia coli of fermenting and producing l-Alanine and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810033876.4A CN108060114B (en) | 2018-01-15 | 2018-01-15 | A kind of Escherichia coli of fermenting and producing l-Alanine and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108060114A true CN108060114A (en) | 2018-05-22 |
| CN108060114B CN108060114B (en) | 2019-03-01 |
Family
ID=62141678
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810033876.4A Active CN108060114B (en) | 2018-01-15 | 2018-01-15 | A kind of Escherichia coli of fermenting and producing l-Alanine and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108060114B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108660142A (en) * | 2018-05-28 | 2018-10-16 | 河北大学 | A kind of gene of L-alanine with high yield and its construction method of engineering bacteria |
| CN109456928A (en) * | 2018-12-13 | 2019-03-12 | 江南大学 | One plant of expression L-Aspartic acid-β-decarboxylase Escherichia coli recombinant strain and its application |
| CN109468254A (en) * | 2018-11-13 | 2019-03-15 | 江南大学 | A kind of method and its application improving l-Alanine production efficiency |
| CN109536429A (en) * | 2018-12-13 | 2019-03-29 | 江南大学 | A kind of production L-Aspartic acid-β-decarboxylase genetic engineering bacterium and its application |
| CN110004102A (en) * | 2019-04-23 | 2019-07-12 | 南京工业大学 | A kind of bacterial strain and method using maleic acid whole-cell catalytic synthesis L-Aspartic acid |
| CN110016459A (en) * | 2019-04-29 | 2019-07-16 | 江南大学 | A method of producing the recombination bacillus coli and its anaerobic fermentation of Beta-alanine |
| EP3960879A1 (en) | 2020-09-01 | 2022-03-02 | Metabolic Explorer | Microorganism and method for the improved production of alanine |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974476A (en) * | 2010-08-31 | 2011-02-16 | 安徽华恒生物工程有限公司 | XZ-A26 bacterial strain for producing L-alanine with high yield as well as construction method and application thereof |
| CN102618570A (en) * | 2012-03-20 | 2012-08-01 | 南京工业大学 | Method for constructing escherichia coli genetic engineering bacteria for producing fumaric acid |
-
2018
- 2018-01-15 CN CN201810033876.4A patent/CN108060114B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974476A (en) * | 2010-08-31 | 2011-02-16 | 安徽华恒生物工程有限公司 | XZ-A26 bacterial strain for producing L-alanine with high yield as well as construction method and application thereof |
| CN102618570A (en) * | 2012-03-20 | 2012-08-01 | 南京工业大学 | Method for constructing escherichia coli genetic engineering bacteria for producing fumaric acid |
Non-Patent Citations (1)
| Title |
|---|
| 徐友强等: "重组大肠杆菌转化富马酸生产L-丙氨酸", 《生物加工过程》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108660142A (en) * | 2018-05-28 | 2018-10-16 | 河北大学 | A kind of gene of L-alanine with high yield and its construction method of engineering bacteria |
| CN109468254A (en) * | 2018-11-13 | 2019-03-15 | 江南大学 | A kind of method and its application improving l-Alanine production efficiency |
| CN109468254B (en) * | 2018-11-13 | 2021-03-26 | 江南大学 | Method for improving production efficiency of L-alanine and application thereof |
| CN109456928A (en) * | 2018-12-13 | 2019-03-12 | 江南大学 | One plant of expression L-Aspartic acid-β-decarboxylase Escherichia coli recombinant strain and its application |
| CN109536429A (en) * | 2018-12-13 | 2019-03-29 | 江南大学 | A kind of production L-Aspartic acid-β-decarboxylase genetic engineering bacterium and its application |
| CN110004102A (en) * | 2019-04-23 | 2019-07-12 | 南京工业大学 | A kind of bacterial strain and method using maleic acid whole-cell catalytic synthesis L-Aspartic acid |
| CN110016459A (en) * | 2019-04-29 | 2019-07-16 | 江南大学 | A method of producing the recombination bacillus coli and its anaerobic fermentation of Beta-alanine |
| CN110016459B (en) * | 2019-04-29 | 2021-03-19 | 江南大学 | Recombinant escherichia coli for producing beta-alanine and anaerobic fermentation method thereof |
| EP3960879A1 (en) | 2020-09-01 | 2022-03-02 | Metabolic Explorer | Microorganism and method for the improved production of alanine |
| WO2022049116A1 (en) | 2020-09-01 | 2022-03-10 | Metabolic Explorer | Microorganism and method for the improved production of alanine |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108060114B (en) | 2019-03-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108060114B (en) | A kind of Escherichia coli of fermenting and producing l-Alanine and its application | |
| CN106148265B (en) | It is a kind of produce chondroitinase recombined bacillus subtilis and its application | |
| CN104830815B (en) | A kind of method that α phenylpyruvic acids are efficiently produced using resting cell | |
| CN108467860B (en) | Method for high yield of gamma-aminobutyric acid | |
| CN106754610B (en) | Recombinant engineering bacterium for surface display expression of glutamate decarboxylase and construction method and application thereof | |
| CN112251428B (en) | Glutamic acid decarboxylase mutant and application thereof in production of gamma-aminobutyric acid | |
| CN106520715B (en) | A kind of short-chain dehydrogenase and its gene, recombinant expression carrier, genetic engineering bacterium and its application in the synthesis of astaxanthin chiral intermediate | |
| CN106957878A (en) | A kind of method that living things catalysis produces 2 benzyl carbinols | |
| CN104046586B (en) | One strain gene engineering bacterium and the application in producing (2R, 3R)-2,3-butanediol thereof | |
| CN114672525B (en) | Biosynthesis method and application of N-acetyl-5-methoxy tryptamine | |
| CN111748535B (en) | Alanine dehydrogenase mutant and application thereof in fermentation production of L-alanine | |
| CN114874959A (en) | Genetically engineered bacterium for producing L-theanine by using glucose from head fermentation, method and application | |
| CN109371070A (en) | A kind of method of high yield α-ketoisovaleric acid | |
| CN106480006A (en) | A kind of L Arabinose isomerase and its application | |
| CN116200353A (en) | Carbonyl reductase mutant, recombinant bacterium and application thereof | |
| CN115838682A (en) | Bacillus licheniformis engineering strain for efficiently producing 2' -fucosyllactose by utilizing mannan | |
| CN110791536B (en) | Biosynthesis method of levodopa | |
| CN106434710A (en) | Heatproof arginase gene expression sequence and application | |
| CN110872595B (en) | Acid-resistant expression cassette and application thereof in fermentation production of organic acid | |
| CN111378682A (en) | Method for reducing ethyl carbamate content in yellow wine by metabolically modifying saccharomyces cerevisiae | |
| CN115011622A (en) | Screening method and application of D-psicose 3-epimerase mutant | |
| CN106148257B (en) | The Klebsiella pneumoniae of transformation and its application for producing gluconic acid | |
| CN114107270B (en) | L-aspartic acid beta-decarboxylase mutant | |
| CN102719463B (en) | A kind of method producing restructuring mixing L-arabinose isomerase | |
| CN114606172B (en) | Bacillus amyloliquefaciens engineering bacteria for improving heme yield and construction method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |