CN108060114B - 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
- CN108060114B CN108060114B CN201810033876.4A CN201810033876A CN108060114B CN 108060114 B CN108060114 B CN 108060114B CN 201810033876 A CN201810033876 A CN 201810033876A CN 108060114 B CN108060114 B CN 108060114B
- Authority
- CN
- China
- Prior art keywords
- escherichia coli
- alanine
- asd
- recombinant strain
- aspa
- 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.)
- Active
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
Abstract
The invention discloses a kind of Escherichia coli of fermenting and producing l-Alanine and its applications, belong to technical field of bioengineering.The present invention can introduce Aspartase AspA and L-Aspartic acid-β-decarboxylase AsD in the Escherichia coli of fermenting and producing fumaric acid, the turn-over capacity of l-Alanine in further overexpression YgaW gene and strengthening recombinant bacterium, construct the new mushroom-seed culturing that can use glucose fermentation production l-Alanine, it is fermented in the fermenter 45h using the recombinant bacterial strain, l-Alanine yield reaches 147g/L, saccharic acid conversion ratio 79.0%.
Description
Technical field
The present invention relates to a kind of Escherichia coli of fermenting and producing l-Alanine and its applications, belong to biotechnology neck
Domain.
Background technique
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 used as acid of the natural sweetener to the sense of taste and organic acid that improve flavoring agent.?
Field of medicaments, l-Alanine are also the primary raw material for synthesizing 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, i.e., by culture rich in the micro- life of 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 high production cost.Xu You is strong etc. to be passed through
Clone Origin has L- asparagus fern ammonia in the asD gene of Comamonas testosteroni (Comonas testosteroni), at one plant
Heterogenous expression in the escherichia coli CICC11022S of sour enzyme (AspA) production capacity, building conversion fumaric acid produce the third ammonia of L-
The recombination engineering of acid, recombinant bacterium 9h convert the l-Alanine that fumaric acid generates 112.7g/L, conversion ratio 93.8%.This method with
Big Escherichia coli is 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
5 competition approach of key that l-Alanine synthesizes, overexpression rate-limiting step enzyme the third ammonia of L- are deleted Deng using genetic engineering means
Acidohydrogenase strengthens production path of the pyruvic acid to l-Alanine, constructs 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 at
Divide simply, anaerobic fermentation power consumption is low, and product yield is high.The construction method of building l-Alanine production bacterial strain is single at present,
It is related to a plurality of contended path, genetic engineering is complicated for operation.
Summary of the invention
To solve the above-mentioned problems, the present invention produces fumaric acid by Escherichia coli fermentation, utilizes Aspartase AspA
It is converted into aspartic acid, further produces l-Alanine, building using L-Aspartic acid-β-decarboxylase AsD conversion aspartic acid
The new mushroom-seed culturing of efficient production l-Alanine.
The first purpose of the invention is to provide a kind of Escherichia coli recombinant strain, the Escherichia coli recombinant strain be by
It can produce in the Escherichia coli of fumaric acid, be overexpressed Aspartase AspA and L-Aspartic acid-β-decarboxylase AsD.
In one embodiment of the invention, the Aspartase AspA and L-Aspartic acid-β-decarboxylase AsD is logical
Nucleotide sequence RBS1 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 gene aspA is such as
Shown in SEQ ID NO.1, the L-Aspartic acid-β-decarboxylase AsD encoding gene asD nucleotide sequence such as SEQ ID
Shown in NO.2.
In one embodiment of the invention, the Escherichia coli for producing fumaric acid are Escherichia coli CICC.23846.
In one embodiment of the invention, pass through Aspartase AspA and L- described in pEtac plasmid expressing in series
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 integration constitutive promoter J23119.
In one embodiment of the invention, the nucleotide of the l-Alanine transport protein AlaE encoding gene YgaW
Sequence is as shown in SEQ ID NO.3.
A second object of the present invention is to provide the construction method of the Escherichia coli recombinant strain, the method is specifically:
(1) pEtac plasmid is used, using nucleotide sequence RBS1 as shown in SEQ ID NO.4 as link peptide, table of connecting
Up to nucleotide sequence Aspartase AspA 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 nucleotide sequence the l-Alanine transport protein AlaE upstream region of gene 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 is obtained.
Third purpose of the present invention is to provide a kind of utilization recombination bacillus coli E.coli P-119 fermenting and producing 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 34~36 DEG C of temperature, stirs 80~120rpm, when OD600 reaches 10 in 6.8~7.2,0.1~1vvm of ventilatory capacity
When~15,2~3g/L lactose induction Aspartase is added and the expression of L-Aspartic acid-β-decarboxylase, fermentation process work as Portugal
When grape sugar concentration is lower than 10~15g/L, 500~700g/L glucose solution is added, every liter of fermentation liquid adds 20~40g grape
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 as follows: 60~80g/L of initial glucose, yeast powder 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 production l-Alanine.
Usefulness of the present invention:
For Escherichia coli as most common cell factory, genetic background is clear, in the large intestine for capableing of fermenting and producing fumaric acid
Aspartase AspA and L-Aspartic acid-β-decarboxylase AsD is introduced in bacillus, and 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 use it is honest and clean
Valence glucose fermentation produces the superior strain of l-Alanine, and the production path there is no relevant report at present at present.
Detailed description of the invention
The building of Fig. 1: pEtac-aspA-asD co-expression plasmid.
Specific embodiment
It is all using routine experiment method, implementing material can obtain from commercial channels in following embodiments.
In following embodiments, escherichia coli (Escherichia coli) is starting strain, is 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 assay: 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 flask respectively, and 0.09gCTAB powder and 2.5mL ion is added
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 rapid centrifugation dilution is taken to carry out liquid phase detection.
L-Aspartic acid-β-decarboxylase AsD Enzyme activity assay: 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 induction 12h is weighed respectively in 250mL conical flask, is added
0.09g CTAB powder and 14mL deionized water, be added 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 reaction was completed, appropriate reaction solution rapid centrifugation dilution is taken to carry out liquid phase detection.
Enzyme activity unit (U): enzyme amount required for 1 μm of oL product of production is defined as 1 enzyme-activity unit per minute.
Glucose assays method: divided using SBA-40 bio-sensing analyzer (Shandong Province academy sciences Biology Research Institute)
Analysis.
Produce the calculating of intensity: production intensity (g/L/h)=l-Alanine yield (g/L)/fermentation time (h).
Embodiment 1: the building of double enzyme coexpression bacterial strains
(1) genome of e. coli k12 and Comamonas testosteroni is extracted using bacterial genomes extracts kit
DNA；
(2) it is cloned from genome of E.coli DNA using primer AspA-up and AspA-down in table 1 and obtains aspA base
Cause is connected to expression vector pETac using SacI and NotI double digestion, 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 base
Cause；
(4) after the 4 sections of asD gene sequencing obtained above-mentioned clone are correct, using HindIII and NotI double digestion, respectively
It is connected to pETac-aspA plasmid 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 medium (100mg/L kanamycin sulfate), 35 DEG C of culture 10h, according to
4% inoculum concentration is inoculated in Medium of shaking flask fermentation, and 2g/L lactose is added when OD600=2.0, continues to cultivate, when glucose disappears
End fermentation is exhausted, l-Alanine yield in centrifugation detection supernatant collects bacterium mud and detects enzyme activity；
Fermentation medium components: initial glucose 60g/L, yeast powder 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 between two genes under same culture conditions
L-Alanine yield is up to 37.6g/L when sequence, and AspA and AsD enzyme activity are respectively 301U/g and 478U/g when fermentation ends.
The building of 1 co-expression plasmid of table uses primer
2 recombinant bacterium fermenting and producing l-Alanine of table
Embodiment 2: the building of L-alanine with high yield Escherichia coli
Constitutive promoter J23119 is added in YgaW genomic upstream using the principle of Red homologous recombination
(BBa23119), strengthen AlaE protein expression, 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, is to draw with Kan-S, Kan-A
Object, pKD4 are template, expand kan gene, and using YgaW-S, YgaW-A as primer, e. coli k12 genome is template, amplification
YgaW gene obtains Kan+YgaW segment using fusion DNA vaccine, is connected to PMD19 and carries out gene sequencing；
(2) using 119-S and YgaW-A as primer, the PMD19 plasmid that correct Kan+YgaW segment is sequenced in connection is template,
PCR obtains having the knockout frame of homology arm, J23119 promoter, Kan gene；
(3) pKD46 plasmid is 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 resistance；
(4) with making its lose pKD46 plasmid after 43 DEG C of positive colony bacterial strain of kan resistance cultures, prepare electric-shock feeling by
State cell, electroporated plasmid pCP20.Conversion fluid is coated on the LB plate containing ampicillin, 30 DEG C of culture 12h, screening
Positive transformant.
(5) transformant is transferred to the recombination of 43 DEG C of cultures 12h screening ampicillins and kalamycin resistance while missing again
Bacterium, J23119 promoter successful integration, imports co-expression plasmid pETac-aspA-asD (RBS1) is constructed 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 is trained in the way of in embodiment 1
It supports, fermentation ends detect l-Alanine content in fermentation liquid, and ferment 48h, and E.coli P-119 produces l-Alanine 49.8g/L,
37.6g/L compared to E.coli PAD RBS1 improves 32.4%.
The gene constructed J23119 promoter of table 3YgaW uses primer
Embodiment 3:E.coli P-119 fermenting and producing l-Alanine
Seed culture based component: yeast powder 5g/L, peptone 10g/L, NaCl 5.0g/L, MgSO4·7H2O 1.0g/L。
Fermentation medium components: initial glucose 70g/L, yeast powder 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 seed culture medium from the appropriate E.coli P-119 of inclined-plane picking, 35 DEG C, 200rpm shaken cultivation 10~
12h, shake-flask seed are inoculated in fermentor according to 10% inoculum concentration, in 5L fermentor culture medium liquid amount be 3L, dilute sulfuric acid and
Ammonium hydroxide control fermentation pH is 6.8~7.2, ventilatory capacity 0.5vvm, 35 DEG C of temperature, 100rpm is stirred, when OD600 reaches 25~30
When, 2g/L lactose induction Aspartase and aspartic acid-beta-decarboxylation expression of enzymes is added, when concentration of glucose is lower than 10~15g/
When L, 600g/L glucose solution is added, every liter of fermentation liquid adds 60g glucose, adds altogether 2 times, ties after glucose consumption is most
Beam fermentation.
As a result: fermentation 45h, l-Alanine yield reach 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: yeast powder 5g/L, peptone 10g/L, NaCl 5.0g/L, MgSO4·7H2O 1.0g/L。
Fermentation medium components: initial glucose 70g/L, yeast powder 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 seed culture medium from the appropriate E.coli P-119 of inclined-plane picking, 35 DEG C, 200rpm shaken cultivation 10~
12h, shake-flask seed are inoculated in fermentor according to 4% inoculum concentration, and culture medium liquid amount is 3L, dilute sulfuric acid and ammonia in 5L fermentor
Water management ferments pH for 6.8~7.2, ventilatory capacity 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 is added, when concentration of glucose is lower than 10~15g/L
When, 600g/L glucose solution is added, every liter of fermentation liquid adds 30g glucose, adds altogether 4 times, ties after glucose consumption is most
Beam fermentation.
As a result: fermentation 45h, l-Alanine yield reach 147g/L, and 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 intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
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 (7)
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, it is overexpressed Aspartase AspA and L-Aspartic acid-β-decarboxylase AsD；The Aspartase AspA and
L-Aspartic acid-β-decarboxylase AsD is connected by nucleotide sequence RBS1 as shown in SEQ ID NO.4；Pass through pEtac matter
Aspartase AspA and L-Aspartic acid-β-decarboxylase AsD described in grain expressing in series；The Escherichia coli recombinant strain is in L- third
Propylhomoserin transport protein AlaE encoding gene YgaW upstream integration constitutive promoter J23119.
2. Escherichia coli recombinant strain according to claim 1, which is characterized in that the Aspartase AspA encoding gene
The nucleotide sequence of aspA is as shown in SEQ ID NO.1, the L-Aspartic acid-β-decarboxylase AsD encoding gene asD nucleosides
Acid sequence is as shown in SEQ ID NO.2.
3. Escherichia coli recombinant strain according to claim 1, which is characterized in that the Escherichia coli for producing fumaric acid are big
Enterobacteria CICC.23846.
4. 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.
5. a kind of 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 being inoculated in fermentation medium, dilute sulfuric acid and ammonium hydroxide control pH are 6.8
~7.2,0.1~1vvm of ventilatory capacity, stir 80~120rpm by 34~36 DEG C of temperature, when OD600 reaches 10~15, addition 2~
3g/L lactose induces Aspartase and the expression of L-Aspartic acid-β-decarboxylase, and fermentation process is lower than 10 when concentration of glucose
When~15g/L, 500~700g/L glucose solution is added, every liter of fermentation liquid adds 20~40g glucose, it adds altogether 2~6 times,
Terminate fermentation after glucose consumption is most.
6. according to the method described in claim 5, it is characterized in that, fermentation medium components are as follows: 60~80g/ of initial glucose
L, 4~6g/L of yeast powder, 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。
7. application of the Escherichia coli recombinant strain described in claim 1 in production l-Alanine.
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 CN108060114A (en) | 2018-05-22 |
| CN108060114B true 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) |
Families Citing this family (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 |
| CN109468254B (en) * | 2018-11-13 | 2021-03-26 | 江南大学 | Method for improving production efficiency of L-alanine and application thereof |
| 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 |
| 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 |
| 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 |
| 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 |
Family Cites Families (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 |
| CN102618570B (en) * | 2012-03-20 | 2014-04-09 | 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
Also Published As
| Publication number | Publication date |
|---|---|
| CN108060114A (en) | 2018-05-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108060114B (en) | A kind of Escherichia coli of fermenting and producing l-Alanine and its application | |
| CN105296456B (en) | A kind of stability-enhanced glutamic acid decarboxylase enzyme mutant of pH and its application | |
| CN106148265B (en) | It is a kind of produce chondroitinase recombined bacillus subtilis and its application | |
| 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 | |
| CN106957878A (en) | A kind of method that living things catalysis produces 2 benzyl carbinols | |
| CN106434510A (en) | Genetically engineered bacterium for producing L-aspartic acid through fermentation | |
| CN104046586B (en) | One strain gene engineering bacterium and the application in producing (2R, 3R)-2,3-butanediol thereof | |
| CN104131041A (en) | Production method for alpha-ketoglutaric acid | |
| CN112359005A (en) | Escherichia coli engineering bacterium with improved acid stress capability and application thereof | |
| CN111748535B (en) | Alanine dehydrogenase mutant and application thereof in fermentation production of L-alanine | |
| CN106566823A (en) | Cloning of novel glutamate decarboxylase gene and application thereof | |
| CN107227284A (en) | A kind of restructuring streptococcus zooepidemicus for the micromolecule hyaluronic acid that ferments | |
| CN112391329B (en) | Escherichia coli engineering bacteria with improved acid stress resistance and application thereof | |
| CN111411066B (en) | Double-way composite neuraminic acid-producing bacillus subtilis and construction method thereof | |
| CN109371070A (en) | A kind of method of high yield α-ketoisovaleric acid | |
| CN102382790A (en) | Recombinant bacillus subtilis for high yield of catalase (CAT), and construction method and application thereof | |
| CN112391330B (en) | Method for improving acid stress resistance of recombinant escherichia coli | |
| CN108504617A (en) | A kind of Escherichia coli recombinant strain and its construction method of high-yield L-lysine | |
| CN110791536B (en) | Biosynthesis method of levodopa | |
| CN114672525A (en) | Biosynthesis method and application of N-acetyl-5-methoxytryptamine | |
| CN106434611A (en) | Method for preparing L-ornithine by means of double-enzyme coupling by taking L-arginine as raw material | |
| 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 |
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 |