CN109251941A - A kind of Escherichia coli of high yield succinic acid and its application - Google Patents

A kind of Escherichia coli of high yield succinic acid and its application Download PDF

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CN109251941A
CN109251941A CN201811156449.1A CN201811156449A CN109251941A CN 109251941 A CN109251941 A CN 109251941A CN 201811156449 A CN201811156449 A CN 201811156449A CN 109251941 A CN109251941 A CN 109251941A
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escherichia coli
succinic acid
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CN109251941B (en
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刘佳
刘立明
罗秋玲
陈修来
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Wuxi Chenming Biotechnology Co ltd
Jiangnan University
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Jiangnan University
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/0104—Pyruvate kinase (2.7.1.40)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Abstract

The invention discloses a kind of screening of high yield succinate producing strain and construction methods, belong to technical field of bioengineering.The present invention produces the wild strain of succinic acid by isolating from camel cud content, and protein engineering transformation is carried out to the pyruvate kinase of the bacterial strain, reduce the enzyme activity of pyruvate kinase, the succinic acid production for obtaining mutant strain improves 26.4% in shaking flask level, anaerobic fermentation 55h in the fermenter, succinic acid production reaches 80.0g/L, and succinic acid acid production intensity reaches 1.45g/L/h.

Description

A kind of Escherichia coli of high yield succinic acid and its application
Technical field
The present invention relates to a kind of Escherichia coli of high yield succinic acid and its applications, belong to technical field of bioengineering.
Background technique
Succinic acid, scientific name succinic acid are a kind of important C4 platform chemicals.Starting as synthesis general chemistry product is former Material, succinic acid are widely used in food, chemistry, medicine and other field, the report of publication in U.S. Department of Energy 2004 Middle first be classified as succinic acid in 12 kinds of most potential large biological-based chemicals.
The conventional production methods of succinic acid are chemical synthesis, mainly there is paraffin oxidation style, methyl chloroacetate cyaniding hydrolysis Method and vanadic anhydride catalytic hydrogenation method etc., but the problems such as reduction due to petroleum resources and environmental pollution are got worse, chemistry The drawbacks of synthetic method, increasingly shows.And succinic acid is produced by fermentation method, it can get rid of to non-renewable strategic resource stone The dependence of oil, using renewable resource, fixed carbon dioxide mitigates greenhouse effects, shows good development prospect.It grinds at present The strain for studying carefully more production succinic acid has: Actinobacillus succinogenes, Anaerobiospirillum succinoproducens and Escherichia coli.Produce succinic acid Actinobacillus is usually to screen from nature, is oriented transformation, is resistant to the succinate of high concentration, Guettler M Et al. using mutant strain Actinobacillus succinogenes FZ53 produce succinic acid production highest, using glucose as carbon source, fermentation 48h produce Amount can achieve maximum output 110g/L, and the research about Actinobacillus succinogenes strain is less, needs further to it Physiological property, fermenting property and genetic background are studied.The utilizable fermentation substrate of Anaerobiospirillum succinoproducens is wider It is general, such as glucose, lactose, glycerol etc., the result of study of Samuelov et al. show under optimum condition, produces succinic acid and detests The yield of oxygen spirillum succinic acid can achieve 1.2mol/1.0mol glucose, maximum output 65.0g/L, but the bacterial strain is sent out Ferment needs stringent anaerobic environment, is difficult to realize in industrial applications.For Escherichia coli as type strain, genetic background is clear, It is easy to operate, strain can be transformed using the technology of various molecular biology, so using Escherichia coli fermentation succinic acid Have become a hot spot, research also achieves numerous progress, and Vemuri G N carries out two using recombination bacillus coli AFP111 Footwork fermentation 76h, the ultimate density of succinic acid can reach 99.2g/L.
The production efficiency of Escherichia coli fermentation is lower at present, up to 1.3g/L/h, usually there is acetic acid, ethyl alcohol in fermentation liquid Equal by-products, and be unable to the fermentation of enduring high-concentration sugar etc..In order to obtain high performance production bacterial strain, it usually needs utilize tradition The method combined is transformed with molecular biology in breeding means, various groups of credit analysis.
Summary of the invention
It is by inhibiting big the first purpose of the invention is to provide a kind of application of Escherichia coli in production succinic acid The activity of pyruvate kinase in enterobacteria, improves the ability that Escherichia coli produce succinic acid, and specific steps include:
(1) the gene encoding for pyruvate kinase pykA in clone E. coli；
(2) to clone obtained gene encoding for pyruvate kinase pykA as parent, the pyruvate kinase for constructing enzyme activity decline is prominent Variant；
(3) the gene encoding for pyruvate kinase pykA of Escherichia coli is replaced with into pyruvate kinase mutant gene, is recombinated Escherichia coli；
(4) recombinant bacterium is activated in seed culture medium；
(5) recombinant bacterium after inoculation activation ferments into fermentation medium.
In one embodiment of the invention, the Escherichia coli include Escherichia coli FMME-SuA (Escherichia Coli), the Escherichia coli were preserved in China typical culture collection center on August 27th, 2018, and deposit number is CCTCC NO:M 2018568, preservation address are Hubei China Wuhan, Wuhan University.
In one embodiment of the invention, pyruvate kinase mutant described in step (3) includes pykA (N22A/N23A), the nucleotide sequence of the enzyme mutant is encoded as shown in SEQ ID NO.9.
In one embodiment of the invention, recombination bacillus coli construction method includes homologous heavy with Red in step (3) Gene encoding for pyruvate kinase pykA in original strain is replaced with the pyruvate kinase mutant gene of enzyme activity decline by group technology.
In one embodiment of the invention, activation described in step (4) is by recombination bacillus coli at 30-40 DEG C, 200rpm shaken cultivation 12-14h.
In one embodiment of the invention, fermentation described in step (5) is to be inoculated with seed by the inoculum concentration of 10-25% Liquid stirs 100-150rpm, anaerobic fermentation 50-65h into fermentation medium at 30-40 DEG C.
It in one embodiment of the invention, include: glucose 90-120g/L in fermentation medium used in step (5), Corn pulp 4-10g/L, Na2HPO4·12H2O 1-2g/L, NaH2PO4·2H2O 2-3g/L, MgSO4·7H2O 0.2-0.3g/L, CaCl2 0.1-0.2g/L。
A second object of the present invention is to provide a kind of Escherichia coli FMME-SuA (Escherichia coli), features It is, the Escherichia coli were preserved in China typical culture collection center on August 27th, 2018, and deposit number is CCTCC NO:M 2018568, preservation address are Hubei China Wuhan, Wuhan University.
Third object of the present invention is to provide a kind of recombination bacillus colis, which is characterized in that is with Escherichia coli FMME- SuA is starting strain, will be pykA (N22A/N23A) gene as shown in SEQ ID NO.9 using Red methods of homologous recombination DNA fragmentation imports in Escherichia coli FMME-SuA, and the gene encoding for pyruvate kinase pykA replaced in Escherichia coli FMME-SuA is obtained 's.
Fourth object of the present invention is to provide application of the above-mentioned recombination bacillus coli in production succinic acid.
Fifth object of the present invention is to provide application of the above-mentioned Escherichia coli FMME-SuA in production succinic acid.
Usefulness of the present invention:
1. further being reduced using protein engineering means by independently screening bacterial strain for high yield of succinic acid from camel cud The enzyme activity of pyruvate kinase PykA can significantly improve succinic acid and produce sour efficiency, the production intensity of succinic acid is made to reach 1.45g/ (Lh), heteroacid rate can also be reduced, the downstream purification of product is conducive to.
2. the Escherichia coli independently screened can not need to add glucose with enduring high-concentration glucose in fermentation process, The bacterial strain mutant strain can use corn pulp and glucose anaerobic fermentation cheap and easy to get, realize the efficient production of succinic acid.
Biomaterial preservation
Escherichia coli FMME-SuA was preserved in China typical culture collection center, preservation address on August 27th, 2018 For Hubei China Wuhan Wuhan University, deposit number is CCTCC NO:M 2018568.
Detailed description of the invention
PykA gene replacement is the flow chart of pykA (N22A/N23A) gene using Red homologous recombination by Fig. 1.
Specific embodiment
In following embodiments, explain is all to implement material from business way using conventional experimental method more Diameter can obtain.
In following embodiments, using Escherichia coli FMME-SuA as starting strain, can also be used Escherichia coli derivative strain or The Escherichia coli of succinic acid can be produced as starting strain after other modifications.
(1) pyruvate kinase Enzyme activity assay
It is detected using Agilent pyruvate kinase kit.
(2) measurement of succinic acid, acetic acid and ethanol content
Succinic acid and acetic acid: high performance liquid chromatography, chromatographic column: Aminex HPX-87H；Mobile phase: 5mmol/L H2SO4；Flow velocity: 0.6mL/min；Temperature: 35 DEG C；Sample volume: 10 μ L；Detector: UV detector.
Ethyl alcohol: high performance liquid chromatography, chromatographic column: Aminex HPX-87H；Mobile phase: 5mmol/L H2SO4；Flow velocity: 0.6mL/min；Temperature: 35 DEG C；Sample volume: 10 μ L；Detector: differential refraction detector.
(3) glucose assays method
It is analyzed using SBA-40 bio-sensing sensing analysis instrument.
(4) culture medium
Enriched medium: yeast powder 20g/L, glucose 25g/L, fumaric acid sodium 8g/L, CaCO32.0g, pH 4.5- 5.5。
Plate isolation base: beef extract 3g/L, peptone 5g/L, NaCl 5g/L, agar 20g/L, bromocresol green 2g/ L, pH 7.0.
Seed culture medium: beef extract 3g/L, peptone 5g/L, NaCl 5g/L.
Medium of shaking flask fermentation: corn pulp 20g/L, glucose 70g/L, Na2HPO42.5g/L, MgCl20.5g/L, CaCO33.0g, pH 4.5-5.5.
(5) calculation formula of intensity is produced
Produce intensity (g/L/h)=succinic acid production (g/L)/fermentation time (h).
(6) calculation formula of glucose yield
Glucose yield (%)=succinic acid production (g/L)/glucose additive amount (g/L) × 100
Embodiment 1: the screening of succinate producing strain
1. the enrichment culture of bacterial strain
Fresh camel cud content 10g is taken, is added in the sterile saline of bead and shakes 10min, so It is gone in the enriched medium that total volume is 200mL afterwards, 32-37 DEG C of Anaerobic culturel is transferred primary according to 30% inoculum concentration after for 24 hours.
2. the primary dcreening operation of bacterial strain
By the bacterium solution gradient dilution after enrichment culture, it is coated on agar medium containing bromocresol green, 32-37 DEG C of anaerobism training It supports, the plate color of part periphery of bacterial colonies becomes yellow after 24-36h, and the big bacterial strain of picking yellowish discoloration circle carries out culture presevation With further anaerobic fermentation；
3. the secondary screening of bacterial strain
37 DEG C of shake cultures in the seed culture medium of 24 deep-well plates are inoculated in biggish 756 plants of bacterial strain of circle of primary dcreening operation discoloration For 24 hours, according to the switching of 10% inoculum concentration in fermentation medium in 24 deep-well plates, 32-37 DEG C of anaerobic fermentation 48-64h, use is thin Layer chromatography (TCL) quantitative detection succinic acid, total 134 plants of bacterium can produce succinic acid, further use high performance liquid chromatography (HPLC) content of detection production succinic acid, obtains the bacterial strain F-121 that a plant height produces succinic acid, and succinic acid production reaches 31.9g/ L。
Embodiment 2: the identification of succinate producing strain
(1) colony characteristics and thalli morphology
Bacterial strain F-121 gradient dilution is coated on LB nutrient agar, and round microprotrusion, surface are formed after overnight incubation Smooth wet, neat in edge white single colonie.Microscopy shows that thallus is short and small rod-shaped, no gemma, and Gram's staining is feminine gender.
(2) physiological and biochemical property
The identification of customary physiological biochemical character is carried out to bacterium, as the result is shown: the strain fermentation glucose produces acid and produces gas, Lactose fermenters, maltose, xylose, mannitol, arabinose, azymic inositol, cellulose；Dynamic experiment, M-P experiment, indoles Experiment, catalase experiment are positive；Citric acid experiment, V-P experiment and urase experiment are negative, the biochemical with escherichia coli Matter matches
(3) 16S rDNA sequence is analyzed
It is extracted using DNA and illustrates extraction genomic DNA by DNA of bacteria extracts kit, using genomic DNA as template, Carry out PCR amplification with bacterial 16 S rDNA universal primer, amplified production send to company and is sequenced, sequencing result in NCBI into Row Blast is compared, and the bacterial strain is Escherichia coli as the result is shown.
Forward primer P16s8: sequence is as shown in SEQ ID NO.1；Reverse primer P16s-1492: sequence such as SEQ ID Shown in NO.2.
Comprehensive colonial morphology feature, Physiology and biochemistry detection and 16S rRNA analysis confirm that the bacterial strain is Escherichia coli, life Entitled FMME-SuA.The bacterial strain is submitted and is preserved in China typical culture collection center, and deposit number is CCTCC NO: M 2018568, the deposit date is on August 27th, 2018, preservation address was Hubei China Wuhan, Wuhan University.Further detection The enzyme activity of critical path during production of succinic acid, wherein lactic dehydrogenase (LDH), acetokinase (ACK) and pyruvate formate Lyases (PFL) Enzyme activity assay is feminine gender.
Embodiment 3: the protein engineering transformation of pyruvate kinase
(1) primed probe pykA-S (sequence is as shown in SEQ ID NO.3) and pykA-A (sequence such as SEQ ID are used Shown in NO.4) it clones to obtain pykA gene from the Escherichia coli FMME-SuA screened, it is SEQ that gene order is obtained after sequencing ID NO.5, amino acid sequence is SEQ ID NO.6, consistent with the gene encoding for pyruvate kinase sequence in e. coli k12 source.
(2) primer pykA-S1 (sequence is as shown in SEQ ID NO.7) and pykA-A1 (sequence such as SEQ ID NO.8 are used It is shown) and fallibility PCR kit clone to obtain pykA genetic fragment, using agarose gel electrophoresis, be tapped and recovered target fragment. Using NdeI and HindIII double digestion purpose band and pET28a plasmid, is connected overnight using T4 ligase, convert Escherichia coli BL21 competent cell is coated with the solid LB plate containing 100mg/L kanamycins, screens positive transformant, and building completes third Pyruvate kinase mutant library.
(3) positive transformant is verified using pykA-S and pykA-A primer PCR, PCR verification result is that positive bacterium colony connects Kind carries out fermented and cultured in LB culture medium, and at 37 DEG C, shake culture is added to OD600 to 0.8-1.0 under the conditions of 200rpm 0.4mmol/L IPTG, temperature are down to 25 DEG C and continue to cultivate 8-10h, and induction pyruvate kinase expression is examined using pyruvate kinase Test agent box detects the enzyme activity of different mutants, and screening obtains the significant decrease of pykA (N22A/N23A) mutant enzyme activity.After sequencing Obtaining gene order is SEQ ID NO.9, and amino acid sequence is SEQ ID NO.10, compared with original amino acid, 22 Asparagine with 23 sports alanine, and as shown in table 1, the enzyme activity after mutation has dropped 2.1 times.
1 mutant enzyme activity determination of table
Embodiment 4: the building of recombinant bacterial strain FMME-SuAP
It, will be in original strain with Red methods of homologous recombination using the Escherichia coli FMME-SuA screened as starting strain PykA gene replacement is pykA (N22A/N23A) gene, and detailed process such as Fig. 1 will be had homologous using Red homologous recombination technique The DNA fragmentation 1 of arm, kan gene and pykA (N22A/N23A) gene imports the Escherichia coli FMME-SuA for having pKD46 plasmid In, the alanine kinases pykA gene in homologous recombination replacement Escherichia coli FMME-SuA is carried out, is eliminated using pCP20 plasmid Kan resistance obtains mutant bacteria FMME-SuAP.
Embodiment 5: in the horizontal original strain FMME-SuA of shaking flask and recombinant bacterial strain FMME-SuAP fermenting and producing succinic acid
Original strain FMME-SuA and mutant strain FMME-SuAP fermenting and producing succinic acid, seed liquor are used in shaking flask level At 37 DEG C, 200rpm shake culture 12-14h, it is seeded to fermentation medium according to 10% inoculum concentration, is terminated after Anaerobic culturel 50h Fermentation takes the yield of glucose residual and succinic acid in sample detection fermentation liquid.The results are shown in Table 2, and ferment 50h, FMME- The succinic acid production of SuA and FMME-SuAP is respectively 33.0g/L and 41.7g/L, mutant strain output increased 26.4%.
Opportunistic pathogen and recombinant bacterium fermenting and producing succinic acid under 2 anaerobic condition of table
Embodiment 5: original strain FMME-SuA and recombinant bacterial strain FMME-SuAP fermenting and producing succinic acid 1 under anaerobic condition
(1) 7.5L fermentative medium formula: glucose 100g/L, corn pulp 4g/L, Na2HPO4·12H2O 1-2g/L, NaH2PO4·2H2O 2-3g/L, MgSO4·7H2O 0.2-0.3g/L, CaCl2 0.1g/L。
(2) original strain FMME-SuA is picked them separately from inclined-plane and recombinant bacterial strain FMME-SuAP is inoculated in seed culture medium In, 37 DEG C, 200rpm shaken cultivation 12h, shake-flask seed liquid is inoculated in 7.5L stirred fermentor according to 10% inoculum concentration respectively Anaerobic fermentation, fermentation medium liquid amount are 5.0L, 37 DEG C of temperature, stir 100rpm, are passed through the CO that gas is filtration sterilization2, Ventilatory capacity is 1-1.5vvm, and control gas outlet pressure is 0.05Mpa, and it is 5.0-5.5 that 4mol/L NaOH, which controls pH,.
(3) it ferments and stops when 65h, detect the concentration of glucose and succinic acid in recombinant bacterial strain FMME-SuAP fermentation liquid, Portugal Grape sugar remains 3.10g/L, succinic acid production 70.2g/L, and glucose yield is 72.4%, and production of succinic acid intensity is 1.40g/ L/h.Through detecting, without by-products such as acetic acid, ethyl alcohol in fermentation liquid.Original strain FMME-SuA glucose remains 5.32g/L, amber Amber acid yield is 57.2g/L, and glucose yield is 72.4%, and production of succinic acid intensity is 1.40g/L/h.The results are shown in Table 3.
Embodiment 6: original strain FMME-SuA and recombinant bacterial strain FMME-SuAP fermenting and producing succinic acid 2 under anaerobic condition
(1) 7.5L fermentative medium formula: glucose 110g/L, corn pulp 8g/L, Na2HPO4·12H2O 1-2g/L, NaH2PO4·2H2O 2-3g/L, MgSO4·7H2O 0.2-0.3g/L, CaCl2 0.1g/L。
(2) original strain FMME-SuA is picked them separately from inclined-plane and recombinant bacterial strain FMME-SuAP is inoculated in seed culture medium In, 37 DEG C, 200rpm shaken cultivation 12h, shake-flask seed liquid is inoculated in 7.5L stirred fermentor according to 20% inoculum concentration respectively, Fermentation medium liquid amount is 5.5L, 37 DEG C of temperature, stirs 200rpm, is passed through the CO that gas is filtration sterilization2, ventilatory capacity 1- 1.5vvm, control gas outlet pressure are 0.05MPa, and it is 5.0-5.5 that 4mol/L NaOH, which controls pH,.
(3) it ferments and stops when 55h, detect the concentration of glucose and succinic acid in fermentation liquid respectively, recombinate in fermented liquid Glucose remains 6.11g/L, succinic acid production 77.8g/L, and glucose yield is 74.9%, and production of succinic acid intensity is 1.41g/L/h.Through detecting, without by-products such as acetic acid, ethyl alcohol in fermentation liquid.Original strain FMME-SuA glucose residual 11.2g/L, succinic acid production 60.1g/L, glucose yield are 60.8%, and production of succinic acid intensity is 1.09g/L/h., knot Fruit is as shown in table 3.
Embodiment 7: tolerance of the recombinant bacterial strain FMME-SuAP to high glucose concentration
(1) 7.5L fermentative medium formula: glucose 120g/L, corn pulp 8g/L, Na2HPO4·12H2O 1-2g/L, NaH2PO4·2H2O 2-3g/L, MgSO4·7H2O 0.2-0.3g/L, CaCl2 0.1g/L。
(2) from inclined-plane, picking original strain FMME-SuA and FMME-SuAP are inoculated in seed culture medium respectively, and 37 DEG C, 200rpm shaken cultivation 12h, shake-flask seed liquid are inoculated in 7.5L stirred fermentor according to 20% inoculum concentration respectively, fermented and cultured Base liquid amount is 5.5L, 37 DEG C of temperature, stirs 200rpm, is passed through the CO that gas is filtration sterilization2, ventilatory capacity 1-1.5vvm, Control gas outlet pressure is 0.05Mpa, and it is 5.0-5.5 that 4mol/L NaOH, which controls pH,.
(3) it ferments and stops when 55h, detect the concentration and acetic acid of glucose and succinic acid, ethyl alcohol in fermentation liquid respectively and contain Amount.
The result shows that glucose is residual after recombinant bacterial strain fermentation in the fermentation medium in the very high situation of concentration of glucose 5.98g/L, succinic acid production 80.0g/L are stayed, and glucose remains 13.8g/L after original strain fermentation, succinic acid production is 60.5g/L.Prove that recombinant bacterial strain FMME-SuAP is able to maintain the efficient life of succinic acid when concentration of glucose is 100-120g/L It produces, production intensity reaches 1.40-1.45g/L/h, is significantly higher than the most high production intensity that current Escherichia coli produce succinic acid (1.30g/L/h), the bacterial strain have stronger tolerance to high glucose concentration, do not need to add glucose in fermentation process, Almost without by-product, production technology is simplified.
Opportunistic pathogen and recombinant bacterium fermenting and producing succinic acid under 3 anaerobic condition of table
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 LISTING
<110>Southern Yangtze University
<120>a kind of Escherichia coli of high yield succinic acid and its application
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ggtctgccaa ttttcgccat gtcgcgccat gaacgtacgc tgaacctgac tgctctctat 1260
cgtggcgtta cgccggtgca ctttgatagc gctaatgacg gcgtagcagc tgccagcgaa 1320
gcggttaatc tgctgcgcga taaaggttac ttgatgtctg gtgacctggt gattgtcacc 1380
cagggcgacg tgatgagtac cgtgggttct actaatacca cgcgtatttt aacggtagag 1440
taa 1443
<210> 6
<211> 480
<212> PRT
<213> Escherichia coli
<400> 6
Met Ser Arg Arg Leu Arg Arg Thr Lys Ile Val Thr Thr Leu Gly Pro
1 5 10 15
Ala Thr Asp Arg Asp Asn Asn Leu Glu Lys Val Ile Ala Ala Gly Ala
20 25 30
Asn Val Val Arg Met Asn Phe Ser His Gly Ser Pro Glu Asp His Lys
35 40 45
Met Arg Ala Asp Lys Val Arg Glu Ile Ala Ala Lys Leu Gly Arg His
50 55 60
Val Ala Ile Leu Gly Asp Leu Gln Gly Pro Lys Ile Arg Val Ser Thr
65 70 75 80
Phe Lys Glu Gly Lys Val Phe Leu Asn Ile Gly Asp Lys Phe Leu Leu
85 90 95
Asp Ala Asn Leu Gly Lys Gly Glu Gly Asp Lys Glu Lys Val Gly Ile
100 105 110
Asp Tyr Lys Gly Leu Pro Ala Asp Val Val Pro Gly Asp Ile Leu Leu
115 120 125
Leu Asp Asp Gly Arg Val Gln Leu Lys Val Leu Glu Val Gln Gly Met
130 135 140
Lys Val Phe Thr Glu Val Thr Val Gly Gly Pro Leu Ser Asn Asn Lys
145 150 155 160
Gly Ile Asn Lys Leu Gly Gly Gly Leu Ser Ala Glu Ala Leu Thr Glu
165 170 175
Lys Asp Lys Ala Asp Ile Lys Thr Ala Ala Leu Ile Gly Val Asp Tyr
180 185 190
Leu Ala Val Ser Phe Pro Arg Cys Gly Glu Asp Leu Asn Tyr Ala Arg
195 200 205
Arg Leu Ala Arg Asp Ala Gly Cys Asp Ala Lys Ile Val Ala Lys Val
210 215 220
Glu Arg Ala Glu Ala Val Cys Ser Gln Asp Ala Met Asp Asp Ile Ile
225 230 235 240
Leu Ala Ser Asp Val Val Met Val Ala Arg Gly Asp Leu Gly Val Glu
245 250 255
Ile Gly Asp Pro Glu Leu Val Gly Ile Gln Lys Ala Leu Ile Arg Arg
260 265 270
Ala Arg Gln Leu Asn Arg Ala Val Ile Thr Ala Thr Gln Met Met Glu
275 280 285
Ser Met Ile Thr Asn Pro Met Pro Thr Arg Ala Glu Val Met Asp Val
290 295 300
Ala Asn Ala Val Leu Asp Gly Thr Asp Ala Val Met Leu Ser Ala Glu
305 310 315 320
Thr Ala Ala Gly Gln Tyr Pro Ser Glu Thr Val Ala Ala Met Ala Arg
325 330 335
Val Cys Leu Gly Ala Glu Lys Ile Pro Ser Ile Asn Val Ser Lys His
340 345 350
Arg Leu Asp Val Gln Phe Asp Asn Val Glu Glu Ala Ile Ala Met Ser
355 360 365
Ala Met Tyr Ala Ala Asn His Leu Lys Gly Val Thr Ala Ile Ile Thr
370 375 380
Met Thr Glu Ser Gly Arg Thr Ala Leu Met Thr Ser Arg Ile Ser Ser
385 390 395 400
Gly Leu Pro Ile Phe Ala Met Ser Arg His Glu Arg Thr Leu Asn Leu
405 410 415
Thr Ala Leu Tyr Arg Gly Val Thr Pro Val His Phe Asp Ser Ala Asn
420 425 430
Asp Gly Val Ala Ala Ala Ser Glu Ala Val Asn Leu Leu Arg Asp Lys
435 440 445
Gly Tyr Leu Met Ser Gly Asp Leu Val Ile Val Thr Gln Gly Asp Val
450 455 460
Met Ser Thr Val Gly Ser Thr Asn Thr Thr Arg Ile Leu Thr Val Glu
465 470 475 480
<210> 7
<211> 36
<212> DNA
<213>artificial synthesized
<400> 7
ggaattccat atgatgtcca gaaggcttcg cagaac 36
<210> 8
<211> 31
<212> DNA
<213>artificial synthesized
<400> 8
cccaagcttt tactctaccg ttaaaatacg c 31
<210> 9
<211> 1443
<212> DNA
<213>artificial synthesized
<400> 9
atgtccagaa ggcttcgcag aacaaaaatc gttaccacgt taggcccagc aacagatcgc 60
gatgcagcac ttgaaaaagt tatcgcggcg ggtgccaacg ttgtacgtat gaacttttct 120
cacggctcgc ctgaagatca caaaatgcgc gcggataaag ttcgtgagat tgccgcaaaa 180
ctggggcgtc atgtggctat tctgggtgac ctccaggggc ccaaaatccg tgtatccacc 240
tttaaagaag gcaaagtttt cctcaatatt ggggataaat tcctgctcga cgccaacctg 300
ggtaaaggtg aaggcgacaa agaaaaagtc ggtatcgact acaaaggcct gcctgctgac 360
gtcgtgcctg gtgacatcct gctgctggac gatggtcgcg tccagttaaa agtactggaa 420
gttcagggca tgaaagtgtt caccgaagtc accgtcggtg gtcccctctc caacaataaa 480
ggtatcaaca aacttggcgg cggtttgtcg gctgaagcgc tgaccgaaaa agacaaagca 540
gacattaaga ctgcggcgtt gattggcgta gattacctgg ctgtctcctt cccacgctgt 600
ggcgaagatc tgaactatgc ccgtcgcctg gcacgcgatg caggatgtga tgcgaaaatt 660
gttgccaagg ttgaacgtgc ggaagccgtt tgcagccagg atgcaatgga tgacatcatc 720
ctcgcctctg acgtggtaat ggttgcacgt ggcgacctcg gtgtggaaat tggcgacccg 780
gaactggtcg gcattcagaa agcgttgatc cgtcgtgcgc gtcagctaaa ccgagcggta 840
atcacggcga cccagatgat ggagtcaatg attactaacc cgatgccgac gcgtgcagaa 900
gtcatggacg tagcaaacgc cgttctggat ggtactgacg ctgtgatgct gtctgcagaa 960
actgccgctg ggcagtatcc gtcagaaacc gttgcagcca tggcgcgcgt ttgcctgggt 1020
gcggaaaaaa tcccgagcat caacgtttct aaacaccgtc tggacgttca gttcgacaat 1080
gtggaagaag ctattgccat gtcagcaatg tacgcagcta accacctgaa aggcgttacg 1140
gcgatcatca ccatgaccga atcgggtcgt accgcgctga tgacctcccg tatcagctct 1200
ggtctgccaa ttttcgccat gtcgcgccat gaacgtacgc tgaacctgac tgctctctat 1260
cgtggcgtta cgccggtgca ctttgatagc gctaatgacg gcgtagcagc tgccagcgaa 1320
gcggttaatc tgctgcgcga taaaggttac ttgatgtctg gtgacctggt gattgtcacc 1380
cagggcgacg tgatgagtac cgtgggttct actaatacca cgcgtatttt aacggtagag 1440
taa 1443
<210> 10
<211> 480
<212> PRT
<213>artificial synthesized
<400> 10
Met Ser Arg Arg Leu Arg Arg Thr Lys Ile Val Thr Thr Leu Gly Pro
1 5 10 15
Ala Thr Asp Arg Asp Ala Ala Leu Glu Lys Val Ile Ala Ala Gly Ala
20 25 30
Asn Val Val Arg Met Asn Phe Ser His Gly Ser Pro Glu Asp His Lys
35 40 45
Met Arg Ala Asp Lys Val Arg Glu Ile Ala Ala Lys Leu Gly Arg His
50 55 60
Val Ala Ile Leu Gly Asp Leu Gln Gly Pro Lys Ile Arg Val Ser Thr
65 70 75 80
Phe Lys Glu Gly Lys Val Phe Leu Asn Ile Gly Asp Lys Phe Leu Leu
85 90 95
Asp Ala Asn Leu Gly Lys Gly Glu Gly Asp Lys Glu Lys Val Gly Ile
100 105 110
Asp Tyr Lys Gly Leu Pro Ala Asp Val Val Pro Gly Asp Ile Leu Leu
115 120 125
Leu Asp Asp Gly Arg Val Gln Leu Lys Val Leu Glu Val Gln Gly Met
130 135 140
Lys Val Phe Thr Glu Val Thr Val Gly Gly Pro Leu Ser Asn Asn Lys
145 150 155 160
Gly Ile Asn Lys Leu Gly Gly Gly Leu Ser Ala Glu Ala Leu Thr Glu
165 170 175
Lys Asp Lys Ala Asp Ile Lys Thr Ala Ala Leu Ile Gly Val Asp Tyr
180 185 190
Leu Ala Val Ser Phe Pro Arg Cys Gly Glu Asp Leu Asn Tyr Ala Arg
195 200 205
Arg Leu Ala Arg Asp Ala Gly Cys Asp Ala Lys Ile Val Ala Lys Val
210 215 220
Glu Arg Ala Glu Ala Val Cys Ser Gln Asp Ala Met Asp Asp Ile Ile
225 230 235 240
Leu Ala Ser Asp Val Val Met Val Ala Arg Gly Asp Leu Gly Val Glu
245 250 255
Ile Gly Asp Pro Glu Leu Val Gly Ile Gln Lys Ala Leu Ile Arg Arg
260 265 270
Ala Arg Gln Leu Asn Arg Ala Val Ile Thr Ala Thr Gln Met Met Glu
275 280 285
Ser Met Ile Thr Asn Pro Met Pro Thr Arg Ala Glu Val Met Asp Val
290 295 300
Ala Asn Ala Val Leu Asp Gly Thr Asp Ala Val Met Leu Ser Ala Glu
305 310 315 320
Thr Ala Ala Gly Gln Tyr Pro Ser Glu Thr Val Ala Ala Met Ala Arg
325 330 335
Val Cys Leu Gly Ala Glu Lys Ile Pro Ser Ile Asn Val Ser Lys His
340 345 350
Arg Leu Asp Val Gln Phe Asp Asn Val Glu Glu Ala Ile Ala Met Ser
355 360 365
Ala Met Tyr Ala Ala Asn His Leu Lys Gly Val Thr Ala Ile Ile Thr
370 375 380
Met Thr Glu Ser Gly Arg Thr Ala Leu Met Thr Ser Arg Ile Ser Ser
385 390 395 400
Gly Leu Pro Ile Phe Ala Met Ser Arg His Glu Arg Thr Leu Asn Leu
405 410 415
Thr Ala Leu Tyr Arg Gly Val Thr Pro Val His Phe Asp Ser Ala Asn
420 425 430
Asp Gly Val Ala Ala Ala Ser Glu Ala Val Asn Leu Leu Arg Asp Lys
435 440 445
Gly Tyr Leu Met Ser Gly Asp Leu Val Ile Val Thr Gln Gly Asp Val
450 455 460
Met Ser Thr Val Gly Ser Thr Asn Thr Thr Arg Ile Leu Thr Val Glu
465 470 475 480

Claims (10)

1. a kind of application of Escherichia coli in production succinic acid, which is characterized in that by inhibiting pyruvic acid in Escherichia coli to swash The activity of enzyme, improves the ability that Escherichia coli produce succinic acid, and specific steps include:
(1) the gene encoding for pyruvate kinase pykA in clone E. coli；
(2) to clone obtained pykA as parent, the pyruvate kinase mutant of enzyme activity decline is constructed；
(3) the gene encoding for pyruvate kinase pykA of Escherichia coli is replaced with into pyruvate kinase mutant gene, obtains recombination large intestine Bacillus；
(4) recombinant bacterium is activated in seed culture medium；
(5) recombinant bacterium after inoculation activation ferments into fermentation medium.
2. application according to claim 1, which is characterized in that the Escherichia coli include Escherichia coli FMME-SuA (Escherichia coli), the Escherichia coli were preserved in China typical culture collection center on August 27th, 2018, Deposit number is CCTCC NO:M 2018568, and preservation address is Hubei China Wuhan, Wuhan University.
3. application according to claim 1, which is characterized in that pyruvate kinase mutant described in step (3) includes PykA (N22A/N23A) encodes the nucleotide sequence of the enzyme mutant as shown in SEQ ID NO.9.
4. application according to claim 1, which is characterized in that recombination bacillus coli construction method includes using in step (3) Gene encoding for pyruvate kinase pykA in the original strain pyruvate kinase for replacing with enzyme activity decline is mutated by Red homologous recombination technique Body gene.
5. application according to claim 1, which is characterized in that activation described in step (4) is that recombination bacillus coli exists 30-40 DEG C, 200rpm shaken cultivation 12-14h.
6. application according to claim 1, which is characterized in that fermentation described in step (5) is the inoculum concentration by 10-25% Seed liquor is inoculated with into fermentation medium, 100-150rpm, anaerobic fermentation 50-65h are stirred at 30-40 DEG C；In used medium It include: glucose 90-120g/L, corn pulp 4-10g/L, Na2HPO4·12H2O 1-2g/L, NaH2PO4·2H2O 2-3g/L, MgSO4·7H2O 0.2-0.3g/L, CaCl2 0.1-0.2g/L。
7. a kind of Escherichia coli FMME-SuA (Escherichia coli) for producing succinic acid, which is characterized in that the large intestine bar Bacterium was preserved in China typical culture collection center on August 27th, 2018, and deposit number is CCTCC NO:M 2018568, Preservation address is Hubei China Wuhan, Wuhan University.
8. a kind of recombination bacillus coli, which is characterized in that be with Escherichia coli FMME-SuA as claimed in claim 7 for bacterium germination out Strain, using Red methods of homologous recombination, the DNA fragmentation of pykA (N22A/N23A) gene as shown in SEQ ID NO.9 is imported In Escherichia coli FMME-SuA, replace what the gene encoding for pyruvate kinase pykA in Escherichia coli FMME-SuA was obtained.
9. application of the Escherichia coli as claimed in claim 7 in production succinic acid.
10. application of the recombination bacillus coli according to any one of claims 8 in production succinic acid.
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