CN109251941A

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

Info

Publication number: CN109251941A
Application number: CN201811156449.1A
Authority: CN
Inventors: 刘佳; 刘立明; 罗秋玲; 陈修来
Original assignee: Wuxi Chenming Biotechnology Co ltd; Jiangnan University
Current assignee: Jiangnan University
Priority date: 2018-09-30
Filing date: 2018-09-30
Publication date: 2019-01-22
Anticipated expiration: 2038-09-30
Also published as: CN109251941B

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

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<120>a kind of Escherichia coli of high yield succinic acid and its application

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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

1. the application of a kind of Escherichia coli in producing succinic acid, it is characterized in that, by suppressing the activity of pyruvate kinase in Escherichia coli, improve the ability of Escherichia coli to produce succinic acid, and concrete steps comprise:

(1) clone the pyruvate kinase gene pykA in Escherichia coli;

(2) using the cloned pykA as a parent, construct a pyruvate kinase mutant with decreased enzyme activity;

(3) replacing the pyruvate kinase gene pykA of Escherichia coli with a pyruvate kinase mutant gene to obtain a recombinant Escherichia coli;

(4) activating the recombinant bacteria in the seed medium;

(5) inoculating the activated recombinant bacteria into the fermentation medium for fermentation.

2. application according to claim 1, is characterized in that, described Escherichia coli comprises Escherichia coli FMME-SuA (Escherichia coli), and described Escherichia coli has been deposited in China Type Culture Collection on August 27, 2018 , the deposit number is CCTCC NO:M 2018568, and the deposit address is Wuhan University, Wuhan, Hubei, China.

3. application according to claim 1 is characterized in that, the pyruvate kinase mutant described in step (3) comprises pykA (N22A/N23A), and the nucleotide sequence encoding the enzyme mutant is such as SEQ ID NO .9 shown.

4. application according to claim 1, is characterized in that, in step (3), recombinant Escherichia coli construction method comprises that the pyruvate kinase gene pykA in original strain is replaced by the pyruvate that enzyme activity declines with Red homologous recombination technology Kinase mutant gene.

5 . The application according to claim 1 , wherein the activation in step (4) is to shake the recombinant Escherichia coli at 30-40° C. and 200 rpm for 12-14 hours. 6 .

6. application according to claim 1, is characterized in that, the fermentation described in step (5) is to inoculate seed liquid to fermentation medium by 10-25% inoculum size, and stir 100-150rpm under 30-40 ℃ , anaerobic fermentation 50-65h; the medium used includes: glucose 90-120g/L, corn steep liquor 4-10g/L, Na ₂ HPO ₄ 12H ₂ O 1-2g/L, NaH ₂ PO ₄ 2H ₂ O 2-3g/L, MgSO ₄ ·7H ₂ O 0.2-0.3g/L, CaCl ₂ 0.1-0.2g/L.

7. a succinic acid-producing Escherichia coli FMME-SuA (Escherichia coli), is characterized in that, described Escherichia coli has been deposited in China Type Culture Collection Center on August 27, 2018, and the deposit number is CCTCC NO:M 2018568, deposited at Wuhan University, Wuhan, Hubei, China.

8. a recombinant Escherichia coli, is characterized in that, is the starting bacterial strain with Escherichia coli FMME-SuA described in claim 7, adopts Red homologous recombination method, as shown in pykA (N22A/N22A/ The DNA fragment of N23A) gene was introduced into Escherichia coli FMME-SuA and obtained by replacing the pyruvate kinase gene pykA in Escherichia coli FMME-SuA.

9. the application of Escherichia coli described in claim 7 in producing succinic acid.

10. The application of the recombinant Escherichia coli of claim 8 in the production of succinic acid.