CN109251941B - A kind of Escherichia coli with high production of succinic acid and its application - Google Patents

Abstract

The invention discloses a screening and constructing method of a high-yield succinic acid production strain, belonging to the technical field of biological engineering. According to the invention, a wild strain for producing succinic acid is separated from camel rumen contents, and protein engineering transformation is carried out on pyruvate kinase of the strain, so that the enzyme activity of the pyruvate kinase is reduced, the succinic acid yield of the obtained mutant strain is improved by 26.4% in a shake flask level, the succinic acid yield reaches 80.0g/L, and the succinic acid production strength reaches 1.45g/L/h through anaerobic fermentation in a fermentation tank.

Description

A kind of Escherichia coli with high production of succinic acid and its application

technical field

The invention relates to a high-yielding succinic acid Escherichia coli and its application, belonging to the technical field of bioengineering.

Background technique

Succinic acid, scientific name succinic acid, is an important C4 platform compound. As a starting material for the synthesis of general chemicals, succinic acid has a wide range of applications in food, chemistry, medicine and other fields. In a report released by the US Department of Energy in 2004, succinic acid was listed as 12 most potential bulk bio-based chemicals in the first place.

The traditional production method of succinic acid is chemical synthesis method, mainly including paraffin oxidation method, methyl chloroacetate cyanidation hydrolysis method and vanadium pentoxide catalytic hydrogenation method, etc., but due to problems such as the reduction of petroleum resources and the increasingly serious environmental pollution, The disadvantages of chemical synthesis methods are increasingly apparent. The production of succinic acid by fermentation can get rid of the dependence on non-renewable strategic resource petroleum, use renewable resources, fix carbon dioxide to reduce the greenhouse effect, and show a good development prospect. At present, the most studied succinic acid-producing strains are: Actinomyces succinic acid-producing, Anaerobic spirochetes succinic acid-producing and Escherichia coli. Actinobacillus succinate producing is usually screened from nature and directional transformation is carried out to tolerate high concentrations of succinate. Guettler M et al. used the mutant Actinobacter succinate producing FZ53 to produce the highest yield of succinic acid, using glucose as the Carbon source, the maximum yield of 48h fermentation can reach 110g/L. There are few studies on the species of Actinobacter succinates, and further research is needed on its physiological characteristics, fermentation performance and genetic background. Anaerobes succinates can utilize a wide range of fermentation substrates, such as glucose, lactose, glycerol, etc. The research results of Samuelov et al. It can reach 1.2mol/1.0mol glucose, and the maximum yield is 65.0g/L, but the fermentation of this strain requires strict anaerobic environment, which is difficult to achieve in industrial application. As a model strain, Escherichia coli has a clear genetic background and is easy to operate. Various molecular biology techniques can be used to transform the strain. Therefore, the use of Escherichia coli to ferment succinic acid has become a hot spot, and many research progress has been made. Vemuri GN uses Recombinant Escherichia coli AFP111 was fermented in two steps for 76h, and the final concentration of succinic acid could reach 99.2g/L.

At present, the production efficiency of E. coli fermentation is low, the highest is 1.3g/L/h, and the fermentation broth usually contains by-products such as acetic acid and ethanol, and cannot tolerate high-concentration sugar fermentation. In order to obtain high-performance production strains, it is usually necessary to combine traditional breeding methods, various omics analysis and molecular biology transformation.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a kind of application of Escherichia coli in producing succinic acid, which is to improve the ability of Escherichia coli to produce succinic acid by inhibiting the activity of pyruvate kinase in Escherichia coli, and the concrete steps include:

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

(2) Using the cloned pyruvate kinase gene 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.

In one embodiment of the present invention, the Escherichia coli includes Escherichia coli FMME-SuA (Escherichiacoli), and the Escherichia coli has been deposited in the China Center for Type Culture Collection on August 27, 2018, and the deposit number is CCTCC NO :M 2018568, deposited at Wuhan University, Wuhan, Hubei, China.

In one embodiment of the present invention, the pyruvate kinase mutant described in step (3) includes pykA (N22A/N23A), and the nucleotide sequence encoding the enzyme mutant is shown in SEQ ID NO.9.

In one embodiment of the present invention, the recombinant Escherichia coli construction method in step (3) comprises using Red homologous recombination technology to replace the pyruvate kinase gene pykA in the original strain with a pyruvate kinase mutant gene with reduced enzymatic activity.

In one embodiment of the present invention, the activation in step (4) is to shake the recombinant Escherichia coli at 30-40° C. and 200 rpm for 12-14 hours.

In one embodiment of the present invention, the fermentation described in step (5) is to inoculate the seed liquid into the fermentation medium according to the inoculum amount of 10-25%, stir at 100-150 rpm at 30-40 ° C, and anaerobic fermentation for 50 -65h.

In one embodiment of the present invention, the fermentation medium used in step (5) includes: glucose 90-120 g/L, corn steep liquor 4-10 g/L, Na ₂ HPO ₄ 12H ₂ O 1-2 g/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.

The second object of the present invention is to provide a kind of Escherichia coli FMME-SuA (Escherichia coli), characterized in that, the Escherichia coli has been deposited in the China Center for Type Culture Collection on August 27, 2018, and the deposit number is CCTCC NO:M 2018568, the deposit address is Wuhan University, Wuhan, Hubei, China.

The third object of the present invention is to provide a kind of recombinant Escherichia coli, which is characterized in that, with Escherichia coli FMME-SuA as the starting strain, using the Red homologous recombination method, pykA (N22A shown in SEQ ID NO. /N23A) gene DNA fragment was introduced into Escherichia coli FMME-SuA and obtained by replacing the pyruvate kinase gene pykA in Escherichia coli FMME-SuA.

The fourth object of the present invention is to provide the application of the above-mentioned recombinant Escherichia coli in the production of succinic acid.

The fifth object of the present invention is to provide the application of the above-mentioned Escherichia coli FMME-SuA in the production of succinic acid.

Advantages of the present invention:

1. By self-selecting high-yielding strains of succinate from camel rumen, and further using protein engineering methods to reduce the enzymatic activity of pyruvate kinase PykA, the acid production efficiency of succinate can be significantly improved, and the production intensity of succinate can reach 1.45g/(L· h), it can also reduce the rate of impurity acid, which is beneficial to the downstream purification of the product.

2. The self-selected Escherichia coli can tolerate high concentrations of glucose, and do not need to add glucose during the fermentation process. The mutant strain of this strain can utilize cheap and readily available corn steep liquor and glucose for anaerobic fermentation to achieve efficient production of succinic acid.

biological material preservation

Escherichia coli FMME-SuA was deposited in the China Center for Type Culture Collection on August 27, 2018, and the deposit address is Wuhan University, Wuhan, Hubei, China, and the deposit number is CCTCC NO:M 2018568.

Description of drawings

Figure 1 is a flow chart of replacing the PykA gene with the pykA (N22A/N23A) gene using Red homologous recombination.

Detailed ways

In the following examples, conventional experimental methods are adopted without further explanation, and implementation materials are all available from commercial sources.

In the following examples, Escherichia coli FMME-SuA is used as the starting strain, and Escherichia coli derived strains or other modified Escherichia coli that can produce succinic acid can also be used as starting strains.

(1) Pyruvate kinase enzyme activity detection

Detection was performed using the Agilent Pyruvate Kinase Kit.

(2) Determination 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/LH ₂ SO ₄ ; flow rate: 0.6 mL/min; temperature: 35°C; injection volume: 10 μL; detector: UV detector.

Ethanol: high performance liquid chromatography, chromatographic column: Aminex HPX-87H; mobile phase: 5mmol/LH ₂ SO ₄ ; flow rate: 0.6mL/min; temperature: 35°C; injection volume: 10μL; detector: differential refractive index detection device.

(3) Glucose determination method

Analysis was performed using an SBA-40 Biosensing Sensing Analyzer.

(4) Culture medium

Enrichment medium: yeast powder 20g/L, glucose 25g/L, sodium fumarate 8g/L, CaCO ₃ 2.0g, pH 4.5-5.5.

Plate separation medium: beef extract 3g/L, peptone 5g/L, NaCl 5g/L, agar 20g/L, bromocresol green 2g/L, pH 7.0.

Seed medium: beef extract 3g/L, peptone 5g/L, NaCl 5g/L.

Shake flask fermentation medium: corn steep liquor 20 g/L, glucose 70 g/L, Na ₂ HPO ₄ 2.5 g/L, MgCl ₂ 0.5 g/L, CaCO ₃ 3.0 g, pH 4.5-5.5.

(5) Calculation formula of production intensity

Production intensity (g/L/h) = succinic acid production (g/L)/fermentation time (h).

(6) Calculation formula of glucose yield

Glucose yield (%) = succinic acid yield (g/L) / glucose added (g/L) × 100

Example 1: Screening of succinic acid producing strains

1. Enrichment culture of strains

Take 10 g of fresh camel rumen contents, add it to sterile physiological saline with glass beads and shake for 10 minutes, then transfer it to enriched medium with a total volume of 200 mL, and culture at 32-37 °C for 24 hours at a rate of 30% The inoculum is transferred once.

2. Primary screening of strains

Gradient dilution of the enriched bacterial liquid, spread on agar medium containing bromocresol green, anaerobic cultivation at 32-37 ℃, after 24-36h, the color of the plate around some colonies turns yellow, pick out the yellow discoloration circle Large strains are used for strain preservation and further anaerobic fermentation;

3. Rescreening of strains

The 756 strains with larger discoloration circles in the primary screening were inoculated into the seed medium of the 24 deep-well plate for 24 hours with shaking at 37°C, and transferred to the fermentation medium in the 24-deep-well plate according to 10% of the inoculum, 32-37 Anaerobic fermentation at ℃ for 48-64h, using thin layer chromatography (TCL) to quantitatively detect succinic acid, a total of 134 strains can produce succinic acid, and further use high performance liquid chromatography (HPLC) to detect the content of succinic acid produced, and get one strain The strain F-121 with high succinic acid production reached 31.9 g/L of succinic acid.

Example 2: Identification of succinic acid producing strains

(1) Colony characteristics and cell morphology

Strain F-121 was diluted and spread on LB nutrient agar medium, and after overnight culture, white single colonies with circular micro-convex, smooth and moist surface and neat edges were formed. Microscopic examination showed that the cells were short rods without spores, and Gram staining was negative.

(2) Physiological and biochemical characteristics

The screening strains were identified by routine physiological and biochemical characteristics, and the results showed that: the strains fermented glucose to produce acid and gas, fermented lactose, maltose, xylose, mannitol, arabinose, but did not ferment inositol and cellulose; kinetic experiments, M-P experiments, Indole test, catalase test were positive; citric acid test, V-P test and urease test were negative, consistent with the biochemical properties of Escherichia coli

(3) 16S rDNA sequence analysis

Use DNA extraction to extract genomic DNA according to the instructions of the bacterial DNA extraction kit. Using genomic DNA as a template, PCR amplification is performed with bacterial 16S rDNA universal primers. The amplified products are sent to the company for sequencing, and the sequencing results are Blast compared in NCBI , the results showed that the strain was Escherichia coli.

The forward primer P16s8: the sequence is shown in SEQ ID NO.1; the reverse primer P16s-1492: the sequence is shown in SEQ ID NO.2.

Comprehensive colony morphological characteristics, physiological and biochemical detection and 16S rRNA analysis confirmed that the strain was Escherichia coli and named FMME-SuA. The strain has been submitted and deposited in the China Center for Type Culture Collection, the preservation number is CCTCC NO:M 2018568, the preservation date is August 27, 2018, and the preservation address is Wuhan University, Hubei, China. The enzyme activities of key pathways in the production of succinate were further detected, and the enzyme activities of lactate dehydrogenase (LDH), acetate kinase (ACK) and pyruvate formate lyase (PFL) were all negative.

Example 3: Protein Engineering of Pyruvate Kinase

(1) Use primer probes pykA-S (sequence shown in SEQ ID NO. 3) and pykA-A (sequence shown in SEQ ID NO. 4) to clone the pykA gene from the screened Escherichia coli FMME-SuA, and sequence The obtained gene sequence is SEQ ID NO.5, and the amino acid sequence is SEQ ID NO.6, which are consistent with the pyruvate kinase gene sequence derived from Escherichia coli K12.

(2) Use primers pykA-S1 (sequence shown in SEQ ID NO. 7) and pykA-A1 (sequence shown in SEQ ID NO. 8) and error-prone PCR kit to clone the pykA gene fragment, use agarose gel Gel electrophoresis, and gel tapping to recover the target fragment. The target band and pET28a plasmid were double digested with NdeI and HindIII, ligated overnight with T4 ligase, transformed into E. coli BL21 competent cells, coated on solid LB plates containing 100 mg/L kanamycin, screened for positive transformants, and constructed Complete the pyruvate kinase mutant library.

(3) Use pykA-S and pykA-A primers to verify the positive transformants by PCR, inoculate the positive colonies in the LB medium for fermentation culture, and shake them to OD600 to 0.8-1.0 at 37°C and 200rpm. , adding 0.4 mmol/L IPTG, and the temperature was lowered to 25 °C for 8-10 h to induce the expression of pyruvate kinase. The enzyme activity of different mutants was detected by pyruvate kinase detection kit, and the pykA(N22A/N23A) mutant was obtained by screening. Enzyme activity was significantly reduced. The gene sequence obtained after sequencing is SEQ ID NO.9, and the amino acid sequence is SEQ ID NO.10. Compared with the original amino acid sequence, the asparagine at positions 22 and 23 are all mutated to alanine, as shown in Table 1, The enzyme activity after mutation decreased by 2.1 times.

Table 1 Mutant enzyme activity assay

Example 4: Construction of recombinant strain FMME-SuAP

Taking the screened Escherichia coli FMME-SuA as the starting strain, the pykA gene in the original strain was replaced by the pykA (N22A/N23A) gene with the Red homologous recombination method. DNA fragment 1 of homology arm, kan gene and pykA (N22A/N23A) gene was introduced into Escherichia coli FMME-SuA with pKD46 plasmid, and homologous recombination was performed to replace the alanine kinase pykA gene in Escherichia coli FMME-SuA, The mutant strain FMME-SuAP was obtained by eliminating kan resistance using pCP20 plasmid.

Example 5: Fermentation of original strain FMME-SuA and recombinant strain FMME-SuAP for succinic acid production at shake flask level

The original strain FMME-SuA and the mutant strain FMME-SuAP were used to ferment succinic acid at the shake flask level. The seed solution was shaken at 37°C and cultured at 200 rpm for 12-14 hours, and then inoculated into the fermentation medium according to 10% of the inoculum. After anaerobic culture for 50 hours After the fermentation, samples were taken to detect the residual glucose and the production of succinic acid in the fermentation broth. The results are shown in Table 2. After 50 h of fermentation, the succinic acid yields of FMME-SuA and FMME-SuAP were 33.0 g/L and 41.7 g/L, respectively, and the yield of mutant strains increased by 26.4%.

Table 2 Fermentation of original bacteria and recombinant bacteria to produce succinic acid under anaerobic conditions

Example 5: Fermentation of original strain FMME-SuA and recombinant strain FMME-SuAP to produce succinic acid 1 under anaerobic conditions

(1) 7.5L fermentation medium formula: glucose 100g/L, corn steep liquor 4g/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 0.1g/L.

(2) The original strain FMME-SuA and the recombinant strain FMME-SuAP were respectively picked from the slant and inoculated into the seed medium, 37°C, 200 rpm shaking culture for 12 hours, and the shake flask seed liquid was inoculated into 7.5 L of stirring fermentation according to 10% of the inoculum respectively. Anaerobic fermentation is carried out in the tank, the liquid volume of the fermentation medium is 5.0L, the temperature is 37°C, the stirring is 100rpm, the gas is filtered and sterilized CO ₂ , the ventilation volume is 1-1.5vvm, and the pressure of the control gas outlet is 0.05Mpa, 4mol/L NaOH controls the pH to be 5.0-5.5.

(3) The fermentation was stopped at 65h, and the concentration of glucose and succinic acid in the fermentation broth of the recombinant strain FMME-SuAP was detected. The residual glucose was 3.10 g/L, the output of succinic acid was 70.2 g/L, and the yield of glucose was 72.4%. The production of succinic acid The strength was 1.40 g/L/h. After testing, the fermentation broth does not contain by-products such as acetic acid and ethanol. The original strain FMME-SuA had a glucose residue of 5.32 g/L, a succinic acid yield of 57.2 g/L, a glucose yield of 72.4%, and a succinic acid production intensity of 1.40 g/L/h. The results are shown in Table 3.

Example 6: Fermentation of original strain FMME-SuA and recombinant strain FMME-SuAP to produce succinic acid 2 under anaerobic conditions

(1) 7.5L fermentation medium formula: glucose 110g/L, corn steep liquor 8g/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 0.1g/L.

(2) The original strain FMME-SuA and the recombinant strain FMME-SuAP were respectively picked from the slant and inoculated into the seed medium, 37°C, 200 rpm shaking culture for 12 h, and the shake flask seed liquid was inoculated into 7.5 L of stirring fermentation according to 20% of the inoculum respectively. In the tank, the filling volume of the fermentation medium is 5.5L, the temperature is 37°C, the stirring is 200rpm, the gas is filtered and sterilized CO ₂ , the ventilation volume is 1-1.5vvm, and the pressure of the control air outlet is 0.05MPa, 4mol/L NaOH controls the pH to 5.0-5.5.

(3) Fermentation was stopped at 55h, and the concentrations of glucose and succinic acid in the fermentation broth were detected respectively. The residual glucose in the recombinant bacterial fermentation broth was 6.11 g/L, the output of succinic acid was 77.8 g/L, the yield of glucose was 74.9%, and the yield of succinic acid was 74.9%. The production intensity was 1.41 g/L/h. After testing, the fermentation broth does not contain by-products such as acetic acid and ethanol. The original strain FMME-SuA had a glucose residue of 11.2 g/L, a succinic acid yield of 60.1 g/L, a glucose yield of 60.8%, and a succinic acid production intensity of 1.09 g/L/h. , the results are shown in Table 3.

Example 7: Tolerance of recombinant strain FMME-SuAP to high glucose concentrations

(1) 7.5L fermentation medium formula: glucose 120g/L, corn steep liquor 8g/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 0.1g/L.

(2) The original strains FMME-SuA and FMME-SuAP were respectively picked from the slant and inoculated into the seed medium, 37 ° C, 200 rpm shaking culture for 12 h, and the shake flask seed liquid was respectively inoculated into a 7.5 L stirring fermenter according to 20% of the inoculum amount. , the filling volume of the fermentation medium is 5.5L, the temperature is 37°C, the stirring is 200rpm, the gas is filtered and sterilized CO ₂ , the ventilation volume is 1-1.5vvm, the control air outlet pressure is 0.05Mpa, 4mol/L NaOH control The pH is 5.0-5.5.

(3) The fermentation was stopped at 55 h, and the concentrations of glucose and succinic acid, and the contents of acetic acid and ethanol in the fermentation broth were detected respectively.

The results showed that in the case of high glucose concentration in the fermentation medium, the recombinant strain had a residual glucose of 5.98 g/L and a succinic acid yield of 80.0 g/L, while the original strain had a residual glucose of 13.8 g/L and a succinic acid yield of 80.0 g/L. The yield was 60.5 g/L. It is proved that the recombinant strain FMME-SuAP can maintain the efficient production of succinic acid when the glucose concentration is 100-120 g/L, and the production intensity reaches 1.40-1.45 g/L/h, which is significantly higher than the current maximum production intensity of succinic acid produced by Escherichia coli (1.30g/L/h), the strain has a strong tolerance to high glucose concentration, does not need to add glucose during the fermentation process, has almost no by-products, and simplifies the production process.

Table 3 Fermentation of original bacteria and recombinant bacteria to produce succinic acid under anaerobic conditions

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

SEQUENCE LISTING

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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> Synthetic

<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 (7)

1. The application of Escherichia coli in succinic acid production is characterized in that the succinic acid production capacity of Escherichia coli is improved by inhibiting the activity of pyruvate kinase in Escherichia coli, and the specific steps comprise:

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

(2) constructing a pyruvate kinase mutant with reduced enzyme activity by using the cloned pykA as a parent;

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

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

(5) inoculating the activated recombinant bacteria into a fermentation culture medium for fermentation;

the Escherichia coli is Escherichia coli FMME-SuA, which has been preserved in China center for type culture Collection in 2018, 8 and 27 months, with the preservation number of CCTCC NO: M2018568, and the preservation address of Wuhan university, Hubei, China;

the pyruvate kinase mutant in the step (3) is pykA, and the nucleotide sequence for coding the pyruvate kinase mutant is shown as SEQ ID NO. 9;

the construction method of the recombinant escherichia coli in the step (3) comprises the step of replacing the pyruvate kinase gene pykA in the original strain with a pyruvate kinase mutant gene with reduced enzyme activity by using a Red homologous recombination technology.

2. The use of claim 1, wherein the activation in step (4) is performed by culturing the recombinant E.coli strain at 30-40 ℃ for 12-14h with shaking at 200 rpm.

3. The use as claimed in claim 1, wherein the fermentation in step (5) is carried out by inoculating the seed solution into the fermentation medium at an inoculum size of 10-25%, stirring at 150rpm at 30-40 ℃ and carrying out anaerobic fermentation for 50-65 h; the culture medium used comprises: glucose 90-120g/L, corn steep liquor 4-10g/L, Na₂HPO₄·12H₂O 1-2g/L，NaH₂PO₄·2H₂O 2-3g/L，MgSO₄·7H₂O 0.2-0.3g/L，CaCl₂0.1-0.2g/L。

4. Escherichia coli FMME-SuA capable of producing succinic acid is preserved in China Center for Type Culture Collection (CCTCC) No. M2018568 in 8 months and 27 days in 2018, and the preservation address is Wuhan university in Hubei of China.

5. A recombinant Escherichia coli, characterized in that, the Escherichia coli FMME-SuA of claim 4 is used as the starting strain, DNA fragment of pykA gene shown as SEQ ID NO.9 is introduced into Escherichia coli FMME-SuA by Red homologous recombination method, and pyruvate kinase gene pykA in Escherichia coli FMME-SuA is replaced to obtain the recombinant Escherichia coli.

6. Use of the Escherichia coli strain according to claim 4 for producing succinic acid.

7. Use of the recombinant E.coli of claim 5 for the production of succinic acid.

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