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Novel constructed high-yield malic acid gene engineering and method for producing malic acid

A technology of genetically engineered bacteria and malic acid, applied in the field of bioengineering, can solve problems such as low production intensity and slow growth of mold

Inactive Publication Date: 2008-09-03
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the growth rate of mold is slow, and the production intensity of products is low. The development of malic acid production strains with simple nutritional requirements, rapid growth and high yield is the key to accelerating the production of biological methods instead of petrochemical methods.

Method used

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  • Novel constructed high-yield malic acid gene engineering and method for producing malic acid
  • Novel constructed high-yield malic acid gene engineering and method for producing malic acid
  • Novel constructed high-yield malic acid gene engineering and method for producing malic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] This example illustrates the process of knocking out the fumarase fum gene in the parental Escherichia coli NZN111 using homologous recombination technology to obtain a strain that eliminates apramycin resistance.

[0035] 1. Using LB medium, cultivate Escherichia coli NZN111 to OD at 37°C under aerobic conditions 600 =0.4~0.6, prepared to be electrotransfer competent.

[0036] 2. Electrotransform the recombinant plasmid into competent Escherichia coli NZN111. The electric shock conditions are: 200Ω, 25μF, electric shock voltage 2.3kV, electric shock time 4-5ms. Immediately after electric shock, the cells were added to pre-cooled 1mL SOC medium, cultured at 150r / min, 30°C for 1h, and then spread on LB medium plate with ampicillin (amp) to screen out the positive transformant NZN111 (pKD46).

[0037] 3. Add 10 mM L-arabinose to LB medium, induce plasmid pKD46 to express λ recombinase at 30°C, and make electroporation competent.

[0038] 4. Using the apramycin resistance...

Embodiment 2

[0052] This example illustrates the process of constructing an expression plasmid overexpressing malic enzyme, restoring the ability of the recombinant strain to metabolize glucose under anaerobic conditions, and obtaining the strain Escherichia coli JM127.

[0053] 1. Construction of an expression plasmid for overexpressing malic enzyme, the process comprising:

[0054] (1) synthesize primers with Nco I and HindIII restriction sites,

[0055] Upstream primer: 5'-CATGCCATGGATATTCAAAAAAAGAGTGAGTG-3'

[0056] Downstream primer: 5'-CCCAAGCTTTTAGATGGAGGTACGGC-3'

[0057] (2) Using Escherichia coli K12 as a template, colony PCR was carried out under the reaction conditions of 95° C. for 1.5 min, 63° C. for 1.0 min, 72° C. for 1.5 min, and a total of 35 cycles. After purifying the amplified sfcA gene, the expression plasmid pTrc99a was digested with Nco I and HindIII respectively, and ligated to obtain the recombinant plasmid pTrc99a-sfcA.

[0058] 2. Introducing the plasmid pTrc...

Embodiment 3

[0060] This example illustrates the comparison of the fermentative acid production capacity of newly constructed recombinant Escherichia coli JM127 and NZN111 (pTrc99a-sfcA).

[0061] When Escherichia coli NZN111 (CGSC7726) was introduced into the plasmid pTrc99a-sfcA and overexpressed the malic enzyme gene, the ability of NZN111 to metabolize glucose under anaerobic conditions was restored, and a high yield of succinate was accumulated. The constructed recombinant E. coli Escherichia coli JM127 produces mixed acids of malic acid, fumaric acid, succinic acid and acetic acid when it grows under anaerobic conditions, and malic acid is the main product. In order to investigate the effect of fumarase activity reduction on acid production, a two-stage fermentation mode was adopted, and the inoculum was transferred from the cryopreservation tube to the Erlenmeyer flask according to 1% (v / v). When the aerobic culture OD 600 Induce to OD by 0.7mM IPTG to about 0.8~1.0 600 When it is ...

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Abstract

The invention discloses a method for constructing strain Escherichia coli JM127 in high product malic acid gene project and acid producing method thereof. The constructing method mainly includes deactivating or knocking out enzyme with ability to make malic acid dehydrate and pyruvate degrade, and over-expressing enzyme regenerated by NAD. A two-stage fermentation experiment using a constructed Escherichia coli is carried out, which includes steps of: (1) inoculating activated Escherichia coli into enrichment medium, aerobically culturing to enhance biomass; (2) after treating the bacteria liquid membrane obtained by aerobic culture, introducing it into LB culture medium containing 20-100 g / L glucose and a definite content of carbonate, anaerobically fermenting to produce malic acid.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to the construction of a high-yielding malic acid recombinant bacterial strain, and also relates to a method for fermenting and producing malic acid using the bacterial strain. Background technique [0002] Malic acid is an important intermediate product in the internal circulation of the human body and is easily absorbed by the human body. Therefore, as an excellent food additive and functional food, it is widely used in food, medical and health care and other fields. At present, the production of malic acid uses fumaric acid or maleic acid as raw material, pressurized and co-heated with water vapor to form DL-malic acid, or furfural as raw material, treated with hydrogen peroxide, and transformed under the action of ultrasonic waves. Due to the complex production process, high process conditions, difficult separation and purification technology, and the fact that the raw mate...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12N9/04C12P7/46C12R1/19
Inventor 姜岷马江锋陈可泉王益娜于丽黄秀梅韦萍欧阳平凯
Owner NANJING UNIV OF TECH
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