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Malic acid-production gene engineering bacteria and its construction and use

A technology of genetically engineered bacteria and malic acid, applied in the field of bioengineering, can solve problems such as growth inhibition, inability to utilize glucose, etc., and achieve good effects and simple and feasible fermentation methods.

Inactive Publication Date: 2014-09-17
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In addition, due to the deletion of the pyruvate formate lyase gene pflB and the lactate dehydrogenase gene ldhA, a large amount of pyruvate is accumulated, which feedback inhibits the transport of glucose by the PTS sugar transport system, making the bacteria unable to utilize glucose under anaerobic conditions. and inhibited growth

Method used

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  • Malic acid-production gene engineering bacteria and its construction and use
  • Malic acid-production gene engineering bacteria and its construction and use
  • Malic acid-production gene engineering bacteria and its construction and use

Examples

Experimental program
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Effect test

Embodiment 1

[0049] This example illustrates the process of knocking out the fumarase gene fumB in the parental Escherichia coli NZN111 using homologous recombination technology to obtain a strain that eliminates apramycin resistance.

[0050] Using homologous recombination technology to knock out the fumarase (FUM) gene:

[0051] 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.

[0052] 2. The plasmid pKD46 was electrotransformed into competent Escherichia coli NZN111, the electric shock conditions were: 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).

[0053] 3. Add 10 mM L-arabinose to LB medium, induce plasmid pKD46 to express λ recombi...

Embodiment 2

[0065] This example illustrates the process of eliminating the apramycin-resistant strain by further knocking out the fumarate reductase gene frdABCD by using homologous recombination technology on the basis of knocking out the fumarase gene fumB in the parental Escherichia coli NZN111.

[0066] Knockout of the fumarate reductase (FRD) gene by homologous recombination:

[0067] 1. Using LB medium, cultivate E. coli lacking ldhA, pflB and fumB at 37°C under aerobic conditions to OD 600 =0.4~0.6, prepared to be electrotransfer competent.

[0068] 2. The plasmid pKD46 was electrotransformed into the competent strain in the above step 1. The electric shock conditions were: 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 positive transformants.

[0069] 3. Add 10 mM L-ara...

Embodiment 3

[0080] This example illustrates the construction process of the expression plasmid pTrc99a-pyc of exogenous pyruvate carboxylase.

[0081] 1. Synthesize primers with NcoI and PstI restriction sites:

[0082] Upstream primer: 5'-CATGCCATGGTCAGCTGATGAGAAACGTCGAGAAG-3'

[0083] Downstream primer: 5'-AAAACTGCAGGGTCATTCTCTTCAAAGCCAAAACGA-3'

[0084] 2. Using Lactococcus lactis cremoris NZ9000 genomic DNA as a template, PCR amplifies the target gene fragment. The reaction conditions are: 94°C, 5min; (94°C for 45s, 53°C for 45s, 72°C for 300s, 35 cycles); 72°C, 10min , the agarose gel electrophoresis of the PCR product pyc is as follows Figure 7 shown. After purifying the amplified pyc gene, it was cut with NcoI and PstI at the same time as the expression plasmid pTrc99a, and then ligated to obtain the recombinant plasmid pTrc99a-pyc. The construction map of the recombinant plasmid is as follows: Figure 8 shown. Figure 9 The result of single enzyme digestion identification of...

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Abstract

The invention provides malic acid-production gene engineering bacteria Escherichiacoli BA043. The malic acid-production gene engineering bacteria is named as BA043 (Escherichiacoli BA043) and has a preservation registration number CCTCC NO: M2014034. The invention also provides a construction method of the malic acid-production gene engineering bacteria and a method for fermentation production of malic acid. Through knockout or inactivation of fumarase and fumarate reductase and through over coexpression of exogenous pyruvate carboxylase and nicotinic acid phosphoribosyl transferase, recombinant escherichia coli can reduce by-product pyruvic acid generation by glucose metabolism growth so that a malic acid yield and production intensity are greatly improved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a genetically engineered strain of malic acid-producing Escherichia coli and its construction and application, in particular to the construction of a genetically engineered strain Escherichia coli BA043 that efficiently utilizes glucose to produce malic acid and a method for producing malic acid . Background technique [0002] Malic acid, also known as 2-hydroxysuccinic acid, is one of the members of the tricarboxylic acid cycle in organisms, and its molecular formula is C 4 h 6 0 5 , There is a chiral carbon atom in the molecule, so there are three forms of existence, namely D-malic acid, L-malic acid and DL-malic acid. L-type malic acid widely exists in nature, while chemically synthesized malic acid is a mixture of L-type and D-type. Since L-malic acid can be directly absorbed and utilized by organisms, while D-malic acid is non-physiologically active and needs to be ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/09C12N15/70C12P7/46C12R1/19
Inventor 姜岷任心怡马江锋张敏朱婧李凤
Owner NANJING UNIV OF TECH
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