Recombinant escherichia coli strain for producing beta-alanine as well as construction method and application thereof

A technology for recombining Escherichia coli and Escherichia coli, which is applied in the biological field, can solve the problems of low production of β-alanine and difficulty in meeting the requirements of industrial production, and achieve the effect of increasing production rate

Active Publication Date: 2014-07-02
ANHUI HUAHENG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The Chinese magazine "Amino Acids and Biological Resources" in 2005, Volume 27, Issue 1, Pages 52-55, published a journal paper titled "Synthesis and Application of β-alanine" (β-alanine is also known as β-alanine), which introduced that Chuan Liyang et al. used microorganisms producing organic nitrile degrading enzymes to catalyze the hydrolysis of β-aminopropionitrile to synthesize β-alanine, and the concentration of the obtained β-alanine product reached 47mmol / L ( About 4.2g / L), Tawaki Shinichiro and others used microorganisms to convert β-aminopropanol to synthesize β-alanine, and the concentration of the obtained product reached 4g / L, but the yield of β-alanine produced by the above biological method was low , it is difficult to meet the requirements of industrial production

Method used

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  • Recombinant escherichia coli strain for producing beta-alanine as well as construction method and application thereof
  • Recombinant escherichia coli strain for producing beta-alanine as well as construction method and application thereof
  • Recombinant escherichia coli strain for producing beta-alanine as well as construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0022] Construction and identification of Escherichia coli strains lacking aspartate amino-lyase gene:

[0023] 1) Amplification of the Apr gene: the DNA of the pIJ773 plasmid was used as a PCR amplification template to amplify the Apr gene (the pIJ773 plasmid was purchased from Hefei Baimai Biotechnology Co., Ltd.). The nucleotide sequences of primers aspA-KO-U and aspA-KO-D used to increase the Apr gene are shown in SEQ ID NO.2 and SEQ ID NO.3. The PCR amplification system is: DNA template 20ng, primer (10uM) 1ul, distilled water 40ul; PCR amplification conditions: 94°C pre-denaturation for 5min, 1 cycle; 94°C denaturation for 45s, 50°C annealing for 45s, 72°C extension for 90s, 10 cycles; 94°C denaturation for 45s, 55°C annealing for 45s, 72°C extension 90s, 15 cycles; 72°C extension for 10 min, 1 cycle.

[0024] 2) Obtain DNA fragments: Digest the PCR product obtained in step 1, that is, digest the Apr gene amplified from the DNA of the pIJ773 plasmid, thereby removing th...

Embodiment 2

[0028] Construction and identification of the recombinant Escherichia coli strain of the present invention:

[0029]1) Construction of recombinant E. coli strains:

[0030] The first step is to double digest the PanD gene of Corynebacterium glutamicum with NdeI and HindIII to obtain a DNA fragment of the PanD gene, which is the aspartic acid-1-decarboxylase gene of Corynebacterium glutamicum , with NdeI and HindIII double digestion plasmid pET24a (+) (Novagen company product), the size of the plasmid pET24a (+) is 5.31kb, which contains kanamycin resistance gene and lactose repressor lacI gene, the promoter is T7lac, and has multiple restriction endonuclease sites, the enzyme digestion system is: 43 μl of DNA, 5 μl of bufferR, 1 μl of HindIII, 1 μl of NdeI; the digestion conditions are: incubate at 37°C for 3 hours, as attached image 3 Shown is the agarose gel electrophoresis image of the digested product. The concentration of the agarose gel is 1.0%. Lanes 1 to 3 are DNA fr...

Embodiment 3

[0035] Fermentation and enzyme activity determination of the recombinant Enterobacter bacterial strain of the present invention:

[0036] 1) Fermentation of recombinant Enterobacter strains:

[0037] The first step is to prepare seed liquid and fermentation liquid. The composition of seed liquid medium is: peptone 1% ~ 1.5%, yeast extract 2% ~ 2.5%, glycerin 0.4% ~ 0.5%, potassium dihydrogen phosphate 0.2% ~ 0.3% , dipotassium hydrogen phosphate 1%~2%, kanamycin 50mg / L, the balance is pure water, adjust the pH to 7.0~7.2 with ammonia water; the composition of the fermentation medium is: peptone 1%~1.5%, yeast Extract 2% to 2.5%, glycerin 0.4% to 0.5%, potassium dihydrogen phosphate 0.2% to 0.3%, dipotassium hydrogen phosphate 1% to 2%, the rest is pure water, adjust the pH to 7.0 to 7.2 with ammonia water .

[0038] In the second step, put 500ml of seed liquid culture medium in a 2L Erlenmeyer flask, sterilize it at 121°C for 20 minutes, inoculate it on an LB plate after coo...

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Abstract

The invention discloses a recombinant escherichia coli strain (Escheerichiacoli) AHB-36 for producing beta-alanine, the preservation number is CCTCC M 2013629, the recombinant escherichia coli strain is a deficiency aspartic acid amino lyase gene and contains an aspartic acid-1-decarboxylase gene. The invention further aims at providing a construction method of the recombinant escherichia coli strain. The recombinant escherichia coli strain can be used for knocking off the aspartic acid amino lyase gene and preventing the escherichia coli from pyrolyzing the aspartic acid into fumaric acid, so that the aspartic acid is completely applied to production of beta-alanine, and meanwhile aspartic acid-1-decarboxylase is expressed in the recombinant escherichia coli strain, and thus the yield of beta-alanine is further increased.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant Escherichia coli strain producing β-alanine and its construction method and application. Background technique [0002] β-alanine is widely used in the fields of medicine and food technology. It is mainly used for the synthesis of pantothenic acid, calcium pantothenate, and food nutritional additives. The production of β-alanine at home and abroad is basically based on chemical synthesis. Mainly, such as acrylic acid method, acrylonitrile method, etc., most of these chemical synthesis methods require conditions such as strong base and strong acid, high temperature and high pressure, and the product purification is cumbersome, causing pollution to the environment, especially the use of nitrile-containing raw materials for synthesis, which is harmful to the environment and biological Therefore, actively seeking green and environmentally friendly production methods of β-ala...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P13/06C12R1/19
Inventor 郭恒华刘洁张冬竹唐思青刘洋
Owner ANHUI HUAHENG BIOTECH
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