Escherichia coli capable of producing L-alanine through fermentation and application of escherichia coli

A technology of Escherichia coli and alanine, applied in the field of bioengineering, can solve problems such as single construction method and complicated genetic engineering operation

Active Publication Date: 2018-05-22
金华利家园生物工程有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the construction method of constructing L-alanine production strain is single

Method used

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  • Escherichia coli capable of producing L-alanine through fermentation and application of escherichia coli
  • Escherichia coli capable of producing L-alanine through fermentation and application of escherichia coli
  • Escherichia coli capable of producing L-alanine through fermentation and application of escherichia coli

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Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1: Construction of dual enzyme co-expression strain

[0031] (1) Use the bacterial genome extraction kit to extract the genomic DNA of Escherichia coli K12 and Comamonas testosteroni;

[0032] (2) Using the primers AspA-up and AspA-down in Table 1 to clone the aspA gene from Escherichia coli genomic DNA, use SacI and NotI double digestion and connect it to the expression vector pETac to obtain pETac-aspA;

[0033] (3) According to the statistical linear relationship between RBS intensity and the expression of the regulated protein, design RBS1, RBS2, RBS3, and RBS4 to regulate the expression intensity of the two enzymes, and use primers AsD-up1 and AsD-down, AsD-up2 and AsD -down, AsD-up3 and AsD-down, AsD-up4 and AsD-down were obtained by cloning the asD gene from the genomic DNA of Comamonas testosteroni;

[0034] (4) After the sequence of the 4 segments of asD gene obtained by the above clone is correct, use HindIII and NotI double restriction enzymes to ...

Embodiment 2

[0042] Embodiment 2: Construction of high-production L-alanine Escherichia coli

[0043] Using the principle of Red homologous recombination, a constitutive promoter J23119 (BBa23119) was added upstream of the YgaW genome to enhance the expression of AlaE protein and promote the conversion of L-alanine from intracellular to extracellular.

[0044] (1) Use the software Primer Premier 5 to design homologous recombination primers (as shown in Table 3), use Kan-S and Kan-A as primers, and pKD4 as a template to amplify the kan gene, and use YgaW-S and YgaW-A as primers, The Escherichia coli K12 genome was used as a template to amplify the YgaW gene, and the Kan+YgaW fragment was obtained by fusion PCR, which was connected to PMD19 for gene sequencing;

[0045] (2) Using 119-S and YgaW-A as primers, connecting and sequencing the correct Kan+YgaW fragment PMD19 plasmid as a template, and PCR to obtain a knockout frame with homology arms, J23119 promoter, and Kan gene;

[0046] (3) T...

Embodiment 3

[0052] Example 3: Fermentation of E.coli P-119 to produce L-alanine

[0053] Seed medium composition: yeast powder 5g / L, peptone 10g / L, NaCl 5.0g / L, MgSO 4 ·7H 2 O 1.0g / L.

[0054] Fermentation medium composition: initial glucose 70g / L, yeast powder 5g / L, tryptone 2g / L, (NH 4 ) 2 SO 4 2g / L, K 2 HPO 4 12H 2 O 4g / L, KH 2 PO 4 4g / L, MgSO 4 ·7H 2 O 0.5g / L, citric acid monohydrate 0.3g / L, MnCl 2 4H 2 O0.08g / L.

[0055] Pick an appropriate amount of E.coli P-119 from the slant and inoculate it in the seed medium, culture it with shaking at 35°C and 200rpm for 10-12 hours, inoculate the shake flask seeds in the fermenter according to 10% inoculum amount, and fill the medium in the 5L fermenter The volume is 3L, dilute sulfuric acid and ammonia water control the fermentation pH to 6.8-7.2, the ventilation rate is 0.5vvm, the temperature is 35°C, and the stirring is 100rpm. When the OD600 reaches 25-30, add 2g / L lactose to induce aspartase and asparagine Amino acid-β-dec...

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Abstract

The invention discloses escherichia coli capable of producing L-alanine through fermentation and application of the escherichia coli and belongs to the technical field of biological engineering. According to the escherichia coli, aspartase AspA and L-aspartic acid-beta-decarboxylase AsD are introduced into the escherichia coli capable of producing fumaric acid through the fermentation; furthermore, a YgaW gene is over-expressed to strengthen the transport ability of the L-alanine in recombinant bacteria, so that a novel strain capable of fermenting glucose to produce the L-alanine is constructed; when the recombinant strain is fermented in a fermentation tank for 45h, the yield of the L-alanine reaches 147g/L and the saccharic acid conversion rate is 79.0 percent.

Description

technical field [0001] The invention relates to an Escherichia coli for fermenting and producing L-alanine and an application thereof, belonging to the technical field of bioengineering. Background technique [0002] L-alanine is a non-essential amino acid and the amino acid with the highest content in human blood. L-alanine is widely used in industries, daily chemicals, food and other fields. In the field of daily chemicals, L-alanine is an important raw material for the synthesis of amino acid surfactants. In the field of food, L-alanine can be used as a natural sweetener to improve the taste of flavoring agents and the acidity of organic acids. In the field of medicine, L-alanine is also the main raw material for the synthesis of vitamin B6 and aminopropanol. [0003] The production process of L-alanine includes extraction method, chemical synthesis method, enzyme catalysis method and fermentation method. At present, the enzymatic method is mainly used in industrial pro...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P13/06C12R1/19
CPCC12N9/88C12P13/06C12Y401/01012C12Y403/01001
Inventor 齐俊平张帆刘佳刘立明
Owner 金华利家园生物工程有限公司
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