Extracellular expression L-aspartate-alpha-decarboxylase engineering bacterium constructing method and application thereof

Abstract

The invention relates to an extracellular expression L-aspartate-alpha-decarboxylase engineering bacterium constructing method and an application of the bacterium to production of beta-alanine. A high-activity L-aspartate-alpha-decarboxylase gene is cloned and co-expressed with a signal peptide gene, a competent escherichia coli cell is transformed, an extracellular secretion engineering bacteriumfor the signal peptide mediated L-aspartate-alpha-decarboxylase is constructed, the extracellular secretion amount of the L-aspartate-alpha-decarboxylase is increased with the matching of the fermenting and regulating measures, and the obtained L-aspartate-alpha-decarboxylase is used for transforming the L-aspartate so as to produce beta-alanine. The implementation of the extracellular release ofan enzyme reduces the toxicity of the excessively stacked protein in the cell on the cell and is favorable for the contact between a substrate and the enzyme; a fermentation liquid is directly used to catalyze the L-aspartate to produce beta-alanine, the reaction is carried out without breaking the cell and extracting the pure enzyme; the beta-alanine is catalytically synthesized by means of substrate fed-batch, the catalytic process is prevented from producing excessive salt, and the product purity is high.

Description

technical field

[0001] The invention relates to a construction method and application of an engineering bacterium expressing L-aspartic acid α-decarboxylase extracellularly, and belongs to the field of biotechnology. Background technique

[0002] In the prior art, most of the proteins expressed by Escherichia coli are intracellular products, which are easy to form inclusion bodies and be degraded by proteases. The protein activity is not high, and membrane permeation treatment is required for catalysis, which is costly.

[0003] L-aspartate α-decarboxylase (L-aspartate-α-decarboxylase, ADC) catalyzes the decarboxylation of L-aspartate to produce β-alanine, which is the synthesis of pantothenic acid in vivo It is an important precursor substance and is the only β-type amino acid that exists in nature. β-alanine and its derivatives are widely used in the fields of medicine, food and chemical industry. The chemical synthesis method is currently the main production method of β...

Examples

Embodiment 1

[0034] An engineering bacterium expressing L-aspartic acid α-decarboxylase extracellularly and a construction method thereof, the steps of the construction method include: cloning a highly active L-aspartate α-decarboxylase gene, and cooperating with the signal peptide gene Express and transform Escherichia coli competent cells, and construct signal peptide-mediated extracellular secretion engineering bacteria of L-aspartic acid α-decarboxylase. Specifically:

[0035] According to the gene sequence information of PanD in the whole genome of B. tequilensis (LGRW01000001) and the cloning vector pET20b, upstream and downstream primers containing adapters (indicated in italics) and restriction endonuclease sites (indicated in bold) were designed. The upstream primer is pelB-PanD -F: 5'- cccagccggcgatgg ccatggatatgtatcgaacaatgatgagc-3'; downstream primer is pelB-PanD-R:5'- gtggtggtggtggtg ctcgagctacaaaattgtacgggcggg-3'. The genomic DNA of B. tequilensis PanD37 (CGMCC NO:10506)...

Embodiment 2

[0037] An engineering bacterium expressing L-aspartic acid α-decarboxylase extracellularly and a construction method thereof, the steps of the construction method include: cloning a highly active L-aspartate α-decarboxylase gene, and cooperating with the signal peptide gene Express and transform Escherichia coli competent cells, and construct signal peptide-mediated extracellular secretion engineering bacteria of L-aspartic acid α-decarboxylase. Specifically:

[0038] The gene encoding the signal peptide torA of trimethylamine-N-oxide reductase was synthesized (Genebank number: BAA36139.1), and amplified using primers F-torA: 5′-AAccatgggcatgaacaataacgatctctttc-3′ and R-torA: 5′-ATCCATGGCCGCTTGCGCCGCAGTC-3′, The torA coding gene and plasmid pET20b were double digested with NdeI and NcoI respectively, and then the two were ligated to construct the plasmid pET20b-torA with torA signal peptide; according to the gene sequence information and Plasmid vector pET20b-torA designed up...

Embodiment 3

[0049] The method for extracellularly expressing L-aspartic acid α-decarboxylase fermentation broth to convert L-aspartic acid to produce β-alanine, the steps are:

[0050] (1) Insert the genetically engineered bacteria E. coli BL21(DE3) / pT20b-pelB-panD constructed in Example 1 into the LB slant medium containing 100 mg / L ampicillin and cultivate it at 37°C for 16 hours, wherein the LB medium formula is: Yeast powder 5g / L, peptone 10g / L, NaCl 5g / L, agar 20g / L, pH7.0, sterilized at 121°C for 30min;

[0051] (2) 50mL of LB liquid medium in a 500mL triangular flask, add 100mg / L ampicillin, connect to 1 ring of slant seed culture medium for 7 hours at 37°C and 200r / min, and the liquid LB medium formula is; yeast powder 5g / L, peptone 10g / L, NaCl 5g / L, pH7.0, sterilized at 121°C for 30min;

[0052] (3) Put 3.0 L medium into a 5 L fermenter, insert the seed solution into the fermentation medium with 5% seed volume, adjust the speed and ventilation to control the dissolved oxygen va...

technical field

[0001] The invention relates to a construction method and application of an engineering bacterium expressing L-aspartic acid α-decarboxylase extracellularly, and belongs to the field of biotechnology. Background technique

[0002] In the prior art, most of the proteins expressed by Escherichia coli are intracellular products, which are easy to form inclusion bodies and be degraded by proteases. The protein activity is not high, and membrane permeation treatment is required for catalysis, which is costly.

[0003] L-aspartate α-decarboxylase (L-aspartate-α-decarboxylase, ADC) catalyzes the decarboxylation of L-aspartate to produce β-alanine, which is the synthesis of pantothenic acid in vivo It is an important precursor substance and is the only β-type amino acid that exists in nature. β-alanine and its derivatives are widely used in the fields of medicine, food and chemical industry. The chemical synthesis method is currently the main production method of β...

Examples

Embodiment 1

[0034] An engineering bacterium expressing L-aspartic acid α-decarboxylase extracellularly and a construction method thereof, the steps of the construction method include: cloning a highly active L-aspartate α-decarboxylase gene, and cooperating with the signal peptide gene Express and transform Escherichia coli competent cells, and construct signal peptide-mediated extracellular secretion engineering bacteria of L-aspartic acid α-decarboxylase. Specifically:

[0035] According to the gene sequence information of PanD in the whole genome of B. tequilensis (LGRW01000001) and the cloning vector pET20b, upstream and downstream primers containing adapters (indicated in italics) and restriction endonuclease sites (indicated in bold) were designed. The upstream primer is pelB-PanD -F: 5'- cccagccggcgatgg ccatggatatgtatcgaacaatgatgagc-3'; downstream primer is pelB-PanD-R:5'- gtggtggtggtggtg ctcgagctacaaaattgtacgggcggg-3'. The genomic DNA of B. tequilensis PanD37 (CGMCC NO:10506)...

Embodiment 2

[0037] An engineering bacterium expressing L-aspartic acid α-decarboxylase extracellularly and a construction method thereof, the steps of the construction method include: cloning a highly active L-aspartate α-decarboxylase gene, and cooperating with the signal peptide gene Express and transform Escherichia coli competent cells, and construct signal peptide-mediated extracellular secretion engineering bacteria of L-aspartic acid α-decarboxylase. Specifically:

[0038] The gene encoding the signal peptide torA of trimethylamine-N-oxide reductase was synthesized (Genebank number: BAA36139.1), and amplified using primers F-torA: 5′-AAccatgggcatgaacaataacgatctctttc-3′ and R-torA: 5′-ATCCATGGCCGCTTGCGCCGCAGTC-3′, The torA coding gene and plasmid pET20b were double digested with NdeI and NcoI respectively, and then the two were ligated to construct the plasmid pET20b-torA with torA signal peptide; according to the gene sequence information and Plasmid vector pET20b-torA designed up...

Embodiment 3

[0049] The method for extracellularly expressing L-aspartic acid α-decarboxylase fermentation broth to convert L-aspartic acid to produce β-alanine, the steps are:

[0050] (1) Insert the genetically engineered bacteria E. coli BL21(DE3) / pT20b-pelB-panD constructed in Example 1 into the LB slant medium containing 100 mg / L ampicillin and cultivate it at 37°C for 16 hours, wherein the LB medium formula is: Yeast powder 5g / L, peptone 10g / L, NaCl 5g / L, agar 20g / L, pH7.0, sterilized at 121°C for 30min;

[0051] (2) 50mL of LB liquid medium in a 500mL triangular flask, add 100mg / L ampicillin, connect to 1 ring of slant seed culture medium for 7 hours at 37°C and 200r / min, and the liquid LB medium formula is; yeast powder 5g / L, peptone 10g / L, NaCl 5g / L, pH7.0, sterilized at 121°C for 30min;

[0052] (3) Put 3.0 L medium into a 5 L fermenter, insert the seed solution into the fermentation medium with 5% seed volume, adjust the speed and ventilation to control the dissolved oxygen va...

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