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Construction, expression and application of genetic engineering bacteria for high-production of beta-alanine

A technology of genetically engineered bacteria and alanine, applied in genetic engineering, application, plant genetic improvement, etc., can solve problems such as low enzyme activity and difficulties

Inactive Publication Date: 2014-07-02
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The present invention aims to overcome the difficulty of screening strains with high enzyme production activity in nature, and the problem that the existing enzyme activity obtained by genetically engineered bacteria using Escherichia coli for heterologous expression is not high, and provides a new enzyme gene bank The excavated enzyme gene obtained a β-alanine-producing engineering bacterium with strong synthetic ability and high enzyme activity, as well as the construction, expression and purification of this high-yielding engineered bacterium, as well as its whole-cell transformation and direct transformation of fermentation broth Method for synthesizing β-alanine

Method used

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  • Construction, expression and application of genetic engineering bacteria for high-production of beta-alanine
  • Construction, expression and application of genetic engineering bacteria for high-production of beta-alanine
  • Construction, expression and application of genetic engineering bacteria for high-production of beta-alanine

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Embodiment 1

[0038] Embodiment 1: Construction of high expression vector

[0039] Primers were designed according to the sequence of the L-aspartic acid-α-alanine decarboxylase PanD gene (NCBI Reference Sequence: NC_004567.2) of Lactobacillus plantarum WCFS1 plant Lactobacillus:

[0040] Forward primer: CGC CATATG TTAATTGATAT, the underlined sequence is the enzyme cutting site NdeI

[0041] Reverse primer: CC CTCGAG AACATATCAATTAA, the underlined sequence is the XhoI restriction site

[0042] The two ends of the gene sequence obtained by PCR have NdeI and XhoI sites, and the gene is inserted into the pET-32a(+) vector to obtain genetically engineered bacteria.

[0043] The protein expressed by the obtained genetically engineered bacteria has a His tag protein at the C-terminus, an XhoI site behind the His tag, and a stop codon TGA before the XhoI site after the His tag.

[0044] The NdeI restriction site in the described NdeI and XhoI restriction site is that NdeI has two restriction...

Embodiment 2

[0046] Embodiment 2: the acquisition of high expression bacterial strain

[0047] The prepared recombinant vector is introduced into Escherichia coli BL21, Rosetta or Origami by conventional methods to construct a genetically engineered bacterium in which the recombinant isomerase exists in the bacterium in a soluble form, and the successfully constructed genetically engineered bacteria are screened out, among which Escherichia coli BL21 The target protein expression of the recombinant bacterium as the host bacterium is relatively good. The engineered bacteria whose expression level of the target protein is not less than 30% shall be used as the engineered strains for production, and shall be preserved in the form of glycerol bacteria or milk freeze-dried strains.

[0048] The specific transformation method is as follows: Take out 100 μL of competent cells from the refrigerator. Thaw cells on ice for 2-5 minutes. After thawing, flick the tube wall 1-2 times to resuspend the ...

Embodiment 3

[0050] Example 3: Purification of L-aspartic acid-α-alanine decarboxylase in highly expressed strains

[0051] (1) Seed Medium Activation Pick a single colony on a petri dish and place it in 5 mL LB (Amp 100 μg / ml) medium, and activate overnight at 37° C. and 200 rpm.

[0052] (2) Fermentation culture Inoculate 800 mL of LB medium (Amp 100 g / mL) with the overnight activated seed solution at a ratio of 1:100, and culture at 37°C and 200 rpm until mid-log phase OD600=0.6-1.

[0053] (3) Induction culture In the mid-log phase, IPTG was added for induction, so that the final concentration was 0.5 mM, and the induction culture was carried out at 25-30° C. and 180 rpm for 4-6 hours.

[0054] (4) SDS-PAGE Take 4 mL of the fermented broth that has been induced, centrifuge at 12,000 rpm for 90 sec, add 800 μL of deionized water, and sonicate for 5 min. Centrifuge at 12000rpm for 10min. Take 20 μL of supernatant after centrifugation and add 5 μL of loading buffer; after centrifugation...

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Abstract

The invention provides new genetic engineering bacteria used for producing beta-alanine, having strong synthetic ability and having higher enzyme activity, construction, expression and purification of the high-production engineering bacteria, and methods for synthesis of beta-alanine respectively through whole cell transformation and fermentation liquid direct transformation. A synthetic method comprises the steps: an L-aspartic-alpha-alanine decarboxylase (PanD) gene of lactobacillus plantarum is obtained by a gene engineering method and has the gene sequence number of NC-004567.2, the gene is constructed into a high-efficiency expression vector, then the high-efficiency expression vector is transformed into recipient bacteria, and thus the genetic engineering bacteria for producing beta-alanine are obtained. The genetic engineering bacteria are subjected to fermentation culture, 80 g / L of a substrate can be transformed by whole cells, and the beta-alanine content reaches 59.7 g / L; and the substrate is directly added into a fermentation liquid, the transformation concentration can reach 10 g / L, the beta-alanine content reaches 6.8 g / L, and the synthetic ability is higher than that of conventional reported genetic engineering bacteria.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, and in particular relates to the construction and expression of a genetically engineered bacterium with high yield of β-alanine, and a method for transforming and synthesizing β-alanine by using the fermented liquid of the genetically engineered bacterium. Background technique [0002] β-alanine is the only β-type amino acid that exists in nature and is the prerequisite for the synthesis of pantothenic acid in organisms. Pantothenic acid is an important precursor substance for the biosynthesis of coenzyme A (CoA) and acyl carrier protein (ACP), and participates in the metabolism of carbohydrates, fatty acids, proteins and energy in organisms and participates in the metabolism of proteins, lipids and sugars. [0003] β-alanine is an important multi-purpose organic raw material. It can be synthesized and polymerized β-alanine for water treatment industry and dye production; it can be used to s...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/60C12N15/63C12P13/06
Inventor 袁京董文玥吴洽庆朱敦明
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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