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Method for constructing engineering bacterium capable of producing beta-alanine and method for producing beta-alanine by adopting engineering bacterium

A technology of engineering bacteria and alanine, which is applied in biochemical equipment and methods, genetic engineering, botany equipment and methods, etc., can solve the problems of low enzyme activity and complicated operation, and achieve low raw material cost and medium composition Simple, high enzymatic effect

Inactive Publication Date: 2017-11-10
LUDONG UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using genetic engineering to construct engineered bacteria is an important way to achieve high-efficiency expression of enzymes. At present, the genes for exogenously expressing L-aspartate α-decarboxylase in engineered strains are mostly derived from Escherichia coli and Corynebacterium glutamicum, and the enzyme activity is not high. , in patent CN104531796A, β-alanine is catalyzed by L-aspartic acid α-decarboxylase engineered bacteria whole cells, the highest catalyzed 48g / L L-aspartic acid, the concentration of β-alanine is about 32g / L L, there is still a distance from industrial application, and the enzyme needs to be put in repeatedly, and the operation is more complicated

Method used

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  • Method for constructing engineering bacterium capable of producing beta-alanine and method for producing beta-alanine by adopting engineering bacterium
  • Method for constructing engineering bacterium capable of producing beta-alanine and method for producing beta-alanine by adopting engineering bacterium
  • Method for constructing engineering bacterium capable of producing beta-alanine and method for producing beta-alanine by adopting engineering bacterium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Construction of Genetic Engineering Bacteria Containing L-Aspartic Acid α-Decarboxylase

[0031] Using the whole genome of Bacillus tequila PanD37 (CGMCC No.10506) as a template, a pair of primers were designed to amplify the L-aspartic acid α-decarboxylase gene PanD by PCR, and the recombinant plasmid pET-32a-PanD (such as figure 2 shown), transform E.coliBL21(DE3) to obtain engineering bacteria producing L-aspartic acid α-decarboxylase.

[0032] According to the sequence information of the L-aspartate α-decarboxylase gene in the whole genome of Bacillus tequila (LGRW01000001), the upstream primer F-PanD containing the restriction endonuclease BamHI restriction site was designed: 5′-AAGGATCCGAAGGAGATATACCATGTATCG- 3'; and a downstream primer R-PanD containing an XhoI restriction site (in bold): 5'-TTCTCGAGCTACAAATTGTACGGGCGGGTTC-3'. Bacillus tequilensis PanD37 (CGMCC No.10506) genomic DNA was extracted by the bacterial genomic DNA extraction kit, and PCR amplificatio...

Embodiment 2

[0034] Acquisition of L-aspartate by expression and secretion α-decarboxylase converts L-aspartate to β-alanine

[0035] (1) inserting the constructed L-aspartic acid α-decarboxylase genetically engineered bacteria into LB slant medium and culturing for 24 hours;

[0036] (2) Connect the 1-ring slant-plane L-aspartate α-decarboxylase genetically engineered bacterial strain to culture in LB liquid seed medium for 12 hours;

[0037] (3) 3.0L medium is loaded into the 5L fermenter, and the seed solution is inserted into the fermentation medium with a seed amount of 5% (volume ratio). The initial rotating speed is 200r / min, and the initial ventilation flow rate is 2L / min. 600 Increase the value and adjust the rotation speed and ventilation flow to maintain the dissolved oxygen value above 20% (measured by the dissolved oxygen electrode), adjust the pH value to be stable at 7.0 with 25% (g / 100ml) ammonia water, incubate at 37°C for 6h, and then cool down to 30°C Express. During t...

Embodiment 3

[0045] Acquisition of L-aspartate by expression and secretion α-decarboxylase converts L-aspartate to β-alanine

[0046] (1) inserting the constructed L-aspartate α-decarboxylase genetically engineered bacteria into LB slant medium and culturing for 18 hours;

[0047] (2) Connect the 1-ring slant L-aspartic acid α-decarboxylase genetically engineered strains to culture in LB liquid seed medium for 8 hours;

[0048] (3) 3.0L medium is loaded into the 5L fermenter, the seed solution is inserted into the fermentation medium with 7% (volume ratio), the initial rotating speed is 200r / min, and the initial ventilation flow rate is 2L / min. 600 Increase the value to adjust the rotation speed and ventilation flow to maintain the dissolved oxygen value above 25%, adjust the pH value to be stable at 7.0 with 25% ammonia water, cultivate at 37°C for 9 hours and cool down to 26°C for expression. During the fermentation process, the pH-stat method was used to feed and supplement the culture...

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Abstract

The invention discloses a method for constructing an engineering bacterium capable of producing beta-alanine and a method for producing beta-alanine by adopting the engineering bacterium, belonging to the field of biotechnologies. The invention provides L-aspartate alpha-decarboxylase gene of a bacillus tequilensis strain, as shown in SEQ ID NO.1, and the engineering bacterium comprising the PanD gene. The invention further provides a constructing method of the engineering bacterium capable of producing the L-aspartate alpha-decarboxylase, L-aspartate alpha-decarboxylase obtained through expression and secretion is adopted for transforming L-aspartic acid, and thus beta-alanine is produced. According to the invention, L-aspartate alpha-decarboxylase derived from bacillus tequilensis is adopted for transforming L-aspartic acid for producing beta-alanine for the first time at home and abroad, after the enzyme is expressed in escherichia coli, the generated L-aspartate alpha-decarboxylase has high enzyme activity, the thallus does not need to be crushed and can be directly used for carrying out transformation, L-aspartic acid of 180g / L can be transformed to the maximum, the yield of beta-alanine can achieve 119.8g / L, and the industrial application has the advantages.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for constructing genetically engineered bacteria and its application in transforming L-aspartic acid to produce β-alanine. Background technique [0002] 1. The medical value and physiological function of β-alanine [0003] β-alanine is the only β-type amino acid that exists in nature. As an important fine chemical and pharmaceutical intermediate, it has a wide range of uses: industrially, it is an important raw material for the synthesis of calcium pantothenate and carnosine, and is also an important raw material for the synthesis of poly β-alanine. The precursor of aminopropionic acid; in medicine, it is used as a raw material to synthesize pamidronate sodium and anti-colitis drug balsalazide for inhibiting bone metastasis of malignant tumors, and it is also an important component of compound amino acids; it can also be used as lead Poisoning antidote and used ...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N1/21C12N15/75C12P13/06
CPCC12N9/88C12P13/06C12Y401/01011
Inventor 冯志彬张娟
Owner LUDONG UNIVERSITY
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