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Engineering bacteria and method for producing beta-alanine

A technology of engineering bacteria and aspartic acid, which is applied in the construction and application field of genetically engineered bacteria, can solve the problems of low yield and low activity of β-alanine, and the inability to achieve industrial production, so as to ensure the stability of enzyme activity, The effect of improving activity, saving cultivation cost and subsequent sewage treatment cost

Active Publication Date: 2015-06-03
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that in these early studies, due to the low activity of the selected L-aspartate α-carboxylase, the yield of β-alanine obtained by biotransformation was extremely low, which could not meet the needs of industrial production.

Method used

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  • Engineering bacteria and method for producing beta-alanine
  • Engineering bacteria and method for producing beta-alanine
  • Engineering bacteria and method for producing beta-alanine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1. Engineering bacteria expressing L-aspartate alpha carboxylase on the construction plasmid

[0048] We selected L-aspartate α-carboxylase from four hosts, Escherichia coli, Corynebacterium glutamicum, Mycobacterium tuberculosis and Bacillus subtilis, respectively. The coding genes of 4 kinds of engineering bacteria were constructed. Wherein, the DNA sequence shown in Sequence 1 of the sequence listing is panD, the encoding gene of L-aspartate α-carboxylase in Escherichia coli BL21(DE)3 E . The DNA sequence shown in SEQ ID NO: 2 of the Sequence Listing is the gene panD encoding L-aspartate α-carboxylase in Corynebacterium glutamicum ATCC13032 C . The DNA sequence shown in Sequence 3 of the sequence listing is panD, the encoding gene of L-aspartate α-carboxylase in Mycobacterium tuberculosis H37Rv M . The DNA sequence shown in SEQ ID NO: 4 of the sequence listing is the gene panD encoding L-aspartate α-carboxylase in Bacillus subtilis 168 B .

[0049] 1. ...

Embodiment 2

[0101] Example 2. Detection of L-aspartate alpha carboxylase activity of engineered bacteria expressing L-aspartate alpha carboxylase on plasmids

[0102] The engineering bacteria A, engineering bacteria B, engineering bacteria C and engineering bacteria D constructed in Example 1 are respectively carried out as follows:

[0103] 1. Pick a single colony of the engineered bacteria, inoculate it in 10 mL of LB liquid medium containing 50 μg / ml kanamycin, and culture with shaking at 37°C and 200 rpm for 12 h.

[0104] 2. Take the entire culture system obtained in step 1, inoculate it into 100 mL of LB liquid medium containing 50 μg / ml kanamycin and 0.2 mM IPTG, and culture with shaking at 30° C. and 200 rpm for 12 h.

[0105] 3. The termination time of step 2 (ie OD 600nm = 3 bacterial solution), take 2 mL of culture solution, add 1 mL of pH 8.0, 0.01 mol / L phosphate buffer, mix well, and then ultrasonically disrupt (power 200W, work for 3 seconds and stop for 3 seconds, total t...

Embodiment 3

[0116] Embodiment 3, utilize engineering bacteria Ding to transform L-aspartic acid to produce β-alanine

[0117] 1. Pick a single colony of the engineered bacterial cells prepared in Example 1, inoculate it in 100 mL of LB liquid medium, and culture with shaking at 37° C. and 200 rpm for 12 h.

[0118] 2. Take the entire culture system obtained in step 1, inoculate it into 1000 mL of LB liquid medium containing 50 μg / ml kanamycin and 0.2 mM IPTG, and culture with shaking at 30° C. and 200 rpm for 12 h.

[0119] 3. Take the culture solution obtained in step 2, centrifuge at 9000 rpm for 10 min, and collect the bacterial cells.

[0120] 4. Take all the cells obtained in step 3, suspend with 300 mL of distilled water, add L-aspartic acid (90 g), biotransform at 37° C., 450 rpm for 15 h, during the transformation process, sample once every hour, using the method in Example 2. Methods The concentration of β-alanine was detected by HPLC.

[0121] For test results see image 3 . ...

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Abstract

The invention discloses engineering bacteria and a method for producing beta-alanine. According to the engineering bacteria, one or more sites of the genome of escherichia coli are integrated with the recombinant bacteria of a DNA sequence, wherein the DNA sequence contains one gene selected from a gene for encoding the alpha-carboxylase of L-aspartic acid of bacillus subtilis and a gene for encoding a polypeptide having at least 60%, preferably 80%, more preferably 90%, further preferably 95%, most preferably 98% and even 99% homology with the enzyme and having the activity of the enzyme. The bacterial strain is capable of greatly expressing the high-activity alpha-carboxylase of L-aspartic acid without adding antibiotics. The bacterial strain can be used for efficiently converting the substrate L-aspartic acid into beta-alanine. The problem of easy plasmid loss in the bacterial strain culture process is solved, the use of the antibiotics in the bacterial culture process and the subsequent treatment of wastewater containing the antibiotics are omitted, and the cost of the industrial production is reduced.

Description

technical field [0001] The invention relates to the field of biocatalysis, in particular to the construction and application of genetically engineered bacteria for producing beta-alanine. Background technique [0002] β-alanine is the only β-alanine naturally occurring in nature, and it is widely used in medical, food and chemical fields. Such as the synthesis of calcium pantothenate and carnosine as chemical raw materials, pamidronate sodium for the treatment of tumor hypercalcemia and non-specific anti-inflammatory drug balsalazide for the treatment of ulcerative colitis. In addition, β-alanine can also be used as a precipitant in the preparation of pharmaceuticals, a water purification flocculant, an electroplating corrosion inhibitor, an antidote for lead poisoning, and a synthetic sweetener. [0003] β-alanine chemical production includes acrylonitrile method, acrylic acid method, succinimide degradation method and β-aminopropionitrile method. Among them, the acryloni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P13/06C12R1/19
CPCC12N9/88C12P13/06C12Y401/01011C12N1/205C12R2001/19
Inventor 蔡真张君丽李寅张冬竹郭恒华
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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