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Recombinant bacteria for producing beta-alanine as well as construction method and application of recombinant bacteria

A construction method and technology of recombinant bacteria, applied in the biological field, can solve the problems of environmental pollution, unsuitable separation and purification, harsh reaction conditions of beta-alanine, etc.

Active Publication Date: 2018-11-02
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] In summary, the production of β-alanine by chemical synthesis generally faces problems such as harsh reaction conditions, unsuitable separation and purification, and easy environmental pollution.

Method used

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  • Recombinant bacteria for producing beta-alanine as well as construction method and application of recombinant bacteria
  • Recombinant bacteria for producing beta-alanine as well as construction method and application of recombinant bacteria
  • Recombinant bacteria for producing beta-alanine as well as construction method and application of recombinant bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0180] Embodiment 1, the construction of recombinant escherichia coli engineering strain FM07

[0181] In this example, a basic strain FM07, which can be used to prepare strains producing β-alanine and 3-hydroxypropionic acid, was prepared. The preparation method of the strain is as follows, and the primers used are shown in Table 1.

[0182] (1) Knockout of fatty acid degradation transcription factor fadR.

[0183] Starting from Escherichia coli BW25113, the fadR gene of Escherichia coli BW25113 was knocked out to obtain the mutant FM01 of Escherichia coli BW25113, the specific steps are as follows:

[0184] (1-a) Prepare a P1 phage containing an Escherichia coli gene fragment having a fadR knockout property.

[0185] The Escherichia coli gene fragment containing the fadR knockout trait comes from Escherichia coli strain JW1176, which is a W3110 series strain containing the fadR knockout trait. JW1176 is a product of the National Institute of Genetics (NIG, Japan) in Japan, ...

Embodiment 2

[0257] Embodiment 2, the preparation of the bacterial strain FM08 that is used to produce β-alanine and the production of β-alanine

[0258] One, for the preparation of the bacterial strain FM08 of producing β-alanine (β-alanine)

[0259] The preparation method of FM08 is as follows, and the primers used are shown in Table 2.

[0260] (1) Construction of a plasmid expressing a truncated gene mcrC of Chloroflexus aurantiacus malonyl-CoA reductase.

[0261] (1-a) PCR amplification of mcrC gene.

[0262] The nucleotide sequence of the transformed Chloroflexus aurantiacus malonyl-CoA reductase truncated gene mcrC is as SEQ ID No.22, and SEQ ID No.23 in the coding sequence list protein shown. Whole gene synthesis of the mcrC gene shown in SEQ ID No.22, and then using the Gibson assembly method (Gibson DG, YoungL, et al.Enzymatic assembly of DNA molecules up to several hundred kilobases.Nat.methods.2009; 6(5):343 -345) The mcrC gene shown in SEQ ID No.22 was connected to the pUC...

Embodiment 3

[0309] Embodiment 3, the preparation of bacterial strain FI08 for producing 3-hydroxypropionic acid and the production of 3-hydroxypropionic acid

[0310] 1. Preparation of bacterial strain FI08 for the production of 3-hydroxypropionic acid

[0311] The preparation method of FI08 is as follows, and the primers used are shown in Table 3.

[0312] (1) Construction of a plasmid expressing the acetyl-CoA carboxylase acc gene cluster of Corynebacterium_glutamicum.

[0313] (1-a) Extraction of genomic DNA of Corynebacterium glutamicum and PCR amplification of the acc gene cluster.

[0314] Genomic DNA of Corynebacterium glutamicum was extracted using a bacterial genome extraction kit (Tiangen Biochemical Technology Co., Ltd., product catalog DP302). Using the total genomic DNA extracted from Corynebacterium glutamicum as a template and accBC-F and accL-R as primers, the gene fragment accBC was amplified by high-fidelity TransStart FastPfu DNA polymerase PCR, and the target fragmen...

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Abstract

The invention discloses recombinant bacteria for producing beta-alanine as well as a construction method and application of the recombinant bacteria. The construction method for the recombinant bacteria disclosed by the invention comprises the following steps: knocking out fadR gene, fabF gene, fabH gene, iclR gene and sucA gene of recipient bacteria; introducing aspC gene or a gene cluster, panDgene, alkL gene and gdh gene into the recipient bacteria; enhancing expression of fadL gene, fadD gene, sthA gene, an atoSC gene cluster and an aceBA gene cluster in the recipient bacteria, wherein the recipient bacteria are bacteria or fungi containing the fadR gene, the fabF gene, the fabH gene, the iclR gene and the sucA gene. Experiments prove that the conversion rate of the beta-alanine produced by using the recombinant bacteria disclosed by the invention with fatty acid as a raw material is 60.87 percent and shows that the recombinant bacteria disclosed by the invention can be used for preparing the beta-alanine.

Description

technical field [0001] The invention relates to a recombinant bacterium producing beta-alanine and its construction method and application in the field of biotechnology. Background technique [0002] β-alanine (English name β-Alanine), also known as β-alanine, is a non-protein amino acid with important value. As a biochemical raw material, β-alanine has broad application prospects in the fields of medicine, feed and food. For example, beta-alanine is used in the synthesis of pantothenic acid (vitamin B5), a component of coenzyme A that is essential for many metabolisms. [0003] At present, the synthesis methods of β-alanine mainly include chemical synthesis and biotransformation, as follows: [0004] 1. Chemical synthesis [0005] (1) Acrylic acid method: β-alanine is obtained mainly by ammoniating acrylic acid (or acrylate, acrylate) and ammonia water at a higher temperature and pressure. The main problem of the acrylic acid method is that there are many by-products, a...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12P13/06C12R1/19
CPCC12N9/0016C12N9/1029C12N9/1096C12N9/88C12N15/70C12P13/06C12Y104/01021C12Y203/01041C12Y203/01179C12Y206/01001C12Y401/01011C07K14/195C07K14/245
Inventor 刘伟丰刘姣刘波薛燕芬陶勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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