Production method for 3-aminoisobutyric acid

A technology of aminoisobutyric acid and alanine, which is applied in the field of metabolic engineering and can solve problems such as high pollution

Active Publication Date: 2018-12-14
ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In order to overcome the high pollution problem of the existing chemical synthesis method of 3-aminoisobutyric acid, the present invention uses metabolic engineering to transform Escherichia coli, which is the most widely used in genetic engineering, and develops a way to produce 3-aminoisobutyric acid by microbial fermentation

Method used

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  • Production method for 3-aminoisobutyric acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1: Construction of the bacterial strain that knocks out pstG gene, fumAC gene cluster and fumB gene

[0047] 1.1 The pCAS plasmid (gifted by the Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, see Jiang, Y., B. Chen, C. Duan, B. Sun, J. Yang and S. Yang. Applied and Environmental Microbiology. 2015,81(7):2506-2514) into Escherichia coli MG1655 by electrotransformation to obtain MG1655pCAS strain. With the pTargetF plasmid (gifted by the Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, see Jiang, Y., B. Chen, C. Duan, B. Sun, J. Yang and S. Yang. Applied and Environmental Microbiology.2015 , 81(7):2506-2514) as a template, using pTargetFΔpstG-F / pTargetFΔpstG-R primers, carry out reverse PCR, and the above PCR products were electrotransformed into Escherichia coli DH5α competent cells (purchased from Shanghai Weidi Biotechnolo...

Embodiment 2

[0082] Embodiment 2: the construction of gene panD and aspA expression plasmid

[0083] The pTrcHis2B plasmid (Thermofisher Company) was digested with BamHI / HindIII to obtain the digested fragment. Using MG1655 genomic DNA as a template, PCR amplification was performed using the panD-Ec-F / PanD-Ec-R primers to obtain the panD gene fragment. Using MG1655 genomic DNA as a template, the aspA gene fragment was obtained by PCR amplification using aspA-Ec-F / aspA-Ec-R primers. PCR primer pairs are:

[0084] PanDec-F:

[0085] 5'-GGAGGAATAAACCATGGAGCTCAGGAGGTAAAAAAACATGCTGCGCACCATCCTCGG-3',

[0086] PanDec-R:

[0087] 5'-CAAATGCATTCTTAAACATGTTTTTTTACCTCTCTAAATGCTTCTCGACGTCA-3',

[0088] aspAec-F:

[0089] 5'-GACGTCGAGAAGCATTTAGAGGAGGTAAAAAAACATGTTTAAGAATGCATTTGC-3',

[0090] aspAec-R: 5'-CTGAGATGAGTTTTTGTTCTAGAATTAGTGGTTACGGATGTACT-3'.

[0091] According to Gibson The operation method of Master Mix shows that the pTrcHis2B plasmid BamHI / HindIII fragment, panD gene fragment an...

Embodiment 3

[0092] Embodiment 3: Construction of gene C24MTgm expression plasmid

[0093] 3.1 Using the pSU2718 plasmid (gifted by the Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) as a template, use pSU2718-F / pSU2718-R primers to obtain the pSU2718 vector fragment by PCR amplification. The lacIqTrc fragment was amplified by PCR using the pTrcHis2B plasmid as a template and using lacIqTrc-F / lacIqTrc-R primers. PCR primer pairs are:

[0094] pSU2718-F: 5'-CGTAATCATGGTCATAGCTG-3',

[0095] pSU2718-R: 5'-GAATGGCGAATGGCGCTGAT-3',

[0096] lacIqTrc-F: 5'-ATCAGCGCCATTCGCCATTCGCCAGTATACACTCCGCTAT-3',

[0097] lacIqTrc-R: 5'-CAGCTATGACCATGATTACGGGAGAGCGTTCACCGACAAA-3'.

[0098] 3.2 Perform Gibson assembly and cloning of the lacIqTrc fragment and the pSU2718 vector backbone PCR fragment to obtain the plasmid pSU-lacIqTrc. S-adenosyl-L-methionine δ24-sterol-C-methyltransferase gene C24MTgm was synthesized according to NCBI'...

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Abstract

The invention discloses a production method for 3-aminoisobutyric acid. A 3-aminoisobutyric acid producing strain is constructed through the following steps: A, knocking out the ptsG, fumAC and fumB genes in the MG1655 genome of Escherichia coli so as to obtain the gene knockout strain MG1655 (delta<ptsG>delta<fumAC>delta<fumB>); B, constructing of expression plasmids expressing the genes panD, aspA and C24MTgm; and C, transforming the plasmids obtained in the step B into the gene knockout strain MG1655 (delta<ptsG>delta<fumAC>delta<fumB>) obtained in the step A so as to obtain a genetically-engineered strain. The Escherichia coli engineered strain constructed in the invention can produce 3-aminoisobutyric acid, and the yield of aminoisobutyric acid produced through shake flask fermentation is as high as 100 mg/L.

Description

technical field [0001] The invention belongs to the field of metabolic engineering, in particular, relates to a method for constructing 3-aminoisobutyric acid production strains, in particular to a method for producing 3-aminoisobutyric acid and / or β-alanine through metabolic engineering strains sour method. Background technique [0002] 3-Aminoisobutyric acid is also called β-aminoisobutyric acid (β-AIB) or 3-amino-2-methylpropionic acid. Its chemical structure is as follows: [0003] [0004] 3-Aminoisobutyric acid is a non-protein amino acid, a metabolite of thymine and valine, which is excreted in urine, in which the R-configuration is from thymine, and the S configuration is from valine. The R-configuration in the liquid accounts for more than 90%. The study found that β-AIB, as the first representative small molecule myokine of the non-adrenergic activator family in the thermogenic program of white adipose tissue, has good druggability for type II diabetes and met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P13/04C12P13/06C12R1/19
CPCC12N9/1007C12N9/88C12P13/04C12P13/06C12Y403/01001
Inventor 范文超高书良王金刚梁岩袁圣伦任亮
Owner ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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