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Recombinant bacteria for producing gamma-aminobutyric acid by glycerin and construction method of recombinant bacteria

A technology of aminobutyric acid and its construction method, which is applied in the fields of synthetic biology and metabolic engineering, can solve problems such as low yield and inability to meet actual production needs, and achieve the effect of improving utilization efficiency

Inactive Publication Date: 2019-09-27
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

Relevant studies were carried out in Escherichia coli, but due to the low yield and the need to add L-glutamic acid exogenously, it could not meet the actual production needs

Method used

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  • Recombinant bacteria for producing gamma-aminobutyric acid by glycerin and construction method of recombinant bacteria
  • Recombinant bacteria for producing gamma-aminobutyric acid by glycerin and construction method of recombinant bacteria
  • Recombinant bacteria for producing gamma-aminobutyric acid by glycerin and construction method of recombinant bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1 improves the strain construction of the Corynebacterium glutamicum of glycerol utilization efficiency

[0024] (1) Design RBS random mutation primers for the three genes glpF, dhaD, and dhaK in the glycerol utilization pathway and the pEC-XK99E plasmid. The primer sequences used are:

[0025] glpF-F: 5'-caccaggtctcacacaaaaggannnnnnnnnatgagtcaaacatcaaccttgaaag-3'

[0026] glpF-R: 5'-caccaggtctcagactgcttacagcgaagctttttgttctg-3'

[0027] dhaD-F: 5'-caccaggtct caagtcaaaggannnnnnnnnatgctaaaagttatcaatctccagc-3'

[0028] dhaD-R: 5'-caccaggtctcacctgggttaacgcgccagccactgctgtc-3'

[0029] dhaK-F: 5'-caccaggtctcacaggaaaggannnnnnnnnatgtctcaattcttttttaaccaacg-3'

[0030] dhaK-R: 5'-caccaggtctcacgttacccttagcccagctcactctccgccag-3'

[0031] pEC-XK99E-F: 5'-caccaggtctcaaacgggcggagagtgagctgggc-3'

[0032] pEC-XK99E-R: 5'-caccaggtctcatgtgaaattgttatccgctcacaattcc-3'

[0033] The pEC-XK99E-glpFDK plasmid was used as a template, and the above primers were used for PCR ampl...

Embodiment 2

[0037] Example 2 Construction of different RBS intensities overexpressing Bacillus megaterium glutamic acid decarboxylase gene gad strain

[0038] (1) First extract the pET21b-Bmgad plasmid from the DH5α / pET21b-Bmgad strain, extract the pXMJ19 plasmid from the DH5α / pXMJ19 strain, and then use the primer pairs gad-F / gad-R and pXMJ19-F / pXMJ19-R to amplify The gad gene fragment and the pXMJ19 vector fragment were obtained by using Golden Gate assembly technology to obtain the plasmid pXMJ19-gad. The gad-H gene fragment was amplified with primers gad-H-F / RBS-gad-R, and the gad-L gene fragment was amplified with primers gad-L-F / RBS-gad-R. Use the GoldenGate assembly technology to connect the above fragments to the vector pXMJ19 to obtain plasmids pXMJ19-gad and pXMJ19-gad H , pXMJ19-gad L . The primer sequences used are:

[0039] gad-F: 5'-gaaggagatatacatatgccacagtggcacccacaccgcg-3'

[0040] gad-R: 5'-gcctttttgcgtttctacaaactcttttgtttatttttc-3'

[0041] pXMJ19-F: 5'-gtttctacaa...

Embodiment 3

[0048] Example 3 Construction of Corynebacterium glutamicum serine / threonine-protein kinase gene pknG deletion strain

[0049] (1) Using the genome of C. glutamicum ATCC 13032 as a template, the pknG-up fragment and pknG- dn fragment. The upper homology arm and the lower homology arm are connected by fusion PCR technology. The fused fragment was treated with HindIII and XbaI enzymes, and connected to the temperature-sensitive knockout vector pCRD206 to obtain the knockout plasmid pCRD206-pknG. The primer sequences used are:

[0050] pknG-up-F: 5'-agtgaagcttattttcggtgaccccaataagg-3'

[0051] pknG-up-R: 5'-caaatagccccaagtcaaaacagcgttttctgtcccttcttcctc-3'

[0052] pknG-dn-F: 5'-ctgttttgacttggggctatttg-3'

[0053] pknG-dn-R: 5'-tctgtctagagaaaatgcccagtttgtccgt-3'

[0054] (3) pknG gene knockout step: the plasmid pCRD206-pknG was electrotransformed into C. glutamicum ATCC 13032 competent cells. Pick the transformant and put it into a test tube of 3mL resistant LBHIS liquid me...

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Abstract

The invention discloses recombinant bacteria for producing gamma-aminobutyric acid by glycerin and a construction method of the recombinant bacteria. Firstly, a glycerin promoting protein gene glpF, a glycerin dehydrogenase gene dhaD and a dihydroxyacetone kinase gene dhaK are subjected to RBS random mutation to obtain corynebacterium glutamicum mutant strains capable of utilizing glycerin efficiently. Base strains capable of producing gamma-aminobutyric acid by glycerin are obtained through glutamate decarboxylase gene gad in over-expression bacillus megaterium. Glutamate decarboxylase is expressed by using RBS with different strength, and high-strength RBS is most favorable for synthesis of gamma-aminobutyric acid; then, the translocator gene gadC of the gamma-aminobutyric acid is over-expressed by increasing the level of precursor L-glutamic acid and knocking out the degradation way of the gamma-aminobutyric acid. Finally, 60 g / L of glycerin is used as a substrate, and the content of the gamma-aminobutyric acid reaches 10.94 g / L after fermentation for 84 h.

Description

technical field [0001] The invention belongs to the technical field of synthetic biology and metabolic engineering. The food safety grade microorganism Corynebacterium glutamicum is used as a host bacterium, and a bacterial strain capable of producing gamma-aminobutyric acid by using glycerol is constructed through a metabolic engineering strategy. Background technique [0002] γ-aminobutyric acid widely exists in plants, microorganisms and animals, and has been widely used in feed, food, health products and industries. Synthesis of GABA by chemical method will have some chemical reagent residues, which is not safe for use in the food and pharmaceutical industries. The microbial fermentation method has mild conditions, simple operation, and low pollution, which is conducive to industrial production. GAD in microbial cells can convert L - Glutamate is converted to GABA. [0003] Corynebacterium glutamicum is a short rod-shaped, single or splayed Gram-positive bacterium that ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/77C12P13/00C12R1/15
CPCC07K14/34C12N9/0006C12N9/1205C12N9/88C12N15/77C12P13/005C12Y101/01006C12Y207/01029C12Y401/01015C12Y401/01031
Inventor 刘君王一然徐宁
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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