Method for fermentation production of L-proline by utilizing recombinant Escherichia coli

A technology of Escherichia coli and proline, applied in the direction of fermentation, recombinant DNA technology, bacteria, etc., to achieve the effect of broad industrial application prospects

Inactive Publication Date: 2015-04-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many high-yield L-proline production strains have been obtained by traditional mutagenesis methods, these traditional methods have many limitations

Method used

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  • Method for fermentation production of L-proline by utilizing recombinant Escherichia coli
  • Method for fermentation production of L-proline by utilizing recombinant Escherichia coli
  • Method for fermentation production of L-proline by utilizing recombinant Escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Acquisition of glutamate kinase gene

[0025] In the synthesis of proline, glutamate kinase (proB) and glutamate dehydrogenase (proA) always complement each other, the expression and effect of the two will affect each other, and the genes of the two are also close to each other, so In this embodiment, the two are designed as a whole to obtain the proBA gene fragment, and a pair of primers P1 (SEQ ID NO: 3), P2 (SEQ ID NO: 3) and P2 (SEQ ID NO: 3) are designed according to the proBA gene sequence published in NCBI (GenBank accession number: x00786. ID NO: 4), the target gene proBA was obtained by PCR amplification.

[0026] In the present embodiment, the template of PCR is Escherichia coli genome, extraction method: get 1.5mL bacterium liquid (overnight culture), 12000rpm centrifuges 30s to collect thalline; Under the situation that does not affect bacterial precipitation, remove supernatant as far as possible; Thalline Resuspend the pellet in 600 μL lysate (...

Embodiment 2

[0027] Example 2: Acquisition of tryptophan tandem promoter gene

[0028] The tryptophan tandem promoter is derived from the tryptophan operon of Escherichia coli K12 strain, and is a strong promoter suitable for industrial production applications. Since two tryptophan tandem promoters can be connected in series to increase the expression intensity, other researchers in the laboratory optimized the tryptophan tandem promoter and then synthesized the tryptophan tandem promoter in the whole gene.

[0029] See SEQ ID NO:1 for the sequence of the tryptophan tandem promoter in this embodiment.

Embodiment 3

[0030] Embodiment 3: the construction of proBA expression plasmid and recombinant bacteria

[0031] First obtain the proBA gene, the proBA gene needs to be amplified by PCR, and the obtained proBA gene 5' end sequence has a HindⅢ (A^AGCTT) restriction site, and the 3' end has an XhoI (C^TCGAG) restriction site , the proBA gene and plasmid vector pET-28a were digested with HindⅢ and XhoI respectively, and the proBA gene and pET-28a plasmid vector after the double-cut enzyme were recovered with an agarose gel kit, and then proBA and pET were digested with T4 DNA ligase. -28a gene fragments were connected. After overnight ligation at 16°C, transfer 10 μL of the ligation solution into Escherichia coli JM109. Pick the transformed single colony culture to extract the plasmid for verification, and then further sequence to verify whether the gene sequence is correct. Thus, the constructed pET-28a-proBA recombinant plasmid was obtained, and the recombinant Escherichia coli JM109 / pET-...

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Abstract

The invention discloses a method for fermentation production of L-proline by utilizing recombinant Escherichia coli. The recombinant Escherichia coli carries recombinant plasmid with gamma-glutamyl kinase genes with site mutagenesis and tryptophan series promoters, wherein the recombinant plasmid is obtained by connecting the cloned gamma-glutamyl kinase genes and the tryptophan series promoter in the lab to the same plasmid and performing whole plasmid PCR site-specific mutagenesis. The invention also discloses an application of the Escherichia coli in production of L-proline.

Description

technical field [0001] The invention discloses a method for fermenting and producing L-proline by recombinant Escherichia coli, which belongs to the field of microbial genetic engineering. Background technique [0002] Among many amino acids that synthesize human protein, L-proline is a non-polar amino acid, and its importance is reflected in many fields such as medicine, agriculture, chemical industry, and food. In medicine, L-proline is the raw material for the synthesis of some special drugs; in agriculture, L-proline can enhance the stress resistance of crops; in the chemical industry, L-proline and some of its derivatives It is also an important chiral molecule and can be used as a catalyst in some reactions; L-proline also plays an important role in food nutrition and other aspects. [0003] The earliest L-proline was mainly obtained by hydrolyzing protein. Due to the complicated production and purification steps, not only the final L-proline yield was low, the utiliz...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/77C12N15/78C12N15/75C12N15/81C12N1/21C12N1/19C12P13/24
Inventor 张震宇胡丹丹范永明
Owner JIANGNAN UNIV
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