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A kind of l-homoserine production strain and its construction method and application

A technology for producing homoserine and bacterial strains, applied in the field of genetic engineering, can solve problems such as low yield, high production cost, and large pollution, and achieve the effects of high yield, low cost, and less environmental pollution

Active Publication Date: 2021-03-30
SICHUAN LIER BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The third technical problem to be solved by the present invention is that the method for producing L-homoserine in the prior art has the problems of high production cost, large pollution and low yield, and then provides a low-cost, high-yield, conditional Method for producing L-homoserine with mildness and less environmental pollution

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Construction of the bacterial strain MG1655 (ΔthrB) of embodiment 1 knockout thrB gene

[0042] The L-homoserine production strain constructed in this example is the thrB gene knockout strain MG1655 (ΔthrB), and its construction method can refer to the literature: Proc Natl Acad Sci US A. 2000 Jun 6; 97 (12): 6640-5, Specifically include the following steps:

[0043] (1) Using pΔthrB-H1-f / pΔthrB-H2-r as primers and using the plasmid containing the Kana resistance cassette as a template, perform PCR amplification, recover and purify the thrB homology arms and A linear DNA fragment containing Kana resistance at the FRT site;

[0044] The primer pΔthrB-H1-f has the sequence shown in SEQ ID No.1, and the primer pΔthrB-H2-r has the sequence shown in SEQ ID No.2, specifically as follows:

[0045] SEQ ID No. 1: 5'-TGGTTAAAGTTTATGCCCCGGCTTCCAGTGCCAATATGAGCGTCGGGTTTGTGTAGGCTGGAGCTGCTTCGAAG-3'

[0046] SEQ ID No.2: 5'-TTAGTTTTCCAGTACTCGTGCGCCCGCCGTATCCAGCCGGCAAATATGAACATGGGAATTA...

Embodiment 2

[0050] Example 2 Bacterial strain MG1655 (ΔthrB, rhtA23) that knocks out the thrB gene and overexpresses the rhtA gene

[0051] The L-homoserine production strain constructed in this example is a strain MG1655 (ΔthrB, rhtA23) in which the thrB gene is knocked out and the rhtA gene is overexpressed. For its construction method, please refer to the literature: Microb Cell Fact.2016Dec 1; 15(1) :205, specifically including the following steps:

[0052] (1) Using Escherichia coli MG1655 as a template, pSOE-rhtA23-H1-f, pSOE-rhtA23-H1-r, pSOE-rhtA23-H2-f, pSOE-rhtA23-H2-r, prhtA23-N20-f, prhtA23 -N20-r is a primer, which is amplified by the overlap extension PCR method (SOE-PCR method), and the immediate adjacent base upstream of the threonine-homoserine resistance gene (transfer pump gene) rhtA is replaced from G to A , obtain the rhtA23 donor DNA fragment, and introduce the cloning sites required for constructing the CRISPR / Cas9 gene editing plasmid at both ends;

[0053] Where...

Embodiment 3

[0067] Example 3 Construction of strain MG1655 (thrB(R235H), rhtA23) that weakens the thrB gene and overexpresses the rhtA gene

[0068] The L-homoserine production strain constructed in this example is the strain MG1655 (thrB(R235H), rhtA23) in which the thrB gene is weakened and the rhtA gene is overexpressed. CRISPR / Cas9 gene editing technology is used to realize the construction of the strain. The R235 position and the upstream base of the rhtA gene were mutated, and the construction method was basically the same as the method in Example 2, the only difference being that the original bacteria MG1655 was used as the operating object, and the gene editing of rhtA23 and thrB (R235H) was realized in two steps. . Specifically: the first step is to repeat the steps described in Example 2 on the MG1655 bacteria to obtain a strain that overexpresses the rhtA gene; the second step is to use the same method on the basis of the transitional strain to realize the R235 position of the ...

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Abstract

In the L-homoserine production strain of the present invention, one or more genes related to the L-homoserine metabolic pathway are knocked out or weakened, and / or one or more genes related to the L-homoserine metabolic pathway are overexpressed Expression or enhancement, and / or mutation of one or more genes involved in the L-homoserine metabolic pathway. The invention also provides the construction method and application of the production strain. The L-homoserine producing bacterial strain of the present invention has many advantages such as high yield, low cost, mild conditions, and less environmental pollution, and has broad application prospects.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to an L-homoserine production strain and its construction method and application. Background technique [0002] L-homoserine is a naturally occurring non-proteinogenic amino acid that occurs in small amounts in many species as an intermediate common to the biosynthesis of threonine, methionine, and lysine. Since L-homoserine has an L-type-α amino acid basic skeleton, and its γ-hydroxyl has various chemical activities, L-homoserine and its derivatives have important applications in pharmaceuticals and physiology as pharmaceutical intermediates. prospect. [0003] At present, there are mainly the following methods for producing L-homoserine at home and abroad: [0004] (1) chemical method; the synthesis of L-homoserine mainly adopts relatively expensive L-methionine as a raw material, and uses methyl iodide or methyl bromide with serious biological toxicity as a methyl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12P13/06C12R1/19
CPCC12N9/1205C12P13/06C12Y207/01039C07K14/245C12N15/70C12R2001/19C12N1/205
Inventor 谢新开徐伟刘婷
Owner SICHUAN LIER BIOTECHNOLOGY CO LTD
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