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L-homoserine production bacterial strain and construction method and application thereof

A technology for producing homoserine and bacterial strains, which is applied in the field of genetic engineering, and can solve the problems of poor stability of strains, etc., and achieve the effects of stable fermentation results, high conversion rate of sugar and acid, and pollution avoidance

Active Publication Date: 2019-04-23
SICHUAN LIER BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current L-homoserine fermentation relies on the gene overexpression system using plasmids as vectors, and its disadvantage is the poor stability of the strain

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Construction of thrB gene knockout, rhtA gene overexpression, thrL gene knockout, and thrA gene enhancement strain MG1655 (ΔthrB, rhtA23, ΔthrL, thrA*(G433R)).

[0033] The L-homoserine production strain constructed in this example is the construction of the strain MG1655 (ΔthrB, rhtA23, ΔthrL, thrA*(G433R)) that knocks out the thrB gene, overexpresses the rhtA gene, knocks out the thrL gene, and mutates the thrA gene. The construction method can be found in Chinese patent application CN201710106474.8.

Embodiment 2

[0035] Construction of genetic engineering strain MG1655 (ΔthrB, rhtA23, ΔthrL, thrA*(G433R), ΔcadA::thrA*-ppc-aspA-pntAB) expressing thrA*, ppc, aspA, pntA and pntB in single copy on chromosomal DNA.

[0036] The L-homoserine production strain constructed in this example is a genetically engineered bacterium MG1655 (ΔthrB, rhtA23, ΔthrL, thrA*(G433R), ΔcadA::thrA) that simultaneously integrates and expresses thrA*, ppc, aspA, pntA and pntB on the chromosomal DNA *-ppc-aspA-pntAB), its construction method can refer to the literature: Microb Cell Fact.2016 Dec 1; 15(1):205, specifically includes the following steps:

[0037] (1) Using the bacterial strain chromosomal DNA obtained in Example 1 as a template, pSOE-thrA-f, pSOE-ppc-thrA-r; pSOE-thrA-ppc-f, pSOE-aspA-ppc-r; pSOE-ppc -aspA-f, pSOE-pntA-aspA-r; pSOE-aspA-pntA-f, pntB-r are primers, amplified by overlap extension PCR method (ie SOE-PCR method), and the template bacteria are located in the corresponding natural ThrA*,...

Embodiment 3

[0070] On the chromosomal DNA, two copies of the genetic engineering strain MG1655 (ΔthrB, rhtA23, ΔthrL, thrA* (G433R), ΔcadA::thrA*-ppc-aspA-pntAB, ΔyidJ: :thrA*-ppc-aspA-pntAB).

[0071] The L-homoserine production strain constructed in this example is the genetic engineering strain MG1655 (ΔthrB, rhtA23, ΔthrL, thrA*(G433R), ΔcadA ::thrA*-ppc-aspA-pntAB, ΔyidJ::thrA*-ppc-aspA-pntAB) construction, using CRISPR / Cas9 gene editing technology to realize the construction of strains, inserting the downstream of the yidJ gene as described in Example 2 The construction method of the SEQ ID No.15 fragment is basically similar to the method in Example 2, the only difference is that: the original bacterial strain of Example 2 is used as the operation object, and the integration position is downstream of the yidJ gene. Specifically: the primers used in the step (2) described in Example 2 were converted from the pSOE-cadA-H1-f, pSOE-cadA-H1-r, pSOE-cadA-H2-f, pSOE-cadA-H2 -r, pcadA-N2...

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Abstract

The invention provides an L-homoserine production bacterial strain. The L-homoserine production bacterial strain is characterized by containing a single and stable chromosome and not containing DNA carrier plasmids or other free DNA carriers, one or more genes related to L-threonine synthesis on the chromosome DNA of the bacterial strain are subjected to knockout or weakening, and / or the genes aresubjected to mutation for enhancement, and / or one or more genes related to the L-homoserine metabolic pathway are integrated and copied on the chromosome DNA of the bacterial strain.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a production strain of L-homoserine 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. Because L-homoserine has the basic skeleton of L-type-α amino acid, and its γ-hydroxyl has various chemical activities, therefore, L-homoserine and its derivatives have important roles in pharmacology and physiology as pharmaceutical intermediates. Application prospects. [0003] At present, there are mainly the following methods for producing L-homoserine at home and abroad: [0004] (1) chemical method; this method adopts relatively expensive L-methionine as a raw material, and uses methyl iodide or methyl bromide with serious biological toxicity as a methylati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P13/06C12R1/19
CPCC12N9/0006C12N9/1096C12N9/1205C12N9/88C12P13/06C12Y101/01003C12Y206/01021C12Y207/01039C12Y401/01031C07K14/245C12N1/20C12N15/09C12N15/52C12R2001/19C12N1/205
Inventor 谢新开徐伟
Owner SICHUAN LIER BIOTECHNOLOGY CO LTD
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