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Recombinant strain and preparation method and application thereof

A technology for recombinant strains and strains, applied in the field of microorganisms, can solve the problems of multiple by-products, easy loss of plasmids, and inability to stably express strains.

Inactive Publication Date: 2016-05-04
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to random mutations in traditional mutation breeding, strains grow slowly and produce more by-products, making it difficult to obtain high-yielding strains
Most of the existing strains transformed by genetic engineering carry engineering plasmids to overexpress the key genes in the amino acid biosynthesis pathway, but because the plasmids are easily lost during the passage process, they cannot be stably expressed and cause a growth burden on the strains

Method used

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  • Recombinant strain and preparation method and application thereof
  • Recombinant strain and preparation method and application thereof
  • Recombinant strain and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Preparation of strain W3110 (thrA*(S345P)) with mutant thrA*(S345P) gene

[0047] (1) Construction of pTargetT-thrA*(S345P) plasmid

[0048]The primer pair gRNAthrAup-f1 / gRNAthrAdn-r1 uses the pTargetT plasmid (from the literature MultigeneEditing in the Escherichia coli Genomeviathe CRISPR-Cas9System, JiangY, ChenB, et al.Appl.EnvironMicrobiol, 2015) as a template to amplify the sgRNA fragment of thrA; the primer pair thrAup-f1 / thrAup- r1 used the W3110 genome as a template to amplify the left fragment of thrA, and the primer pair thrAdn-f1 / thrAdn-r1 used the W3110 genome as a template to amplify the right fragment of thrA; the three fragments were amplified by OE-PCR to obtain the gRNA-thrA fragment ( The full length is 0.7kb), digested with speI / PstI, and inserted into the same restriction site of plasmid pTargetT to obtain pTargetT-thrA*(S345P) plasmid.

[0049] (2) Competent cell preparation and electroporation of pTargetT-thrA*(S345P) plasmid

[0050]...

Embodiment 2

[0055] Example 2: Preparation of the strain W3110 (thrA*(S345P), Δtdh) for knocking out the tdh gene

[0056] (1) Construction of pTargetT-tdh plasmid

[0057] The primer pair gRNAtdhup-f1 / gRNAtdhdn-r1 used the pTargetT plasmid as a template to amplify the sgRNA fragment of tdh; the primer pair tdhup-f4 / tdhup-r4 used the W3110 genome as a template to amplify the left half of tdh knockout; the primer pair tdhdn- f4 / tdhdn-r4 uses the W3110 genome as a template to amplify the right half of the tdh knockout; OE-PCR amplifies the three fragments to obtain a gRNA-tdh knockout fragment (full length 0.9kb), which is digested with speI / PstI , inserted into the same restriction site of plasmid pTargetT to obtain pTargetT-tdh plasmid.

[0058] (2) Competent cell preparation and electroporation of pTargetT-tdh plasmid

[0059] Transform the pCas plasmid into W3110(thrA*(S345P)) competent cells (refer to "Molecular Cloning III" for the transformation method and competent preparation meth...

Embodiment 3

[0063] Example 3: Preparation of bacterial strain W3110 (thrA*(S345P), which strengthens the thrA*BC gene,

[0064] Δtdh::thrA*BC)

[0065] (1) Construction of pTargetT-thrA*BC plasmid

[0066] The primer pair gRNAtdhup-f1 / gRNAtdhdn-r1 used the pTargetT plasmid as a template to amplify the sgRNA fragment of tdh; the primer pair tdhup-f4 / tdhup-r1 used the W3110 genome as a template to amplify the left homology arm of tdh; the primer pair tdhdn-f1 / tdhdn-r4 uses W3110 genome as template to amplify tdh right homology arm; The thrA*BC fragment was added, and the gRNA-thrA*BC-tdh fragment (full length 5.6kb) was amplified by OE-PCR, which was double-digested with speI / PstI and inserted into the same restriction site of the plasmid pTargetT to obtain pTargetT-thrA*BC plasmid.

[0067] (2) Competent cell preparation and electroporation of pTargetT-thrA*BC plasmid

[0068] Transform the pCas plasmid into W3110 (thrA*(S345P), Δtdh) competent cells (refer to "Molecular Cloning III" ...

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Abstract

The invention relates to the field of microorganisms, in particular to a recombinant strain and a preparation method and an application thereof. According to the recombinant strain and the preparation method and the application thereof, wild W3110 is taken as an original strain, a genome of the strain is subjected to relevant modification, and engineering plasmid loads are avoided; key genes in a threonine metabolic pathway are subjected to corresponding overexpression or inactivation, for example, strengthening of thrA*BC and knock-out of tdh, wherein the overexpression of thrA*BC enhances biosynthesis of L-threonine, and knock-out of tdh stops formation of glycine, an L-threonine degradation product. Experimental results show that a W3110 strain does not produce threonine in fermentation, and an MHZ-0213-3 strain produces threonine with the concentration of 4.2g / L in a conversion ratio of about 8.9%. Novel escherichia coli is free of plasmid loads, the creating method is easy and convenient, the yield of L-threonine is higher than that of the contrast strain, and the escherichia coli can be applied to fermentation and subsequent research of L-threonine.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to recombinant strains and their preparation methods and applications. Background technique [0002] L-threonine is one of the 8 kinds of amino acids necessary for the growth of humans and animals. It is widely used in feed, food additives and preparation of pharmaceutical auxiliary materials. At present, L-threonine is mainly produced by microbial fermentation, and various bacteria can be used for L-threonine production, such as wild-type induced mutants of Escherichia coli, Corynebacterium, Serratia, etc. as production strains. Specific examples include mutants resistant to amino acid analogues or various auxotrophs such as methionine, lysine, and isoleucine (Japanese Patent Application Publication No. 224684 / 83; Korean Patent Application Publication No. 8022 / 87) [0003] With the continuous increase of the global threonine demand, the construction and transformation of high-threoni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P13/08C12R1/19
CPCC12N9/1217C12N9/0006C12N9/1205C12P13/08C12Y101/01C12Y101/01003C12Y207/01039C12Y207/02004
Inventor 程江红毛贤军刁刘洋
Owner MEIHUA BIOTECH LANGFANG CO LTD
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