Recombinant strain, as well as preparation method and application thereof

A technology for recombining strains and genes, applied in the field of genetic engineering, can solve the problems that the metabolic operation and process control of the strain cannot be optimized, the strain cannot reach the highest conversion rate, etc., and achieve the effect of wide industrial application prospects and high conversion rate of sugar and acid.

Active Publication Date: 2016-07-06
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Further strain metabolic operations and process control cannot be optimized to the most perfect state, and the strain can never reach its highest conversion rate

Method used

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  • Recombinant strain, as well as preparation method and application thereof
  • Recombinant strain, as well as preparation method and application thereof
  • Recombinant strain, as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1: Recombinant plasmid pK18mobsacB-lysC T311I Construction of and introduction of point mutations in ATCC13032

[0079] Using the ATCC13032 genome as a template, PCR amplification was performed with lysC-1f / lysC-1r primers to obtain the upstream fragment lysC-up; using the ATCC13032 genome as a template, PCR amplification was performed with lysC-2f / lysC-2r primers to obtain Downstream fragment lysC-dn. Using the mixture of lysC-up and lysC-dn fragments as a template, PCR amplification was performed with lysC-1f / lysC-2r primer pair to obtain mutated lysC T311I fragment. lysC T311I The fragment was double-digested with BamHI and NheI, and pK18mobsacB was double-digested with the same enzymes. The two digestion products were ligated with T4DNALigase, transformed into Trans1T1 competent cells, and the recombinant plasmid pK18mobsacB-lysC was obtained T311I .

[0080] ATCC13032 competent cells were prepared according to the classical method of glutamicum (C. gl...

Embodiment 2

[0081] Example 2: Recombinant plasmid pK18mobsacB-hom V59A Construction of and introduction of point mutations in MHZ-0912-1

[0082] Using the ATCC13032 genome as a template, PCR amplification was performed with the primer pair hom-1f / hom-1r to obtain the upstream fragment hom-up; using the ATCC13032 genome as a template, PCR amplification was performed with the primer pair hom-2f / hom-2r to obtain Downstream fragment hom-dn. Using the mixture of hom-up and hom-dn fragments as a template, PCR amplification was performed with the primer pair of hom-1f / hom-2r to obtain the mutant hom V59A fragment. hom V59A The fragment was double-digested with BamHI and NheI, and pK18mobsacB was double-digested with the same enzymes. The two digested products were ligated with T4DNALigase, transformed into Trans1T1 competent cells, and the recombinant plasmid pK18mobsacB-hom was obtained V59A .

[0083] MHZ-0912-1 competent cells were prepared according to the classical method of Guban. ...

Embodiment 3

[0084] Example 3: Construction of recombinant plasmid pK18mobsacB-dapBdapAddh and introduction of point mutations in MHZ-0912-2

[0085]Using the ATCC13032 genome as a template, the dapB-1f / dapA-1r primer pair was used for PCR amplification to obtain the full-length fragment of dapBdapA-up. Using the ATCC13032 genome as a template, the dapB-2f / dapA-2r primer pair was used for PCR amplification to obtain the full-length fragment of dapBdapA-dn. Using the ATCC13032 genome as a template, the ddh-f / ddh-r primer pair was used for PCR amplification to obtain the full-length ddh fragment. The three fragments of dapBdapA-up, dapBdapA-dn and ddh were used as templates, and the primer pair of dapB-1f / dapA-2r was used for PCR amplification, and large fragments were recovered. This fragment was double-digested with SbfI and NheI, and pK18mobsacB was double-digested with the same enzymes. The two digested products were ligated with T4DNALigase, transformed into Trans1T1 competent cells, ...

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Abstract

The invention relates to the technical field of genetic engineering, in particular to a recombinant strain, as well as a preparation method and application thereof. By constructed L-lysine genetically engineered bacteria, effective accumulation of L-lysine in a fermentation process can be implemented, and a higher glucose acid conversion rate is achieved. A genetically engineered strain constructed from the beginning is favorable for achieving high acid yield and high conversion rate, and has broad industrial application prospect, and side effects caused by random mutation are avoided.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant bacterial strain and its preparation method and application. Background technique [0002] Corynebacterium glutamicum (Corynebacterium glutamicum) is a Gram-positive microorganism with the characteristics of fast growth, non-pathogenicity, and weak ability to degrade its own metabolites. As a traditional industrial microorganism, Corynebacterium glutamicum is widely used to produce L-amino acids, nucleotides and other organic acids. [0003] L-Lysine is the second largest amino acid production variety in the world and is widely used in animal feed, medicine and food industry. About 90% of them are used in the feed industry, and 10% are used in the food and pharmaceutical industries. Used as an animal feed additive, L-lysine can help the body absorb other amino acids, thereby improving feed quality. [0004] Corynebacterium glutamicum synthesizes L-lys...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P13/08C12R1/15
CPCC12N9/0006C12N9/0008C12N9/001C12N9/0016C12N9/1217C12N9/93C12P13/08C12Y101/01003C12Y101/01049C12Y102/01051C12Y103/01026C12Y104/01016C12Y207/02004C12Y604/01001
Inventor 胡丹赵丽彬方真刁刘洋毛贤军
Owner MEIHUA BIOTECH LANGFANG CO LTD
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