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Recombinant dna, bacterial strain and application thereof for fermentative production of l-lysine

A technology of lysine and its production method, which is applied in the field of microbial engineering and can solve problems such as L-lysine producing bacteria that have not yet been seen

Active Publication Date: 2021-05-04
CATHAY R&D CENT CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no relevant report on obtaining L-lysine producing bacteria by overexpressing the 8-amino-7-oxononanoate synthase gene

Method used

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  • Recombinant dna, bacterial strain and application thereof for fermentative production of l-lysine
  • Recombinant dna, bacterial strain and application thereof for fermentative production of l-lysine
  • Recombinant dna, bacterial strain and application thereof for fermentative production of l-lysine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The mutant strain that the L-lysine production level that embodiment 1 mutagenesis obtains improves

[0052] Starting from Escherichia coli MG1655 (E.coli MG1655K12, purchased from Beijing Tianenze Gene Technology Co., Ltd.), screening was carried out by means of ARTP physical mutagenesis and adding L-lysine analogue plates.

[0053] Inoculate the MG1655 strain into 50mL of LB medium, and wait until the cell concentration reaches OD 600 When =0.6, the bacteria were collected. Transfer the thalli to the ARTP instrument (Wuxi Yuanqing Tianmu Biotechnology Co., Ltd.), select 70 seconds to carry out mutagenesis treatment (lethal rate reaches 98%), collect the thalline after mutagenesis, and dilute 10 2 and 10 3 After doubling, the M9 medium (in which 4 mg / L of L-lysine analogue AEC was added to the medium) was completely plated. Spread non-mutated bacteria (dilution 10 5 times the bacteria solution), and the bacteria after mutagenesis treatment (diluted 10 3 times the ...

Embodiment 2

[0060] Example 2 Construction of expression vectors pUC18-plac-ppc, pUC18-plac-ddh, pUC18-plac-ppc-plac-bioF and pUC18-plac-ddh-plac-bioF

[0061] First, the recombinant expression plasmids pUC18-plac-ppc and pUC18-plac-ddh were constructed. Primers were designed according to the product manual of the multi-fragment one-step cloning kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.), and a DNA sequence of 20 to 30 bp overlapped with the vector SacI / XbaI double digestion was introduced at both ends of the ppc gene sequence ( Seq ID No: 3) and (Seq ID No: 4), the primer pair ppc-F / R (SEQ ID NO: 5 and 6) was obtained; the two ends of the ddh gene sequence were introduced and overlapped with the carrier SacI / XbaI after double digestion The 20-30 bp DNA sequences (Seq ID No: 7) and (Seq ID No: 8) obtained the primer pair ddh-F / R (SEQ ID NO: 9 and 10). Using Escherichia coli MG1655 genomic DNA as a template, using the primer pair ppc-F / R (SEQ ID NO: 5 and 6), the ppc gene...

Embodiment 3

[0063] Embodiment 3 prepares L-lysine production bacterial strain and produces L-lysine

[0064]First, an L-lysine producing strain was prepared. The M11-A3 mutant strain obtained in Example 1 was used to screen and prepare competent, and the expression vectors pUC18-plac-ppc, pUC18-plac-ddh, pUC18-plac-ppc-plac-bioF and pUC18- plac-ddh-plac-bioF were transferred into the mutant strain of recipient strain M11-A3 respectively. Selection was spread on LB resistance plates containing 100 [mu]g / ml ampicillin. Pick 12 single colonies into 600 μl LB medium supplemented with ampicillin, culture at 37°C for 3 hours, take 1 μl of the bacteria as a template for PCR verification to screen the correct mutant strains, and keep them in glycerol.

[0065] Then, select 3 pUC18-plac-ppc transformants, 3 pUC18-plac-ddh transformants, 3 pUC18-plac-ppc-plac-bioF transformants and 3 pUC18-plac-ddh transformants obtained in Example 1 -plac-bioF transformants and originating strain M11-A3 were sp...

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Abstract

The invention discloses a recombinant DNA, bacterial strain and application for fermenting and producing L-lysine. The present invention increases the content of 8-amino-7-oxononanoic acid synthase in microorganisms to accelerate the first step of catalyzing the synthesis of biotin, thereby increasing the synthesis rate of biotin and further increasing the yield of metabolites. Further, in the mutant strains obtained by mutagenesis to increase the production level of L-lysine, a strong promoter or a high-copy plasmid is used to express the ppc or ddh gene and the 8-amino-7-oxononanoic acid synthase encoding The gene bioF accelerates the first step of catalyzing the synthesis of biotin, thereby increasing the synthesis rate of biotin, thereby increasing the production of L-lysine.

Description

technical field [0001] The invention belongs to the technical field of microbial engineering, and in particular relates to a recombinant DNA for fermenting and producing L-lysine, a bacterial strain and an application thereof. Background technique [0002] The bacteria of the genus Corynebacterium and Escherichia that have the ability to produce L-lysine are transformed by DNA recombination technology, and industrial production has now been realized. The improvement of L-lysine productivity in these bacteria was achieved by overexpression of genes related to L-lysine synthesis pathway and feedback inhibition desensitization, or enhancement of energy supply pathway starting from glucose metabolism. In Escherichia coli, phosphoenolpyruvatecarboxylase (PPC) encoded by the ppc gene is an enzyme that catalyzes the reaction of phosphoenolpyruvate and carbon dioxide to generate oxaloacetate in an irreversible reaction. [0003] In addition to continuously strengthening the metabol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N15/60C12N15/53C12N1/21C12P13/00C12P13/08C12R1/19C12R1/01C12R1/125C12R1/15
CPCC12N9/0016C12N9/1029C12N9/88C12P13/001C12P13/08C12Y104/01016C12Y203/01047C12Y401/01031
Inventor 雷云凤周豪宏陈玲刘修才
Owner CATHAY R&D CENT CO LTD