Corynebacterium constitutive expression vector promoter, expression vector containing promoter and lrp gene

A constitutive expression, coryneform bacteria technology, applied in the biological field, can solve the problems of complex screening methods, easy loss of expression plasmids, affecting host growth and metabolism, etc. High stability effect

Active Publication Date: 2017-09-15
WUHAN GRAND HOYO
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The technical problem to be solved by the present invention is: the current constitutive promoter screening method is complex, and the expr...

Method used

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  • Corynebacterium constitutive expression vector promoter, expression vector containing promoter and lrp gene
  • Corynebacterium constitutive expression vector promoter, expression vector containing promoter and lrp gene
  • Corynebacterium constitutive expression vector promoter, expression vector containing promoter and lrp gene

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Experimental program
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Effect test

Embodiment 1

[0018] 1. Sample preparation for transcriptome sequencing and analysis of transcription information:

[0019] Bacterial culture: pick a ring of isoleucine-producing Corynebacterium glutamicum (H5 strain, obtained from the depository center, and the deposit number is CCTCC NO: M2016609) without a plasmid, and culture it overnight at 31°C in LB medium, Take out the bacterial solution and centrifuge it, resuspend it with an equal volume of sterile water, put it into the LBG freshly sterilized medium with 5% inoculation amount, cultivate it at 31°C and 200rpm until the middle logarithmic phase and the early stage of the stable phase, take it out and ferment liquid, quickly with an equal volume Bacteria Reagent (QIAGEN) was mixed, centrifuged at 5000rpm for 10min, and 100mg of wet bacteria was weighed, and quickly put into liquid nitrogen for preservation.

[0020] RNA extraction: Put the sample stored in liquid nitrogen into a mortar pre-cooled with liquid nitrogen, add an appro...

Embodiment 2

[0108] Example 2 The expression vector constructed by using the promoter fragment RS07910seq2 and its application

[0109] In the process of cell metabolism, not all genes over-expressed can increase the flow of metabolism to target products. There are many biological enzymes produced after gene expression, which can play a role when a moderate expression balance point needs to be reached in metabolism. . Studies have shown that the extensive regulatory protein Lrp (its coding nucleotide sequence is shown in SEQ ID NO: 3) in Corynebacterium glutamicum can improve the synthesis and transport of related genes of L-isoleucine in Corynebacterium glutamicum. The transcription level can also increase the transcription level of the self-encoding gene lrp, but the overexpression of Lrp can inhibit the growth of Corynebacterium glutamicum. We use the promoter fragment PRS07910seq2 as the promoter and lrp as the target gene to construct exogenous expression The plasmid HY-P19-RS07910se...

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Abstract

The invention discloses a method for constructing a corynebacterium constitutive expression vector promoter based on transcriptome sequence. The invention further discloses the corynebacterium constitutive expression vector promoter constructed based on the transcriptome sequence, an expression vector containing the promoter and a recombination strain obtained by converting host cell corynebacterium glutamicum by the expression vector. According to the expression vector constructed by the promoter obtained by the method, the target gene protein expression quantity of the logarithmic phase is low, but the target gene protein expression quantity of the stable phase is greatly improved relative to the logarithmic phase, and the effect to the host bacteria growth is low, and the stability of the plasmid passage is high. The expression vector is suitable for constructing the vector of the target gene of which the logarithmic phase does not need the expression, but the stable phase needs the high expression, and is particularly suitable for constructing engineered strains for amino acid fermentation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a coryneform bacillus constitutive expression vector promoter and a construction method thereof, and also relates to an expression vector containing the promoter and a recombinant bacterial strain. Background technique [0002] Corynebacterium is a class of Gram-positive bacteria. The three main representatives of Corynebacterium: Corynebacterium glutamicum, Brevibacterium flavum and Brevibacterium lactose fermentum have been widely used to produce various chemical substances such as amino acids and nucleotides. (Liebl et al., 1991). The coryneform bacterium mutants obtained through physical or chemical mutagenesis have a strong ability to synthesize useful substances, and through genetic engineering and metabolic engineering to transform the coryneform bacterium wild strains or mutants, can obtain higher production intensity strains. Metabolic engineering is to find key...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/77C12N1/21C12P13/06C12R1/15
Inventor 邢盼盼苏海霞王炯梅雪臣万坤宋盟军李敬刘爱福
Owner WUHAN GRAND HOYO
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