A mutant hypertonic inducible promoter pprop and its application

A promoter and polynucleotide technology, applied in the fields of genetic engineering and molecular biology, can solve the problems of bacterial strain toxicity and achieve high-efficiency production

Active Publication Date: 2021-10-08
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The operative connection of the promoter mutant polynucleotide with the protein-coding gene related to amino acid synthesis or the gene encoding the gene expression regulation protein can realize the high-efficiency expression of the protein-coding gene in the environment of high salt and high osmotic pressure, effectively solving the problem of The current inducible promoter needs to add a high inducer, and the inducer is toxic to the strain

Method used

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  • A mutant hypertonic inducible promoter pprop and its application
  • A mutant hypertonic inducible promoter pprop and its application
  • A mutant hypertonic inducible promoter pprop and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Example 1. Contains endogenous proP Plasmid construction of gene promoter sequence

[0114] ProP is a proline uptake protein, its expression is induced under hyperosmotic conditions, and the tolerance of the strain to the hyperosmotic environment is enhanced by increasing the uptake of compatible substance proline. It has been reported in the literature that the expression of ProP will be significantly up-regulated under hyperosmotic conditions, indicating that the promoter of this gene may also be a hyperosmotic-inducible promoter (Franzel, B., et al., Adaptation of Corynebacterium glutamicum to salt-stress conditions, Proteomics, 2010, 10(3): 445–457.). According to the Corynebacterium glutamicum published by NCBI ( Corynebacterium glutamicum ) genome sequence of ATCC 13032 (NC_003450.3), and designed primers proP-F (SEQ ID NO: 4) and proP-R (SEQ ID NO: 5). Using the ATCC 13032 genome as a template, amplified by PCR with proP The promoter of the gene (P pro...

Embodiment 2

[0115] Example 2. High salt pair proP Induction of gene promoters

[0116] The above-mentioned strains with different promoter recombinant vectors were inoculated into TSB medium containing 5 μg / mL chloramphenicol, and cultured overnight at 30°C and 220 r / min. Among them, the composition of TSB liquid medium is (g / L): glucose, 5 g / L; yeast powder, 5 g / L; soybean peptone, 9 g / L; urea, 3 g / L; succinic acid, 0.5 g / L L; K 2 HPO 4 •3H 2 O, 1 g / L; MgSO 4 •7H 2 O, 0.1g / L; Biotin, 0.01 mg / L; Vitamin B1, 0.1 mg / L; MOPS, 20 g / L. Transfer with or without 0.6 M Na according to initial OD 0.5 2 SO 4 CGXIIY medium, the culture system is 1 mL of 24-well plate, 30°C, 800 r / min for 18 h, and then detect the GFP fluorescence intensity and OD of different strains 600 , using the fluorescence intensity per unit cell (subtracting the fluorescence intensity per unit cell of the control strain under the same conditions) to characterize the relative intensities of different promoters under ...

Embodiment 3

[0117] Example 3. proP Promoter modification and characterization plasmid construction

[0118]In order to further improve the starting strength and retain its higher high-salt-induced activity, the present invention, under the condition of retaining the possible regulatory regions, respectively, the 5'-UTR and the core regions of the -35 and -10 regions of the promoter and the 5 '-UTR undergoes sequence transformation and replacement to obtain P proP-1 (SEQ ID NO: 2) and P proP-2 (SEQ ID NO: 3) Two mutant promoters.

[0119] to pXM-P proP - gfp As a template, use primers proP-1-F (SEQ ID NO: 8) and proP-1-R (SEQ ID NO: 9) to amplify by PCR to replace proP In the 5'-UTR region behind the promoter, after recovering the above PCR fragment, use T4 PNK to phosphorylate the vector fragment, and construct pXM-P by self-circularization proP-1 - gfp . At the same time, with pXM-P proP - gfp As a template, using primers proP-2-F (SEQ ID NO: 10) and proP-2-R (SEQ ID NO: 1...

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Abstract

The present invention provides a polynucleotide mutant with promoter activity, which has enhanced promoter activity compared with wild-type promoter under the environment of elevated salt concentration and osmotic pressure. The polynucleotide is operably linked to the target gene, which can significantly increase the expression intensity of the target gene under the stress environment of high salt and high osmotic pressure, and then produce downstream products stably and efficiently, effectively solving the problem of adding expensive inducers such as IPTG. , and cause toxicity to the strain.

Description

technical field [0001] The invention belongs to the field of genetic engineering and molecular biology, and specifically relates to a polynucleotide mutant with promoter activity, including a transcription expression cassette with a promoter, a recombinant expression vector, a recombinant host cell, and a method for regulating target gene transcription, Methods of producing target compounds. Background technique [0002] Promoters are an important tool for regulating the expression of target genes, and can be divided into two categories: constitutive promoters and inducible promoters. Constitutive promoters are generally not affected by external factors, and express the target gene at a substantially constant level. For example, a series of constitutive promoters have been reported in the current literature (Rytter, J. V., et al., Synthetic promoter libraries for Corynebacterium glutamicum . Appl. Microbiol. Biotechnol., 2014, 98, 2617-2623.). However, for the synthesis of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/77C12N1/21C12R1/15
CPCC12N15/113C12N15/77
Inventor 郑平陈久洲黄婧文孙际宾周文娟石拓马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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