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Mutant of pyruvate carboxylase gene promoter and application thereof

A pyruvate carboxylase and promoter technology, applied in the biological field, can solve problems such as genome instability, and achieve the effects of high application value, high promoter activity and improving amino acid production efficiency

Active Publication Date: 2021-12-07
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

At present, the overexpression of the pyc gene is mostly achieved by increasing its copy number, but the increase in the copy number may lead to the instability of the genome of the strain, and the promoter does not have the above defects in the expression regulation of the gene, and the high-performance promoter can also Can be used to express different target genes

Method used

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  • Mutant of pyruvate carboxylase gene promoter and application thereof
  • Mutant of pyruvate carboxylase gene promoter and application thereof
  • Mutant of pyruvate carboxylase gene promoter and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0052] Embodiment 1. Construction of Corynebacterium glutamicum pyc gene promoter strength characterization plasmid

[0053] In order to characterize the strength of the pyc gene promoter of Corynebacterium glutamicum, the present invention first constructs a characterization vector, and on the basis of the pEC-XK99E plasmid backbone, expresses the N-terminal 60 amino acids of the pyc gene, a connecting peptide and red fluorescent protein gene. According to the published genome sequence and pyc gene annotation information of Corynebacterium glutamicum ATCC13032, the primer pyc-F / R was designed, and the pyc gene promoter and N-terminal 180bp DNA fragment were obtained by PCR amplification using the ATCC13032 genome as a template. pEC-XK99E-rfp reported in literature (Wang Yingchun et al. Screening endogenous high-efficiency constitutive promoters of Corynebacterium glutamicum based on time-series transcriptome [J]. Acta Biological Engineering, 2018, 34(11): 1760-1771) The plas...

Embodiment 2

[0056] Embodiment 2. Corynebacterium glutamicum pyc gene promoter mutant screening and intensity characterization

[0057] (1) Construction of Corynebacterium glutamicum pyc gene promoter mutant library

[0058] The core region of the present invention to Corynebacterium glutamicum pyc gene promoter:

[0059] "CGATGT TTGATT GGGGGAATCGGGGGT TACGAT ACTAGG" is mutated, and the underlines are the main sequences of the -35 region and -10 region of the promoter respectively. The present invention mutates "NNNNNN at the corresponding position of the above core region TTGA TT NNNNNNNNNNNNNNNN TANNAT NNNNNN", use pyc-M1 / M2 and pyc-M3 / M4 primers to amplify the two fragments of the plasmid respectively, and use Novizym's one-step recombination kit to clone and connect, collect all the cloned bacteria obtained and extract the plasmids to obtain pyc gene promoter mutant library. The above library and the wild-type control pEC-XK99E-Ppyc-rfp transformation obtained in Example 1 we...

Embodiment 3

[0067] Embodiment 3. Corynebacterium glutamicum pyc gene promoter mutant is applied to target product production

[0068] (1) Recombinant vector construction of Corynebacterium glutamicum pyc gene promoter mutant

[0069] According to the reported genome sequence of Corynebacterium glutamicum ATCC13032, using the ATCC13032 genome as a template, pyc-UF / pyc-UR1 and pyc-DF1 / pyc-DR, pyc-UF / pyc-UR9 and pyc-DF9 / pyc -DR, pyc-UF / pyc-UR16 and pyc-DF16 / pyc-DR, pyc-UF / pyc-UR20 and pyc-DF20 / pyc-DR as primers, PCR amplification pyc -1, P pyc -9, P pyc -16 and P pyc The upstream and downstream homology arms of the -20 promoter mutation; at the same time, the backbone of pK18mobsacB was amplified with pK18-1 / 2 primers. After the above PCR fragments were recovered, they were cloned and ligated by Novizym’s one-step recombination kit to obtain the recombinant vector pK18-P with promoter mutation pyc -1, pK18-P pyc -9, pK18-P pyc -16 and pK18-P pyc -20. The sequences of the primers use...

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Abstract

The invention discloses a mutant of a corynebacterium glutamicum pyruvate carboxylase gene promoter and application of the mutant. The mutant has improved promoter activity in comparison with that of a wild type promoter. Therefore, the mutant can be used for enhancing the expression of a target gene, for example, the mutant is operably connected with a pyruvate carboxylase gene, so that the expression intensity of pyruvate carboxylase is enhanced, and the amino acid production efficiency of the strain is improved.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a mutant of a pyruvate carboxylase gene promoter and its application in biological amino acids. Background technique: [0002] Amino acids, including lysine, threonine, glutamic acid, etc., are the basic substances that constitute protein required for animal nutrition, and are widely used in industries such as medicine, health, food, animal feed, and cosmetics, and are mainly produced by microbial fermentation. Production, at present, the main production strains include microorganisms of the genus Enterobacter, Corynebacterium, etc., and due to the physiological superiority of Corynebacterium, Corynebacterium has become the most important production strain in the industry. With the continuous development of biotechnology, reports on metabolic engineering of Corynebacterium to increase its amino acid production have gradually increased. These modifications include enhanci...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/67C12N15/77C12N1/21C12P7/44C12P13/00C12P13/06C12P13/08C12P13/10C12P13/12C12P13/14C12P13/24C12R1/15
CPCC12N9/93C12Y604/01001C12N15/67C12N15/77C12P13/08C12P13/12C12P13/06C12P13/14C12P13/24C12P13/10C12P13/005C12P13/001C12P7/44C12N15/63C12R2001/15
Inventor 孙际宾刘娇郑平石拓周文娟陈久洲郭轩马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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