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Optimized mutant of maltose promoter and application of mutant

A technology of promoter and maltose, applied in the biological field, can solve the problems of high price of xylose, increased production cost, leaky expression of promoter and decrease of inducible expression intensity, etc.

Active Publication Date: 2019-12-20
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The commonly used promoter systems in Bacillus subtilis are Pspac, Pxyl and PsacB systems. These three promoter systems are widely used in the research work of Bacillus subtilis, but they also have their own shortcomings: the inducer IPTG of Pspac promoter not only costs High and toxic; the price of xylose, the inducer of Pxyl promoter, is high, which is easy to increase the production cost; the expression level of PsacB promoter is relatively low
However, the transformation of the above strategies often results in a simultaneous decrease in the leaky expression of the promoter and the decrease in the intensity of the induced expression.

Method used

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  • Optimized mutant of maltose promoter and application of mutant
  • Optimized mutant of maltose promoter and application of mutant
  • Optimized mutant of maltose promoter and application of mutant

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preparation example Construction

[0023] 3. Preparation and transformation of DNA:

[0024] DNA was isolated from E. coli and B. subtilis or from agarose gels using DNA preparation kits from Tiangen or Omega according to the manufacturer's instructions. Standard molecular techniques were used in all examples. E. coli was transformed using plasmid DNA as described by Chung C.T. et al., Proc. Natl. Acad. Sci. USA 86, 1989, 21722175. According to a modified "Paris method" (Harwood C.R. Molecular Biological Methods for Bacillus, 1990, John Wiley & Sons Ltd., England), plasmid DNA or DNA fragments were used to transform Bacillus subtilis.

[0025] 4. Determination of green fluorescent protein fluorescence intensity RFU:

[0026] Centrifuge at 4°C and 4000rpm for 10 minutes to collect the bacterial cells cultured to an OD600 of about 0.6-0.8, wash the bacterial cells twice with pre-cooled PBS solution, and transfer 150 μL to a 96-well black-bottomed microtiter plate (Corning , USA), placed in a microplate Spectra...

Embodiment 1

[0035] Example 1 Mutant Construction of Carbon Metabolism Responsive Elements in Maltose Promoter

[0036] Due to the existence of the CCR effect in the maltose metabolic pathway of Bacillus subtilis, the application of the expression system based on the maltose promoter PmalA in the fermentation containing glucose carbon source is restricted. In order to relieve the CCR effect, the carbon metabolism response element cre (cataboliteresponse element, cre) in the maltose promoter PmalA responsible for interacting with the carbon metabolism repressor protein ccpA can be mutated to partially relieve the CCR effect. The construction method is as follows. The GFP fluorescent protein gene was inserted into the pMATE vector through the NdeI / BamHI restriction site to construct the pMATE-GFP vector. Furthermore, using the pMATE-GFP vector as a template, cre-del1.for / cre-del1.rev and cre-del2.for / cre-del2.rev as primers, the 13bp cre element sequence in the Bacillus subtilis maltose prom...

Embodiment 2

[0038] Example 2 Construction of Bacillus subtilis ptsG knockout strain

[0039] In addition, by knocking out the glucose transporter gene ptsG in Bacillus subtilis, the intracellular transport of glucose can be weakened to relieve the effect of CCR. The ptsG gene in the Bacillus subtilis 1A751 expression host strain was knocked out by using the AraR-based Bacillus subtilis traceless knockout technology, and the 1A751△ptsG genetically engineered strain was constructed. The wild-type maltose promoter expression vector pMATE-GFP and the carbon metabolism response element deletion mutants pMATE-cre-del1-GFP, pMATE-cre-del2-GFP and pMATE-cre-comp-GFP constructed above were transformed into 1A751△ptsG In the genetically engineered strains, induce culture at 37°C, 220rpm, 1% maltose and add glucose with gradient concentration (0.25-2%), then use a microplate reader to measure the fluorescence intensity of GFP, and evaluate the ability of knocking out the glucose transporter gene pts...

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Abstract

The invention uses Bacillus as a starting strain, uses a carbon metabolism response element cre in a maltose promoter to transform a target, a lysine ribose switch is fused at the same time, a mutantof the maltose promoter is constructed, the mutant that can reduce the leaky expression of the maltose promoter and improve the induced expression strength of the maltose promoter is obtained, and anexpression vector and host cell which contain the mutant of the promoter are constructed, so as to be conducive to the application of the maltose promoter in protein expression.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a maltose promoter mutant, and an expression vector and a host bacterial strain containing the maltose promoter mutant. Background technique [0002] The Bacillus subtilis promoter is one of the key elements to achieve high gene expression. In recent years, a lot of work has been carried out in the research of promoters and considerable progress has been made. A number of promoters that can be applied to Bacillus subtilis have been cloned. However, the existing promoters of Bacillus subtilis have many problems in terms of quantity, expression level and regulation mode. Further research and improvement are needed to obtain more promoter elements with high expression intensity and convenient induction and regulation. The commonly used promoter systems in Bacillus subtilis are Pspac, Pxyl and PsacB systems. These three promoter systems are widely used in the research work ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/75C12N1/21C12R1/125
CPCC07K14/32C12N15/75C12N1/205C12R2001/125
Inventor 张大伟付刚
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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