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Method for improving nisin induction efficiency in lactic acid bacteria NICE expression system

An expression system, lactic acid bacteria technology, applied in the field of improving nisin induction efficiency in lactic acid bacteria NICE expression system, can solve the problems of harming host bacteria, host cell damage, increasing production costs, etc.

Active Publication Date: 2016-09-21
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since nisin has a certain inhibitory effect on Gram-positive bacteria, high-concentration use may harm the host bacteria and lead to a decrease in protein expression. Usually, a sublethal dose is used to induce the expression of the target protein in the NICE system
In industrial production, the need to maximize the expression of the target protein is often achieved by increasing the inductive concentration of nisin, but this not only easily damages the host cells, but also increases the production cost

Method used

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  • Method for improving nisin induction efficiency in lactic acid bacteria NICE expression system

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Embodiment 1

[0053] Embodiment 1, the acquisition of NisK mutant

[0054] NisK is the receptor protein of nisin, which contains two transmembrane domains and is a membrane protein ( figure 2 ), and its extracellular region is the key region for sensing signals. The coding gene sequence of NisK protein is sequence 1, and the amino acid sequence of NisK protein is sequence 2. NisK is mutated to replace a single amino acid in the extracellular region of NisK with alanine. According to the specific mutation position, the following three types are respectively obtained. mutant:

[0055] 1. NisK mutant E46A: NisK mutant E46A is a protein obtained by mutating the 46th glutamic acid shown in sequence 2 to alanine; the coding gene sequence of NisK mutant E46A is sequence 3.

[0056] 2. NisK mutant E108A: NisK mutant E108A is a protein obtained by mutating the 108th glutamic acid shown in sequence 2 to alanine; the coding gene sequence of NisK mutant E108A is sequence 4.

[0057] 3. NisK mutant ...

Embodiment 2

[0058] Embodiment 2, the application of NisK mutant in improving the nisin induction efficiency in the lactic acid bacteria NICE expression system

[0059] 1. Acquisition of plasmids

[0060] 1. Acquisition of plasmid pMG36e-RKNlacZ

[0061] The plasmid pMG36e-RKNlacZ was constructed using the pMG36e vector (containing the P32 promoter). Plasmid pMG36e-RKNlacZ is a vector obtained by inserting the DNA fragment shown in sequence 6 between the restriction sites XbaI and HindIII of the pMG36e vector, and keeping other sequences of the pMG36e vector unchanged. Wherein, the 8th-695th nucleotide in the DNA fragment shown in sequence 6 is the coding gene of nisR, the 687th-2030th nucleotide is the coding gene of NisK, and the 5094-5330th is the nisA promoter (starting Sub P nisA ), the 2037-5093 nucleotide is the reporter gene lacZ. In the plasmid pMG36e-RKNlacZ, the P32 promoter was used to control the constitutive expression of nisRK, and the nisA promoter P nisA Controls indu...

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Abstract

The invention discloses a method for improving a nisin induction efficiency in a lactic acid bacteria NICE expression system. According to the method disclosed by the invention, the sensing activity of nisin is enhanced by conducting mutation transformation on receptor histidine kinase NisK of nisin molecules of an inducer. Experiments prove that through nisin induction of a same concentration, the expression amount of reporter protein is obviously increased and the nisin induction efficiency is remarkably enhanced after the NisK mutation transformation; therefore, the method disclosed by the invention has a quite important significance for improving the expression amount of heterologous protein, declining the concentration of the inducer and reducing the complexity of constructing the system by virtue of the NICE system.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for improving nisin induction efficiency in a lactic acid bacteria NICE expression system. Background technique [0002] Nisin is a lantibiotic produced by Lactococcus lactis, which is a biologically active antimicrobial peptide synthesized by ribosomes and post-translationally modified. Due to its excellent antibacterial activity, it has been widely used as a natural and safe food preservative in more than 80 countries and regions in the world for more than 40 years. The genes related to the biosynthesis of Nisin are arranged in clusters, forming gene clusters. The propeptide protein of Nisin is first coded and synthesized by nisA, modified by NisBC, transported by NisT outside the cell, and finally the leading peptide is excised by NisP to form mature and active nisin. NisFEGI is responsible for the immunity to nisin molecules, so that the own cells are not d...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12P21/02C12N1/21C12N9/38
CPCC12N9/12C12N9/2471C12Y207/13003C12Y302/01023
Inventor 钟瑾葛晓璇滕坤玲
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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