Corynebacteria inducible promoter, expression vector comprising same, and application thereof

A coryneform, inducible technology, applied in vectors, nucleic acid vectors, viruses/phages, etc., can solve the problems of low yield, increased steps, by-products, cost, and high cost, and achieves the effect of solving low efficiency.

Active Publication Date: 2018-09-11
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reaction process of the chemical synthesis method is relatively long, and some toxic and expensive raw materials must be inevitably used in the reaction process, and at the same time, it will inevitably cause environmental pollution
In addition, the structure of 5-aminolevulinic acid contains a

Method used

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  • Corynebacteria inducible promoter, expression vector comprising same, and application thereof
  • Corynebacteria inducible promoter, expression vector comprising same, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Inositol-inducible promoter P iol build

[0035] Using PCR technology to use Corynebacterium glutamicum 13032 genomic DNA as a template,

[0036] Utilize primers iolR-F and iolR-R and iolT-F and iolT-R to amplify iolR gene and iolT upstream 500bp sequence P iolT500 , PCR conditions were 94°C for 5min for one cycle, 94°C for 30s, 55°C for 30s, 72°C for 40s for 30 cycles, and 72°C for 10min for one cycle, and the reaction system was 50 μL. The PCR product was purified and recovered by 1% agarose gel electrophoresis.

[0037] iolR-F: 5'TCAGTTAACATGACCACCGAAGCTCCCA3'

[0038]iolR-R: 5'GCGGGTTCAGGGGGTGATTACCTGGCCACCAGGTGG3'

[0039] iolt-F: 5' TCACCCCCTGAACCCGC 3'

[0040] iolt-R: 5'CTAGAAGCTTCTTGTCTCCTAAGTTTGTCGTGCC 3'

[0041] Using the above sequence as a template, use primers iolR-F and iolT-R to carry out overlapping PCR. The conditions are 94°C for 5min for 1 cycle, 94°C for 30s, 55°C for 30s, 72°C for 40s for 30 cycles, and 72°C for 10min for 1 cycle....

Embodiment 2

[0042] Example 2: Construction of inositol-induced expression plasmid pIol

[0043] After double-cutting Example 1 with restriction endonucleases Hpa I and Hind III, pXMJ19 was double-cut with restriction endonucleases Hpa I and Hind III to remove its original promoter P tac and lacIq, and then the two were connected by T4 ligase and then transformed into E. coli DH5α, and the inositol-induced expression plasmid pIol was obtained after screening. The schematic diagram of the construction of inositol-induced expression plasmid pIol is as follows: figure 1 shown.

Embodiment 3

[0044] Example 3: Construction of 5-aminolevulinic acid production plasmid pIolhemAL

[0045] Using the genomic DNA of Corynebacterium glutamicum 13032 as a template, the hemA and hemL gene fragments were amplified using primers hemA-F, hemA-R, hemL-F and hemL-R. The PCR conditions were 1 cycle at 94°C for 5 min, 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 40 s, and 1 cycle at 72°C for 10 min, and the reaction system was 50 μL. Purify and recover the PCR product after 1% agarose gel electrophoresis to obtain hemA and hemL.

[0046] hemA-F: 5'CGCGGATCCATGGTGAGTGTACTCATCGTAGGG3'

[0047] hemA-R: 5'TTACTCCCTCGTTTGTGTGGC 3'

[0048] hemL-F:

[0049] 5'GCCACACAAACGAGGGAGTAAGCACCCAAAAACACTATTGACCA3'

[0050] hemL-R: 5'CGGGGTACCTCATGATGCCTTCGCTTCTGC 3'

[0051] The gene fragments hemA and hemL were used as templates, and primers hemA-F and hemL-R were used to carry out overlapping PCR to obtain hemA-hemL fragments.

[0052] After hemA-hemL was double-cut by BamH I a...

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Abstract

The invention relates to a Corynebacteria inducible promoter, an expression vector comprising same, and an application thereof, wherein the expression vector is converted to corynebacterium glutamicumto obtain a 5-aminolevulinic acid producing bacterial strain, by that 5-aminolevulinic acid can be produced through a fermentation method. The expression vector constructed from the promoter, throughcontrollable expression of gene through inositol induction, can overcome problems of inhibition effect on growth of bacterial cells with and high production cost due to high price of an inducing agent, (isopropyl[beta]-D-thiogalactoside, IPTG). The expression vector has characters such as stability. By integrating the promoter with genome of a host or converting the expression vector into the host, corynebacterium glutamicum, the 5-aminolevulinic acid producing bacterial strain is acquired, yield of the 5-aminolevulinic acid reaching 24.2 g/L.

Description

technical field [0001] The invention relates to the field of biotechnology production, in particular to a coryneform bacterium inducible promoter, an expression vector containing the promoter and application thereof. Background technique [0002] At present, expression plasmids induced by isopropyl-β-D-thiogalactoside IPTG are often used in industrial production, and IPTG needs to be added in the production process, which is expensive and not conducive to industrial production, and IPTG has a serious impact on the growth of bacterial cells. The fermentation broth contains a large amount of residual IPTG, which brings difficulties to the separation and extraction of later products. Although lactose can metabolize IPTG, for most coryneform bacteria, lactose cannot enter the cell, further limiting its application in coryneform bacteria. [0003] Inositol, also known as cyclohexanol, belongs to the group of vitamins and is necessary for the synthesis of phosphatidylinositol, a ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/77C12N1/21C12P13/00
CPCC07K14/34C12N15/77C12N2800/101C12P13/005
Inventor 张成林战俊杰王道安李智祥陈宁孟静何继龙李英滋朱福周赵磊徐庆阳谢希贤李燕军范晓光马倩
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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