A coryneform expression system independent of antibiotics as selection pressure

An expression system, coryneform bacteria technology, applied in the direction of microorganism-based methods, bacteria, microorganisms, etc., can solve the problems that are not conducive to transformation, limit the use range of fermentation products, etc., and achieve the effect of easy industrial application, stable existence, and convenient scientific research

Active Publication Date: 2016-08-17
JIANGNAN UNIV
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

However, the addition of antibiotics not only limits the scope of use of specific fermentation products, but also accounts for a large proportion of the cost of fermentation production due to the large amount of antibiotics added in the fermentation medium.
The single use of antibiotics as vectors for selection markers is not conducive to the transformation of laboratory research results to industrial production

Method used

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  • A coryneform expression system independent of antibiotics as selection pressure
  • A coryneform expression system independent of antibiotics as selection pressure
  • A coryneform expression system independent of antibiotics as selection pressure

Examples

Experimental program
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Effect test

Embodiment 1

[0033]The construction of embodiment 1 expression vector pJYW-4, pJYW-5

[0034] Step 1: Digest vector pEC-XK99E (Oliver Kirchner, Andreas Tauch, 2003. Tools for genetic engineering in the amino acid-producing bacterium Corynebacterium glutamicum.) with restriction enzymes Bstz17I and SmaI to remove the lacI-containing q The DNA fragment of the gene and the Ptrc promoter is self-circularized to construct a recombinant plasmid with a size of 5306 bp, which is named pJYW-1;

[0035] Step 2: Using the C. glutamicum ATCC13032 genome as a template, alr-P-(+) and alr-P-(-) as primers, amplify the 1598bp alr gene and its original promoter by PCR, and separate the two ends of the fragment BglII and PstI were introduced. The PCR product was digested with BglII and PstI, and connected to pJYW-1 digested with BamHI and PstI. The constructed plasmid was 6880bp in size and named pJYW-2;

[0036] Step 3: Using MCS-F and MCS-R as primers, the oligonucleotide chain annealing reaction artifi...

Embodiment 2al

[0038] Construction of embodiment 2 alr knockout vector pJYW-6

[0039] Using the genome of C. glutamicum ATCC13032 as a template, the upstream and downstream fragments alr-U, alr-U, alr-D; using pDTW-202 (Jinyu Hu, Yanzhen Tan, YanyanLi, Xiaoqing Hu, Daqing Xu, Xiaoyuan Wang, 2013. Construction and application of an efficient multiple-gene-deletion system in Corynebacterium glutamicum.) as a template, kan-lox -F / R are primers for amplifying the kan resistance gene fragment. alr-U was digested with XhoI and BamHI, alr-D was digested with XbaI and PstI, the kan fragment was digested with BamHI and XbaI, and the three fragments were ligated together into pBluescriptIISK(+) digested with XhoI and PstI to construct the completed plasmid Named pJYW-6 ( figure 2 ).

Embodiment 3

[0040] Verification of the availability of the expression system constructed in Example 3

[0041] In a valine-producing strain YTW-102 (Jinyu Hu, Yanzhen Tan, Yanyan Li, Xiaoqing Hu, Daqing Xu, Xiaoyuan Wang, 2013. Construction and application of an efficient multiple-gene-deletion system in Corynebacterium glutamicum.) Based on this, the knockout vector was used to knock out its alr, and the constructed strain was named YTW-103. The growth of three strains of bacteria YTW-102, YTW-103, YTW-103 / pJYW-4 was measured in the basic medium, and the results were as follows: image 3 shown. At the same time, the maintenance of the plasmid in the bacteria after fermentation and cultivation under the condition of no antibiotics was analyzed. The method used was that the bacteria were cultured on the basic medium under the condition of no antibiotics and then diluted to an appropriate multiple, and then coated with 30mg / L or no kana LBHIS (Jinyu Hu, Yanzhen Tan, Yanyan Li, Xiaoqing Hu...

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Abstract

The invention discloses a corynebacteria expression system without depending on antibiotic being selection pressure, and belongs to the microbe gene engineering field. Alr knock out carrier and expression carrier used for plasmid anaplerosis are constructed to bring conveniences for lab research and utilization, and a fermentation process no longer needs antibiotic. Through test results, the expression system realizes thalline inner plasmid stability in fermentation without adding the antibiotic; when the expression system is used to construct overexpression vector of branched chain amino acid-valine synthesis path main gene ilvBNC, valine output can be obviously improved; the corynebacteria expression system has wide application value.

Description

technical field [0001] The invention discloses a coryneform bacterium expression system that does not rely on antibiotics as a selection pressure, in particular an Escherichia coli-coryneform bacterium shuttle expression system that does not rely on antibiotics as a selection pressure, its construction method and application, and belongs to the field of microbial genetic engineering. Background technique [0002] Corynebacteria are a class of Gram-positive bacteria that belong to the actinomycetes with moderate to high GC content. Since Corynebacterium glutamicum was first isolated to produce L-glutamic acid (Kinoshita et al., 1957), the three main representatives of Corynebacterium: Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum have been widely For the production of amino acids. In particular, it is pointed out that there are only small differences among the three different species through DNA-DNA hybridization experiments. [0003] ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/77C12N1/20C12N15/66C12P13/08C12R1/15
Inventor 王小元胡瑾瑜李颜颜胡晓清谭延镇
Owner JIANGNAN UNIV
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