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Method for constructing recombinant strain capable of producing target gene product at high yield, and recombinant strain and application thereof

A construction method and technology of recombinant bacteria, applied in the field of genetic engineering, can solve the problems of restricting the industrial application of lycopene, organic waste pollution, etc., and achieve the effects of eliminating interference, increasing production and reducing production costs

Inactive Publication Date: 2015-12-23
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the raw materials are affected by seasons and geography, the complexity of chemical synthesis, and the pollution of organic waste all limit the industrial application of lycopene

Method used

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  • Method for constructing recombinant strain capable of producing target gene product at high yield, and recombinant strain and application thereof
  • Method for constructing recombinant strain capable of producing target gene product at high yield, and recombinant strain and application thereof
  • Method for constructing recombinant strain capable of producing target gene product at high yield, and recombinant strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, construction of pIJ8660-Promoters-RiboJ-crtEIB expression plasmid vector

[0043] 1. Cloning of crtE, crtI and crtB genes

[0044] Due to the need for multiple fragment insertion of the plasmid in the later stage, it is necessary to synonymously mutate three BsaI restriction enzyme site sequences within the crtI and crtB genes, design primers to mutate these three sites, and use them to amplify Streptomycesavermitilis MA4680 in Streptomyces avermitilis (ATCC31267), available through ATCC. crtE (SEQ ID NO: 1) [BA000030.3 (1289024 ... 1290265)], crtI (SEQ ID NO: 2) [BA000030.3 (1290262 ... 1291803)] and crtB (SEQ ID NO: 3) [BA000030.3 (1291800 ... ) on chromosome 1292828)] gene.

[0045] Use primer crtF (SEQ ID NO: 4) and primer crtM1R (SEQ ID NO: 5) to amplify fragment E, use primer crtM1F (SEQ ID NO: 6) and primer crtM2R (SEQ ID NO: 7) to amplify fragment I, use primer crtM2F (SEQ ID NO: 8) Fragment B1 was amplified with primer crtM3R (SEQ ID NO: 9), an...

Embodiment 2

[0052] Embodiment 2, transformation of recombinant plasmid

[0053]Due to the strong restriction modification in Streptomyces avermitilis, using Escherichia coli DH5α to directly combine with Streptomyces avermitilis for transfer, the transformation efficiency is extremely low, and sometimes even no transformant can be obtained. However, using E.coliET12567 (PUZ8002) without restriction modification for binding transfer, the transformation efficiency was significantly improved. Therefore, the constructed recombinant plasmid was transformed into E. coliET12567 (PUZ8002) (KieserT, BibbMJ, ButtnerMJ, et al. Practical Streptomyces Genetics, 2000, Norwich: The John Innes Foundation.) to obtain unmethylated DNA, and then combined transfer.

[0054] In this example, Streptomycesavermitilis MA4680 is selected as the starting strain, which produces off-white spores on the plate.

[0055] The negative control plasmid pIJ8660-crtEIB constructed in Example 1, the negative control plasmid...

Embodiment 3

[0056] Embodiment 3, the fermentation research of recombinant bacterial strain

[0057] 1. Shake flask fermentation of Streptomyces avermitilis

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Abstract

The invention relates to a method for constructing a recombinant strain capable of producing a target gene product at high yield, which comprises the following steps: (1) selecting an initial strain and a target gene; (2) constructing an expression vector comprising the target gene; (3) inserting a sequence with insulator function before a ribosome binding site of the target gene in the step (2), and inserting a heterogenous or artificial promoter sequence before the sequence with insulator function; and (4) transforming the expression vector constructed in the step (3) into the initial strain. The invention also relates to a recombinant strain constructed according to the method and a method for producing lycopene by fermentation by using the recombinant bacterium.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for constructing recombinant bacteria producing or high-yielding target gene products, and the constructed recombinant bacteria and their application. Background technique [0002] Animals, plants and microorganisms in nature can synthesize many organic small molecular compounds with structural and functional diversity. Compared with biological macromolecules such as proteins, nucleic acids, and lipids, they do not directly participate in the growth and development of organisms, but they have important biological activities and physiological functions. People call such active small molecule compounds as natural products. Many natural products have important uses in medicine or in industrial and agricultural production. [0003] Traditional methods of developing and mining active natural products, such as high-throughput screening and OSMAC ("onestrain many compounds"...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/76C12P5/02C12R1/465
Inventor 张立新胡逸灵娄春波白超弦苗靳向四海黄佩
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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