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Construction method and application of Arthrobacter simplex engineering strain high in organic solvent tolerance

A technology of Arthrobacter simplex and engineering strains, which is applied in the field of genetic engineering and microbial transformation, and can solve problems such as undiscovered Arthrobacter simplex engineered strains

Active Publication Date: 2017-02-22
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] So far, no simple Arthrobacter engineering strains with high organic solvent tolerance have been constructed by expressing the irrE gene and trehalose synthesis genes otsA and otsB, and the use of the engineered strains for C 1,2 The method of dehydrogenation reaction

Method used

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  • Construction method and application of Arthrobacter simplex engineering strain high in organic solvent tolerance
  • Construction method and application of Arthrobacter simplex engineering strain high in organic solvent tolerance
  • Construction method and application of Arthrobacter simplex engineering strain high in organic solvent tolerance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Construction and application of embodiment 1 irrE engineering strain

[0045] 1 Preparation of genetically engineered bacteria containing recombinant plasmid pART2-irrE

[0046] (1) Construction of recombinant plasmid pART2-irrE

[0047] Deinococcus radiodurans (purchased from China General Microorganism Culture Collection Center, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip code 100101. Strain number: 1.633) The genome is used as a template, and primers irrE3-F (as shown in SEQ ID: 1) and irrE3-R (as shown in SEQ ID: 2) are used to carry out PCR to amplify and obtain the global transcription factor irrE of Deinococcus radiodurans (as shown in SEQ ID: 3) gene sequence,

[0048] SEQ ID: 1 cgggatccca gtgccaacgt cagccc

[0049] SEQ ID: 2 tgctctagac tgtgcagcgt cctgcg

[0050] The PCR reaction system is as follows:

[0051]

[0052] The PCR reaction conditions were: pre-denaturation ...

Embodiment 2

[0101] Embodiment 2: Construction and application of otsA engineering strain

[0102] 1 Preparation of genetically engineered bacteria containing recombinant plasmid pART2-otsA

[0103] (1) Construction of recombinant plasmid pART2-otsA

[0104] Using the genome of Arthrobacter simplex ACCC 12070 as a template, PCR was performed using primers otsA-F (shown in SEQ ID: 4) and otsA-R (shown in SEQ ID: 5) to obtain Arthrobacter simplex Trehalose synthesis gene otsA gene sequence (as shown in SEQ ID: 6),

[0105] SEQ ID: 4 cgggatccag tgggccatga cctcgtgatc

[0106] SEQ ID: 5 tgctctagat caggagcgtg ttgctggtcg

[0107] The PCR reaction system is as follows:

[0108]

[0109] The PCR reaction conditions were: pre-denaturation at 95°C for 5 minutes, 30s at 94°C, 30s at 63°C, 90s at 72°C, and 10 minutes at 72°C after 30 cycles.

[0110] Trehalose synthesis gene otsA sequence and plasmid pART2 were treated with restriction endonucleases BamH Ⅰ and Xba Ⅰ respectively (37°C, 4h), and...

Embodiment 3

[0156] Embodiment 3: Construction and application of otsB engineering strain

[0157] 1 Preparation of genetically engineered bacteria containing recombinant plasmid pART2-otsB

[0158] (1) Construction of recombinant plasmid pART2-otsB

[0159] Using the genome of Arthrobacter simplex ACCC 12070 as a template, PCR was performed using primers otsB-F (shown in SEQ ID: 7) and otsB-R (shown in SEQ ID: 8) to obtain Arthrobacter simplex Trehalose synthesis gene otsB gene sequence (as shown in SEQ ID: 9),

[0160] SEQ ID: 7 cgggatccag tgaggttccc gacgcgcg

[0161] SEQ ID: 8 tgctctagac tagccgagcc ggcggacc

[0162] The PCR reaction system is as follows:

[0163]

[0164] The PCR reaction conditions were: pre-denaturation at 95°C for 5 minutes, 30s at 94°C, 30s at 63°C, 90s at 72°C, and 10 minutes at 72°C after 30 cycles.

[0165] Trehalose synthesis gene otsB sequence and plasmid pART2 were treated with restriction endonucleases BamH Ⅰ and Xba Ⅰ respectively (37°C, 4h), and the...

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Abstract

The invention relates to a construction method and application of an Arthrobacter simplex engineering strain high in organic solvent tolerance. The Arthrobacter simplex engineering strain is obtained by guiding a gene segment into Arthrobacter simplex; experiments verify that tolerance of the engineering strain obtained by utilizing the construction method to methanol and alcohol is improved obviously. By utilizing the engineering strain for steroid C1, 2 dehydrogenation reaction, adding amount of an organic solvent in a system is doubled, feeding concentration of a substrate is increased by three times, and generation amounts of products are increased in different degrees. By constructing and applying the converted engineering strain high in organic solvent tolerance, the adding amount of the organic solvent in the conversion system can be increased, the feeding concentration of the substrate is increased, the generation amount of the products is increased, and the engineering strain has important application value in increasing the conversion efficiency of high-concentration substrates of steroid hydrophobic compounds.

Description

technical field [0001] The invention belongs to the field of genetic engineering and microbial transformation, and in particular relates to the construction of a simple Arthrobacter engineering strain with high tolerance to organic solvents and the transformation of the strain in steroid compounds, such as A ring C 1,2 Applications in dehydrogenation reactions. Background technique [0002] Steroid drugs have important physiological activities and are the second largest class of drugs after antibiotics. C 1,2 The dehydrogenation reaction is the most valuable reaction for the industrial production of prednisone hydrochloride and its homologues, and it is also a typical representative of the conversion method used in the steroid drug industry. steroid drug nucleogenesis C 1,2 The anti-inflammatory activity can be multiplied after the double bond is introduced, and the C of cortisone acetate 1,2 Prednisone acetate is generated after the double bond is introduced, the anti-i...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N1/21C12P33/02C12R1/06
CPCC07K14/195C12N15/74C12N2800/101C12P33/02
Inventor 骆健美王敏薛海洁崔芳芳刘家家申雁冰郑宇
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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