Mutant strains and engineering bacteria of Arthrobacter simplex with stress tolerance

A simple Arthrobacter, mutant strain technology, applied in the field of genetics and breeding, to achieve the effect of improving tolerance

Active Publication Date: 2020-12-29
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The level of tolerance to organic solvents of strains still has great limitations, and needs to be further improved through techniques such as mutation breeding and molecular biology.

Method used

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  • Mutant strains and engineering bacteria of Arthrobacter simplex with stress tolerance
  • Mutant strains and engineering bacteria of Arthrobacter simplex with stress tolerance
  • Mutant strains and engineering bacteria of Arthrobacter simplex with stress tolerance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: Breeding of Arthrobacter simplex mutant strain TCCC11307-UV15X1-2

[0046] 1.1 Cell wall weakening treatment

[0047] The preserved starting bacterial cells were activated on a slant (glucose 10g / L, yeast extract 10g / L, agar 20g / L, pH=7.2). Pick a ring full of bacterial lawn from the activated slant, inoculate in 30mL seed medium (glucose 10g / L, corn steep liquor 10g / L, peptone 5g / L, KH 2 PO 4 2.5g / L, pH=7.2) in a 250mL seed bottle, placed on a rotary shaker at 160r / min, cultured at 32°C for 18h, OD 600 When = 3.0, add the cell wall treatment agent lysozyme 30 μg / mL, and continue shaking culture for 1 h. The culture medium was collected, centrifuged at 4500r / min for 10min, and the supernatant was discarded. The cells were washed twice with normal saline, and then resuspended in 10 mL of normal saline. Shake well to disperse the cells and make a concentration of 10 8 cells / mL of bacterial suspension.

[0048] 1.2 Mutagenesis treatment

[0049] Take ...

Embodiment 2

[0058] Example 2: Evaluation of organic solvent tolerance of mutant strain TCCC11307-UV15X1-2

[0059] 2.1 Comparison of the survival rate of mutant strains and starting strains under high concentration organic solvent shock conditions

[0060] Pick the mutant strain TCCC11307-UV15X1-2 obtained in Example 1 from the slope, and use the starting strain as the control strain, and inoculate the above two strains into shake flasks containing 50mL of seed medium, 32°C, 160r / min Culture under shaking for 24h, transfer into the fresh above-mentioned liquid medium, and change the initial OD 600 The value was adjusted to 0.2, 32°C, 160r / min shaking culture to the late logarithm. Centrifuge at 6000r / min for 10min to collect the bacterial cells, suspend the collected bacterial cells with the fresh above-mentioned liquid medium and measure the OD of the bacterial liquid 600 The values ​​were all adjusted to 1.0, and then 16% (v / v) ethanol and 20% (v / v) methanol were added to shock for 1 ...

Embodiment 3

[0067] Example 3: Determination of ethanol tolerance-related proteins

[0068] The cells of the Arthrobacter simplex mutant strain TCCC11307-UV15X1-2 were collected and cultured for 3 hours under the condition of adding 8% ethanol and without adding ethanol, and the intracellular protein was extracted. Through the comparison of proteomics technology, it was found that compared with that without adding ethanol, , the addition of 8% ethanol led to a significant increase in the expression of a series of proteins, including: heat shock protein GroEL, DnaK; DNA recombination repair protein RecA, DNA helicase UvrD; catalase KatG, superoxide dismutase SOD; trehalose synthesis Enzyme TreS, these proteins may be related to the stress tolerance of bacteria. qRT-PCR was used to further verify the transcription levels of the seven stress-resistance-related protein-encoding genes groEL, dnaK, recA, uvrD, katG, sod, and treS. The results showed that groEL, dnaK, recA , uvrD, katG, sod, tre...

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Abstract

The invention belongs to the field of genetic breeding, and in particular relates to a simple Arthrobacter mutant strain with stress tolerance and a genetic engineering bacterium constructed therefrom. Among them, the survival rate of the mutant strains under 16% ethanol and 20% methanol shock conditions increased by 3.74 times and 2.10 times respectively compared with the starting strain; overexpression of groEL, dnaK, recA, uvrD, katG, sod or treS genes from the mutant strains The tolerance of the engineered bacteria to high concentrations of organic solvents is significantly improved, and they also show higher tolerance to salt stress and oxidative stress. The above engineered bacteria are used in the steroid C1,2 dehydrogenation reaction to put The ethanol concentration was increased to 8-10%, the substrate CA concentration was increased to 6-8g / L, and the product yield was increased by 0.41-3.56 times, which laid a good foundation for the subsequent research on the resistance mechanism and the molecular transformation and application of strains.

Description

Technical field: [0001] The invention belongs to the field of genetic breeding, and in particular relates to a simple Arthrobacter mutant strain with stress tolerance and a genetic engineering bacterium constructed therefrom. Background technique: [0002] Steroid hormone drugs are the second largest class of drugs in clinical use after antibiotics. The dehydrogenation reaction of C1 and 2 positions of steroidal compounds is a typical representative of industrial production of steroidal drugs by microbial transformation, and it is also the most valuable reaction for the production of prednisolone and its homologues. Compared with chemical synthesis methods, microbial transformation reactions have high stereoselectivity and regioselectivity, which can greatly reduce the synthesis steps and shorten the production cycle. However, the poor solubility of the substrate (generally 10 -5 -10 -6 mol / L) and the catalytic environment of the aqueous phase prevent the substrate from b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N1/21C12N15/70C12N15/74C12P33/02C12R1/06
CPCC07K14/195C12N9/0065C12N9/0089C12N9/1051C12N9/14C12P33/02C12Y111/01006C12Y115/01001C12Y204/01245C12Y306/04012C12N1/205C12R2001/06
Inventor 骆健美王敏申雁冰宋昭玉薛海洁崔芳芳王艳霞夏梦雷
Owner TIANJIN UNIV OF SCI & TECH
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