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Compound mutagenesis breeding method of mutant strain for highly yielding beta-farnesene

A technology for compound mutagenesis and mutant strains, applied in microorganism-based methods, mutant preparation, biochemical equipment and methods, etc., can solve the problems of narrow mutation spectrum, saturated resistance, low mutation efficiency, etc., and achieve increased damage degree, compensate for hot spot saturation, improve the effect of the effect

Pending Publication Date: 2019-11-05
山东泓达生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, long-term repeated use of traditional mutagenesis methods often leads to problems such as low mutation efficiency, narrow mutation spectrum, and resistance saturation.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] A compound mutagenesis breeding method for high-yielding β-farnesene mutant strains, comprising the steps of:

[0021] (1) Preparation of bacterial suspension: select the EC-16 slant bacterial lawn cultured at 37°C for 2 days in a ZXMP-A1 full-temperature constant temperature and humidity chamber, wash the bacterial lawn with 10mL normal saline, and put it into a glass container. Place in a ZWY-B3222 constant temperature culture shaker at 30°C and 250r / min for 10min to disperse the bacteria, centrifuge the bacteria suspension at 3000r / min for 15min, discard the supernatant, and use Suspend and wash in normal saline for 2-3 times, count with a bacterial counting plate, and adjust the concentration of the bacterial suspension to about 10 8 A / mL, spare;

[0022] (2) Plasma-ultraviolet compound mutagenesis treatment: take 3mL of bacterial suspension in the ultra-clean workbench and add it to a sterile plate with a diameter of 60mm with a magnetic stirring rotor, and use an...

Embodiment 2

[0032] A compound mutagenesis breeding method for high-yielding β-farnesene mutant strains, comprising the steps of:

[0033] (1) Preparation of bacterial suspension: select the EC-16 slant bacterial lawn cultured at 37°C for 2 days in a ZXMP-A1 full-temperature constant temperature and humidity chamber, wash the bacterial lawn with 10mL normal saline, and put it into a glass container. Place in a ZWY-B3222 constant temperature culture shaker at 30°C and 220r / min for 6 minutes to disperse the bacteria, centrifuge the bacteria suspension at 2500r / min for 10min, discard the supernatant, and use Suspend and wash in normal saline for 2-3 times, count with a bacterial counting plate, and adjust the concentration of the bacterial suspension to about 10 6 A / mL, spare;

[0034] (2) Plasma-ultraviolet compound mutagenesis treatment: take 3mL of bacterial suspension in the ultra-clean workbench and add it to a sterile plate with a diameter of 60mm with a magnetic stirring rotor, and us...

Embodiment 3

[0044] A compound mutagenesis breeding method for high-yielding β-farnesene mutant strains, comprising the steps of:

[0045] (1) Preparation of bacterial suspension: select the EC-16 slant bacterial lawn cultured at 37°C for 2 days in a ZXMP-A1 full-temperature constant temperature and humidity chamber, wash the bacterial lawn with 10mL normal saline, and put it into a glass container. Place in a ZWY-B3222 constant temperature culture shaker at 30°C and 300r / min for 15min to disperse the bacteria, centrifuge the bacteria suspension at 5000r / min for 20min, discard the supernatant, and use Suspend and wash in normal saline for 2-3 times, count with a bacterial counting plate, and adjust the concentration of the bacterial suspension to about 10 9 A / mL, spare;

[0046] (2) Plasma-ultraviolet compound mutagenesis treatment: take 3mL of bacterial suspension in the ultra-clean workbench and add it to a sterile plate with a diameter of 60mm with a magnetic stirring rotor, and use an...

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Abstract

The invention relates to the technical field of biological mutagenesis breeding, and in particular, relates to a compound mutagenesis breeding method of a mutant strain for highly yielding beta-farnesene. The method includes the steps: preparation of a bacterial suspension, plasma-ultraviolet compound mutagenesis, flat plate primary screening, shake flask re-screening and fermentation tank verification. According to the compound mutation breeding method of the mutant strain for highly yielding beta-farnesene, through plasma-ultraviolet compound mutagenesis, flat plate primary screening and shake flask re-screening are adopted, and finally, the fermentation unit of beta-farnesene of the mutant strain for highly yielding beta-farnesene in the fermentation tank is 169.10% higher than that ofa starting strain, and the mutant strain still has good hereditary stability through 8 times of consecutive passage.

Description

technical field [0001] The invention relates to the technical field of biological mutation breeding, in particular to a compound mutation breeding method for high-yielding β-farnesene mutant strains. Background technique [0002] β-Farnesene (also known as β-Farnesene) is a chain sesquiterpene compound composed of three isoprenes, which is widely used in daily chemical, agricultural, chemical, energy and pharmaceutical industries. The field has wide application. Especially in the field of medicine, β-farnesene is an important raw material for the synthesis of isophytic alcohol, the precursor of vitamin E. Vitamin E is one of the vitamin products with the largest market capacity in the world, with an annual global production of about 60,000 tons. The raw material supply of vitamin E precursor isophytic alcohol has been monopolized by BASF in Germany and DSM in the Netherlands. They use citral as an intermediate Synthesize isophytol in vivo, and then use the total chemical s...

Claims

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Application Information

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IPC IPC(8): C12N13/00C12N15/01C12N1/02C12N1/20C12R1/19
CPCC12N13/00C12N15/01C12N1/02C12R2001/19C12N1/205
Inventor 寇凤雨王一然刘伟马杰希
Owner 山东泓达生物科技有限公司
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