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Fragmentation chytrid mutagenesis method and variant produced by fragmentation chytrid mutagenesis method

A technology of Schizochytrium and mutant strains, which is applied in the field of microbial genetic breeding technology and fermentation biology, and can solve the problems of unclear whether there is quizalofop-resistant Schizochytrium and low EPA content

Inactive Publication Date: 2012-12-26
法国罗凯特兄弟公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Schizochytrium is known to be distinct from Nannochloropsis or diatom Nitzschia and to be low in EPA, so far it is unclear whether quizalofop-resistant mutagenic Schizochytrium exists

Method used

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  • Fragmentation chytrid mutagenesis method and variant produced by fragmentation chytrid mutagenesis method
  • Fragmentation chytrid mutagenesis method and variant produced by fragmentation chytrid mutagenesis method
  • Fragmentation chytrid mutagenesis method and variant produced by fragmentation chytrid mutagenesis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Mutation effect of ultraviolet rays on Schizochytrium

[0057] Spread Schizochytrium SR21 on a petri dish and place it under ultraviolet light for irradiation. The irradiation time is divided into 0 seconds (S) (control group), 30S, 40S, 50S, 60S, 70S, 80S, 90S and 100S (experimental group). Place it in the dark for 24 hours after irradiation, and count the number of colonies when they appear. The number of colonies in the control group was 100%, and the lethality of each experimental group was calculated ( figure 1 ). From figure 1It can be seen that with the extension of ultraviolet irradiation time, the lethality of Schizochytrium increased, showing a significant dose effect. The irradiation time of 70S-90S is selected (the lethality rate of Schizochytrium is 60%-80%), and effective mutagenesis treatment can be carried out on Schizochytrium.

[0058] The media used were:

[0059] Glucose 55g / L

[0060] Yeast extract 10g / L

[0061] Sodium glutamate 5...

Embodiment 2

[0067] Example 2 The screening of quizalofop to Schizochytrium

[0068] Add the inhibitor quizalofop (Quizalofop ethyl) {(R)-2-[4-(6-chloro-2-quinoxaline) of fat synthesis key enzyme (acetyl-CoA carboxylase) in the Schizochytrium culture medium phenoxy)-phenoxy]-acrylate}, the added concentrations were 0 μmol / L, 10 μmol / L, 30 μmol / L, 50 μmol / L, 70 μmol / L, 80 μmol / L and 90 μmol / L. With the number of colonies in the control group (0 μmol / L) as 100%, count the number of colonies in each group to calculate the lethal rate ( figure 2 ). From figure 2 It can be seen that within the experimental range, the lethality of Schizochytrium was positively correlated with the concentration of quizalofop. The concentration of quizalofop was 50μmol / L to 80μmol / L for selection of resistant strains.

[0069] For the convenience of operation, quizalofop was added to the culture medium, and Schizochytrium was coated for further directional screening of ultraviolet resistant strains.

[0070...

Embodiment 3

[0081] Embodiment 3 Differences in growth rate and DHA content of control strains and different mutant strains

[0082] After screening and confirmation in Example 2, more than 200 strains with quizalofop-resistant were selected. Then, the selected quizalofop-resistant strains were further screened by using the growth rate and DHA content of the strains as indicators. 20-50 strains were selected in the first re-screening, and 3 strains were selected in the second re-screening. The growth rate and DHA content of the three strains were all higher than the control by more than 10%.

[0083] The conditions for its cultivation are

[0084] a. The medium formula is (g / L):

[0085] Glucose 60g / L

[0086] Yeast extract 15g / L

[0087] Sodium glutamate 4g / L

[0088] Sodium chloride 3g / L

[0089] Magnesium sulfate 5g / L

[0090] Ammonium sulfate 5g / L.

[0091] b. Culture temperature: 22-27°C.

[0092] c. Initial pH: 5.0-7.0.

[0093] image 3 Represents the biomass and DHA cont...

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Abstract

The invention relates to a new fragmentation chytrid variant. Especially, the invention relates to a fragmentation chytrid variant with improved DHA (docosahexaenoic acid) content and / or rapid growth speed and a method for producing DHA by utilizing the variant. Besides, the invention also relates to a method for producing the variant, comprising the steps of carrying out mutagenesis on fragmentation chytrid by adopting ultraviolet rays and screening a strain with rapid growth speed and high DHA content under selective pressure of an inhibitor (such as quizalofop) for fat synthesis key enzyme (acetyl coenzyme A carboxylase).

Description

technical field [0001] The invention belongs to the field of microbial genetic breeding technology and fermentation biology technology, and particularly relates to a mutagenic strain for producing DHA (docosahexaenoic acid, 22:6, n-3). Background technique [0002] DHA (Docosahexaenoic acid, docosahexaenoic acid, 22:6, n-3) is an essential fatty acid, that is, a long-chain fatty acid that cannot be synthesized by the human body and has important physiological functions. DHA is the main component of the human brain and retina, with a content of 20% in the cerebral cortex and 50% in the retina. Therefore, it plays an important role in the development of the nervous system and visual system of the fetus and is important for maintaining normal intelligence. It also has important physiological functions such as preventing and treating cardiovascular diseases, anti-cancer, and anti-inflammation. In addition, DHA is also an essential fatty acid required for the growth and developm...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N13/00C12N15/01C12P7/64A23C9/152A23L1/30C12R1/645C12P7/6434
CPCA23C9/152C12N13/00C12R1/645C12N1/12C12N15/01A23L1/337C12P7/64C12P7/6427C12R1/89A23L1/30A23L1/3006C12N1/14A23L1/296A23C9/1528C12P7/6434A23L33/40A23L33/115A23L17/60C12R2001/89C12N1/125C12N1/20C12N15/03
Inventor 伯翰周洁
Owner 法国罗凯特兄弟公司
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