Schizochytrium limacinum strain as well as building method and application thereof

A technology of Schizochytrium and construction methods, applied in the direction of microorganism-based methods, biochemical equipment and methods, fungi, etc., can solve the problems of unfavorable DHA accumulation and unfavorable cell growth, etc.

Active Publication Date: 2017-07-14
南京脂禾生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

High oxygen is beneficial to the growth of Schizochytrium during the fermentation process, but it is not conducive to the accumulation of DHA
Low temper

Method used

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  • Schizochytrium limacinum strain as well as building method and application thereof
  • Schizochytrium limacinum strain as well as building method and application thereof
  • Schizochytrium limacinum strain as well as building method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 This embodiment illustrates the biological material source information of the present invention

[0036] Schizochytrium sp. HX-308 has been preserved in China Center for Type Culture Collection (CCTCC), and its preservation number is CCTCC No: M 209059.

[0037] The domesticated Schizochytrium sp.HX-RS genetically engineered strain is preserved in the China Center for Type Culture Collection, and its preservation number is CCTCC No: M2017046.

[0038] Carrier PBS-Zeo: self-constructed by our laboratory.

[0039] Vector PMD19-T (simple): commercial vector, purchased from TaKaRa Company.

Embodiment 2

[0040] Example 2 This example illustrates the construction of the recombinant plasmid PBS-Zeo-AT for AT functional domain knockout

[0041] The construction steps of plasmid PBS-Zeo-AT are as follows: figure 1 shown.

[0042] 1. Obtain the upper and lower homology arms of the Schizochytrium AT gene

[0043] (1) Obtain the upstream homology arm of the Schizochytrium AT gene

[0044] According to the AT gene sequence obtained by sequencing, primers ATup-S and ATup-A were designed to amplify the upstream homology arm of Schizochytrium sp.HX-308 and ligated to the PMD19-T vector to obtain a recombinant plasmid pMD19-ATup. in,

[0045] ATup-S sequence:

[0046] 5'AAGGAAAAAAGCGGCCGCTGCTATTCCGTGCTCCTCTC 3',

[0047] ATup-A sequence:

[0048] 5' GCTCTAGAGCGCCTTGGGCGTGATGTTGAG 3'.

[0049] (2) Obtain the downstream homology arm of Schizochytrium AT gene

[0050] Primers ATdown-S and ATdown-A were designed according to the obtained AT sequence of Schizochytrium to amplify the d...

Embodiment 3

[0059] Example 3 This example illustrates the oil production of acyltransferase AT-deficient Schizochytrium strains

[0060] The plasmid PBS-Zeo-AT was double-digested with NotI and KpnI restriction enzymes, and then the fragments containing the upstream and downstream homology arms of AT and bleomycin were purified using the Takara gel recovery kit, and transformed into In Schizochytrium, spread the transformed bacterial solution on a plate containing 20 μg / mL bleomycin, culture at 30°C in the dark for 2 days, pick a positive single colony for shake flask culture, and extract the genome for PCR verification. And the correct defective strain was named Schizochytriumsp.HX-DS.

[0061] The engineered bacteria obtained above were inoculated into the seed medium under the following culture conditions: temperature 30° C., rotation speed 170 rpm, and cell dry weight and fatty acid content were measured respectively. Compared with the parameters of the original strain under the same...

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Abstract

The invention discloses a schizochytrium limacinum strain as well as a building method and application thereof. The strain disclosed by the invention is classified and named as schizochytrium sp. HX-RS, and the preservation number of the schizochytrium sp. HX-RS is CCTCC NO: M2017046. According to the strain, an acyltransferase functional domain originating from shewanella oneidensis PKS enzyme, instead of an acyltransferase functional domain originating from schizochytrium sp. PKS enzyme, is adopted; the strain is obtained through flat panel screening and acclimation screening with high rotation speed and low temperature. The strain can be used for co-producing DHA and EPA; the contents of the EPA and the DHA obtained by carrying out culture fermentation of the strain respectively reach 4.68% and 51.3%, and compared with an original strain, the contents of the EPA and the DHA are respectively increased by 69.9% and 14.3%; moreover, the fermentation production efficiency is high and the cost is relatively low.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and in particular relates to a method for producing oil by using microorganisms, in particular to a Schizochytrium strain capable of fermenting and co-producing DHA and EPA. Background technique [0002] EPA and DHA are omega-3 long-chain polyunsaturated fatty acids that are very important for human growth and development. They have the effects of lowering blood fat and improving intelligence. At the same time, they can also be used to prevent and treat some diseases, such as hypertension, arteriosclerosis, Cardiovascular disease, Alzheimer's disease, anti-inflammation, anti-cancer, etc. These fatty acids can regulate lipid transport, storage and metabolism in many tissues by activating nuclear peroxisome proliferator-activated receptors (PPARs) to reduce serum triglyceride levels. It is difficult for the human body to meet the body's demand for EPA and DHA through its own synthesis, and it n...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12P7/64C12R1/645
CPCC12N9/1025C12P7/6463C12P7/6472
Inventor 黄和任路静陈胜兰耿灵君纪晓俊胡学超
Owner 南京脂禾生物科技有限公司
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