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Method for increasing yield of phaffia rhodozyma astaxanthin

A Phaffia rhodozyme and astaxanthin technology, applied in the biological field, can solve the problems of not fully revealing the method of astaxanthin production, and achieve the effects of reducing feedback inhibition, reducing metabolic competition, and improving metabolic flow and production

Active Publication Date: 2021-03-02
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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Problems solved by technology

However, the above-mentioned single gene knockout did not fully reveal the way to increase astaxanthin production by regulating sterol synthesis

Method used

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  • Method for increasing yield of phaffia rhodozyma astaxanthin

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Embodiment

[0043] 1. Materials and Methods

[0044] 1.1 Strains and culture conditions

[0045] Phaffia rhodozyme strain CBS 6938 (purchased from the Dutch CBS Culture Collection) was used as the material. Routine culture uses YPD liquid medium (glucose 20g / L, peptone 20g / L, yeast extract 10g / L), and the culture conditions are 22°C, 250rpm.

[0046] 1.2 Construction of gene knockout vector

[0047] All sterol synthesis genes were knocked out by homologous recombination, and the knocked out sterol synthesis genes had been annotated with corresponding enzymes in the GenBank genome database. In this example, except for CYP51 and CYP61, knockout vectors for other 10 genes as shown in Table 1 were constructed.

[0048] Table 1. GenBank ID of sterol synthesis genes and Scaffold

[0049]

[0050] The specific vector was constructed using the Gibson assembly method, and the DNA fragment was obtained by the PCR method, and the primers used are shown in Table 2. All PCR amplifications use ...

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Abstract

The invention relates to the technical field of biology, and in particular, relates to a method for knocking out sterol synthesis genes to increase the yield of phaffia rhodozyma astaxanthin. The fermentation yield of phaffia rhodozyma astaxanthin is increased by knocking out a recombinant strain obtained by knocking out one or more genes in phaffia rhodozyma sterol synthase encoding genes; or, the fermentation yield of phaffia rhodozyma astaxanthin is increased by knocking out one or more of phaffia rhodozyma sterol synthetases and knocking out the combination of the phaffia rhodozyma sterolsynthetases and a CYP61 gene. The phaffia rhodozyma sterol synthesis gene is knocked out, so that the metabolic competition of sterol generation for astaxanthin synthesis is reduced or the feedback inhibition of sterol on a mevalonic acid pathway is reduced, and the metabolic flux and yield of astaxanthin synthesis are improved. The astaxanthin content of a single-gene knockout strain can be 2.2 times that of a control strain; the astaxanthin yield of a double-gene knockout strain can be increased by 38% on the basis of the single-gene knockout strain. The method can be applied to constructionof astaxanthin high-producing strains.

Description

technical field [0001] The invention relates to the field of biological technology, in particular to a method for knocking out a sterol synthesis gene to increase the output of Phaffia rhodozyma astaxanthin. Background technique [0002] Astaxanthin is a terpene compound that belongs to carotenoids. Astaxanthin has strong antioxidant, coloring and anti-inflammatory functions, and also has certain anti-cancer effects (Park J, Chyun J, Kim Y, et al. Nutr Metab, 2010, 7:18). According to BCC research, the market for astaxanthin will reach US$1.5 billion by 2020. Moreover, with the continuous expansion of new uses of astaxanthin, its market will continue to expand. [0003] The proportion of astaxanthin synthesized by Phaffia rhodozyma can account for 50% to 85% of cell carotenoids, and it has outstanding advantages in growth speed, cell density, anti-pollution, etc., and is considered an important cell factory for astaxanthin production (Bellora N, Moliné M, David-Palma M, e...

Claims

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Application Information

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IPC IPC(8): C12P23/00C12N15/52C12N15/81C12N15/90C12N1/19C12R1/645
CPCC12P23/00C12N9/00C12N15/815C12N15/905
Inventor 王士安张宁李福利
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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