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Astaxanthin-producing Rhodosporidium kratochvilovae gene engineering strain

A technology for genetically engineered strains and Rhodosporidium spores, which is applied in the field of biotechnology and can solve problems such as high price and the like

Active Publication Date: 2020-07-28
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the source of astaxanthin on the market is mainly chemical synthesis, which is not only expensive, but also significantly different from natural astaxanthin in terms of structure, function, application and safety. Therefore, the demand for astaxanthin from natural sources is increasing

Method used

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  • Astaxanthin-producing Rhodosporidium kratochvilovae gene engineering strain
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  • Astaxanthin-producing Rhodosporidium kratochvilovae gene engineering strain

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Example 1: Construction of recombinant plasmid pRHcrtW-crtZ by seamless cloning

[0021] Using Vazyme's ClonExpress ® II One Step Cloning Kit kit for the construction of recombinant plasmid pRHcrtW-crtZ; designed for seamless cloning according to the instructions crtW, crtZ Gene-specific primers, using the target fragment synthesized by Shuoqing Biotechnology Co., Ltd. as a template, perform PCR amplification on a PCR instrument (BIOER company). The primers, components and amplification conditions used in the reaction are as follows:

[0022] pRHcrtW-F: 5'- ACAACACCAGATCACTCA CCATGG ATGAGCGCACATGCC -3', underlined as Nco I restriction site;

[0023] pRHcrtW-R: 5'-ATCCCGGTCGGCATCTAC GATATC TCATGCGGTGTCCCC -3' , underlined as EcoR V restriction site;

[0024] pRHcrtZ-F: 5'-ATACATTATACGAAC GGTACC TGTACAGTGACCGGT -3', underlined Kpn Ⅰ Restriction site;

[0025] pRHcrtZ-R: 5'-CTGATCCAAGCTCAAGCT AAGCTT GCATGCCTGCAG -3', underlined Hind Ⅲ Restriction site; ...

Embodiment 2

[0029] Example 2: Construction of Rhodosporidium genetically engineered strains and synthesis of astaxanthin and carotenoids in Rhodosporidium yeast

[0030] 1. Agrobacterium-mediated transformation of Rhodosporidium YM25235

[0031] The recombinant plasmid pRHcrtW-crtZ was transformed into Rhodosporidium YM25235 by the Agrobacterium-mediated method, and the transformants were selected with the YPD medium containing the final concentration of Hygromycin B (Hygromycin B) at a final concentration of 150 µg / mL. Genomic DNA of yeast transformants was extracted according to the steps in the instructions of the DNA extraction kit of Bioengineering Co., Ltd., and then verified by PCR. The results are as follows: Figure 6 shown;

[0032] 2. Analysis of astaxanthin and carotenoid synthesis content in Rhodosporidium yeast after transformation of recombinant plasmid pRHcrtW-crtZ by HPLC

[0033] The strain transformed with the recombinant plasmid pRHcrtW-crtZ was cultured at 28°C for ...

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Abstract

The invention discloses an astaxanthin-producing Rhodosporidium kratochvilovae gene engineering strain which contains a nucleotide sequence of beta-carotene hydroxylase gene crtZ shown as SEQ ID NO:1,and a nucleotide sequence of beta-carotene ketolase gene crtW shown as SEQ ID NO:3. The beta-carotene ketolase gene crtW and beta-carotene hydroxylase gene crtZ are transformed into Rhodosporidium kratochvilovae YM25235 through an agrobacterium-mediated method for construction of the Rhodosporidium kratochvilovae gene engineering strain YM25235 / pRHcrtW-crtZ; and the crtZ and crtW genes expressedby the strain can further transform the beta-carotene in the YM25235 strain into astaxanthin, after fermentation culturing is carried out and comparing with a starting strain is carried out, the astaxanthin output of the engineering strain can reach 0.637 mg / g dry strain body, and a foundation is laid for large-scale commercial production of astaxanthin.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a rhodosporidium genetically engineered strain producing astaxanthin. In the invention, the β-carotene ketolase gene is mediated by Agrobacterium wxya and β-carotene hydroxylase gene crtZ Transformed into Rhodosporidium ( Rhodosporidium kratochvilovae ) in YM25235 to construct a Rhodosporidium genetically engineered strain for functional expression. The protease encoded by the two genes can convert β-carotene in the YM25235 strain into astaxanthin. Background technique [0002] Astaxanthin (chemical name: 3,3′-dihydroxy-4,4′-diketo-β-carotene), molecular formula C 40 h 52 o 4 , with a molecular weight of 596.86; it is a ketone carotenoid that widely exists in nature. Astaxanthin is the highest level product of carotenoid synthesis. Astaxanthin has strong antioxidant activity in nature, can effectively remove oxygen free radicals in cells, and also has anti-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/53C12R1/645
CPCC12N9/0077C12N9/0083C12Y114/15C12Y114/99
Inventor 张琦崔志城肖兴明郭彩娜魏云林林连兵季秀玲
Owner KUNMING UNIV OF SCI & TECH
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