Recombinant saccharomyces cerevisiae for producing astaxanthin, and application of recombinant saccharomyces cerevisiae

A technology for recombining Saccharomyces cerevisiae and Saccharomyces cerevisiae, which is applied in the field of microorganisms, can solve the problems of limiting astaxanthin production, achieve good industrial development prospects, improve metabolic flux, increase yield and purity

Active Publication Date: 2021-11-26
WANHUA CHEM (SICHUAN) CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, due to the accumulation of a large number of intermediates in the two-step network metabolic pathway from β-carotene to astaxanthin synthesis, the production of astaxanthin is limited, so the metabolic flux of the downstream network metabolic pathway is dredged, reducing The accumulation of intermediates is of great significance for improving the synthesis of astaxanthin in Saccharomyces cerevisiae

Method used

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  • Recombinant saccharomyces cerevisiae for producing astaxanthin, and application of recombinant saccharomyces cerevisiae
  • Recombinant saccharomyces cerevisiae for producing astaxanthin, and application of recombinant saccharomyces cerevisiae
  • Recombinant saccharomyces cerevisiae for producing astaxanthin, and application of recombinant saccharomyces cerevisiae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Construction of recombinant plasmid PRS416-ADH1t-Gal1-TDH3p-TDH2t

[0043] 1. The genomic DNA of Saccharomyces cerevisiae S228C (Saccharomyces cerevisiae S228C) was used as a template and ADH1t_F and ADH1t_R were used as primers to perform PCR amplification to obtain ADH1t. The sequence of ADH1t is shown in SEQ ID NO:3.

[0044] ADH1t_F: 5'-agctttggacttcttcgccagagg-3' (SEQ ID NO: 1);

[0045] ADH1t_R: 5'-catgccggtagagaggtgtggtcaataag-3' (SEQ ID NO: 2).

[0046] 2. Using the genomic DNA of Saccharomyces cerevisiae S228C as a template and using Gal1_F and Gal1_R as primers to perform PCR amplification to obtain Gal1, the sequence of Gal1 is shown in SEQ ID NO:6.

[0047] Gal1_F: 5'-tatagttttttctccttgacgttaaagtatag-3' (SEQ ID NO: 4);

[0048] Gal1_R: 5'-ttatattgaattttcaaaaattcttactttttttttggatgg-3' (SEQ ID NO: 5).

[0049] 3. Using the genomic DNA of Saccharomyces cerevisiae S228C as a template and using TDH3p_F and TDH3p_R as primers to perform PCR amplifica...

Embodiment 2

[0068] Example 2: Insertion of foreign genes

[0069] 1. Using the AaCrtZ gene as a template and using Primer 5_F and Primer 5_R as primers to perform PCR amplification to obtain BsaI-AaCrtZ-BsaI, the sequence of BsaI-AaCrtZ-BsaI is shown in SEQ ID NO: 25, and the AaCrtZ gene is shown in SEQ ID NO: 25 Shown in the 9-497 position.

[0070] Primer 5_F: 5'-ggtctccaatgactaacttcttgatcgttgttg-3' (SEQ ID NO: 23);

[0071] Primer 5_R: 5'-ggtctccattaagttctttcttgagcttcag-3' (SEQ ID NO: 24).

[0072] 2. Using the BDC263CrtW gene as a template and using Primer 6_F and Primer 6_R as primers to perform PCR amplification to obtain BsaI-BDC263CrtW-BsaI, the sequence of BsaI-BDC263CrtW-BsaI is shown in SEQ ID NO: 28, and the BDC263CrtW gene is shown in SEQ ID NO: 28 in the 9-734 shown.

[0073] Primer 6_F: 5'-ggtctccaatgtccgctgttactccaatg-3' (SEQ ID NO: 26);

[0074] Primer 6_R: 5'-ggtctccattattgaaaataaagaccaccaaggc-3' (SEQ ID NO: 27).

[0075] 3. Using the AaCrtZ gene as a template and u...

Embodiment 3

[0086] Example 3: Construction of recombinant yeast strains producing astaxanthin

[0087] The recombinant expression vectors PRS416-ADH1t-AaCrtZ-Gal1-TDH3p-BDC263CrtW-TDH2t and PRS416-ADH1t-BDC263CrtW-Gal1-TDH3p-AaCrtZ-TDH2t obtained in Example 2 were transformed into strains producing β-carotene by the lithium acetate method Scy10026 Saccharomyces cerevisiae, and through SC-Ura solid medium (synthetic yeast nitrogen source YNB 6.7g / L, glucose 20g / L, mixed amino acid powder 2g / L excluding uracil, 2% agar powder) Transformants were screened to obtain astaxanthin-producing recombinant Saccharomyces cerevisiae strains S1 and S2, respectively.

[0088] The construction method of the Scy10026 Saccharomyces cerevisiae is as follows: transfer the CYC1t-BtCrtI-HXT7p-TDH3p-PaCrtB-ADH1t fragment (SEQ ID NO: 37) into Saccharomyces cerevisiae S288C, perform homologous recombination and replace gal1, gal7, gal10 genes, and integrate into On the chromosome; then transfer the CYC1t-PaCrtY-...

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Abstract

The invention discloses recombinant saccharomyces cerevisiae for producing astaxanthin, and application of the recombinant saccharomyces cerevisiae. According to the recombinant saccharomyces cerevisiae disclosed by the invention, a [beta]-carotene ketolase gene CrtW is subjected to constitutive expression, a [beta]-carotene hydroxylase gene CrtZ is subjected to inducible expression, and an intermediate product [beta]-carotene is synthesized into the astaxanthin. According to the recombinant saccharomyces cerevisiae, a constitutive promoter is used for regulating and controlling the CrtW gene to be firstly expressed, the inducible promoter is used for regulating and controlling the CrtZ gene to be expressed subsequently, so that the metabolic flux of the synthesis path of the astaxanthin of the saccharomyces cerevisiae is obviously improved, the yield of the astaxanthin reaches 464.90 mg/L, and the recombinant saccharomyces cerevisiae has a good industrial development prospect.

Description

technical field [0001] The invention belongs to the field of microorganisms, and relates to a recombinant brewer's yeast producing astaxanthin and its application. Background technique [0002] Astaxanthin (3,3'-dihydroxy-β,β'-carotene-4,4'-dione, C 40 h 52 o 4 , 596.84) has lipophilicity and hydrophilicity, and is also one of the strongest antioxidants found in nature so far. Its antioxidant capacity is 800 times that of coenzyme Q10, 700 times that of anthocyanins, vitamin E 550 times. Astaxanthin has two asymmetric carbons, which are located at the 3 and 3' positions of the β-ionone ring. According to the difference of the two chiral carbons, it is divided into (3R, 3'R), (3S, 3'S), ( 3R, 3'S) three different configurations, among which (3S, 3'S) configuration has the strongest antioxidant capacity. Astaxanthin has multiple conjugated double bonds, which can scavenge free radicals inside and outside the cell membrane, reduce protein and lipid oxidation and DNA damage...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P23/00C12R1/865
CPCC12N9/0069C12N9/0073C12Y114/13129C12P23/00
Inventor 杨祖明王竞辉张雅萍张稳姜西娟黎源
Owner WANHUA CHEM (SICHUAN) CO LTD
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