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Recombinant yeast for producing xanthophyll and application of recombinant yeast

A technology of recombinant yeast and lutein, applied in the biological field, can solve problems such as limiting substrate conversion efficiency and product synthesis, and achieve the effects of solving competitive utilization problems, promoting synthesis, and good application prospects

Active Publication Date: 2021-03-30
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For substrates accumulated on the cell membrane, such as carotenoids, the free protein has less chance of contacting the substrate, which limits the conversion efficiency of the substrate and the synthesis of the product

Method used

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  • Recombinant yeast for producing xanthophyll and application of recombinant yeast
  • Recombinant yeast for producing xanthophyll and application of recombinant yeast
  • Recombinant yeast for producing xanthophyll and application of recombinant yeast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Cloning and expression of enzyme genes required for lutein biosynthesis

[0024] 1. The enzymes required in the lutein synthesis pathway of plants or algae, combined with the existing enzyme library in the laboratory: phytoene synthase, phytoene dehydrogenase, lycopene β-cyclase (W.Xie, X.Lv, L.Ye, P.Zhou, H.Yu, Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering. Metab.Eng.30,69-78, doi:10.1016 / j .ymben.2015.04.009 (2015)), found the rest of the corresponding nucleic acid sequence or protein sequence in NCBI (see Table 1), through the codon optimization website, according to the corresponding yeast host codon optimization, the optimized gene sequence Send it to Gene Company for synthesis, and obtain three enzymes that can be successfully expressed in yeast, namely: lycopene ε-cyclase, carotene β-hydroxylase, and carotene ε-hydroxylase.

[0025] Table 1: Genes required for synthesis

[00...

Embodiment 2

[0037] Example 2: Introduction of a temperature-sensitive Gal4 mutant (Gal4M9)

[0038] 1. Knock out the GAL4 gene by homologous recombination in Saccharomyces cerevisiae. In this process, a HIS3 knockout cassette for GAL4 is constructed: GAL4 upstream homologous segment-HIS3 expression cassette-GAL4 downstream homologous segment. Fragment 1 expressing HIS3 was amplified from PUG23 (GenBank: AF298783.1) with GAL4-HisF1 and GAL4-HisXF1, and the upstream homology arm was amplified from the genome of Saccharomyces cerevisiae BY4741 with GAL4-CyF and GAL4-HISR1, which is the fragment 2. Use GAL4-His-XR1 and GAL4-XinF1 to amplify the downstream homology arm from the Saccharomyces cerevisiae BY4741 genome, which is fragment 3, and perform overlap extension PCR on the three fragments to obtain the HIS3 knockout GAL4 fragment (see Table 2 for primers) . The transcription activator GAL4 of Saccharomyces cerevisiae YY00 constructed in Example 1 was knocked out by using the HIS3 express...

Embodiment 3

[0043] Example 3: Verification of Cell Membrane Localization Sequences

[0044] 1. Primer EGFP-F and EGFP-R Using PUG23 (GenBank: AF298783) as a template to amplify the EGFP fragment by PCR, use NotI and SpeI for the EGFP fragment and the PESC-URA-pPGK1-tADH1 plasmid (see figure 2 ) for double enzyme digestion, transformation after ligation, and cloning to obtain PESC-URA-pPGK1-EGFP-tADH1.

[0045] 2. Utilize the plasmid obtained by cloning in step 1, the primers in Table 4, and use high-fidelity enzyme (Prime STAR TM HSDNA polymerase) to construct plasmids by CPEC (Circular polymerase extension cloning) method. The CPEC reaction system (10 μl) is: Prime Star buffer: 2 μl; dNTPs (2.5mM): 1 μl; primer 1: 0.4 μl; primer 2: 0.4 μl; template: 0.2 μl; DNA polymerase: 0.1 μl; Water: 5.9 μl.

[0046] The PCR program was as follows: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at Tm±5°C for 20 s, extension at 72°C at 1 kb / min, a total of 20 cyc...

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Abstract

The invention discloses a recombinant yeast for producing xanthophyll. An octahydro-lycopene synthase gene, an octahydro-lycopene dehydrogenase gene, a lycopene epsilon cyclase gene, a lycopene beta cyclase gene, a carotene beta hydroxylase gene and a carotene epsilon hydroxylase gene are exogenously introduced into the yeast. The six enzymes capable of being successfully expressed in yeast are obtained through gene cloning and codon optimization, and a genetically engineered yeast strain for producing xanthophyll is constructed. The delta carotene is produced under the catalysis of phytoene synthase, phytoene dehydrogenase and lycopene epsilon cyclase, and then the delta carotene is converted into xanthophyll under the catalysis of lycopene beta cyclase, carotene beta hydroxylase and carotene epsilon hydroxylase.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant yeast for producing lutein and its application. Background technique [0002] The chemical name of Lutein is 3,3'-diol-α-carotene, which can be synthesized in higher plants and algae, and plays an important role in physiological metabolism. As an important part of the plant photosystem, lutein captures light energy in photosynthesis, regulates plant growth and development, and participates in the interaction between plants and the environment as a biological signal molecule. Lutein is also an important component of retinal macular pigment, which has the function of absorbing blue light to protect the eyes. Since humans cannot produce lutein themselves, they need to be ingested from food. Studies have shown that lutein can prevent and treat retinal damage, including age-related macular degeneration, glaucoma, and diabetic retinopathy. As a nutraceutical, lutein can imp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/53C12N15/54C12N15/61C12P23/00C12R1/865
CPCC12N9/001C12N9/1085C12N9/90C12N9/0083C12N9/0073C12P23/00C12Y103/05005C12Y205/01032C12Y505/01018C12Y505/01019C12Y114/99045C12Y114/13129C12N2800/22
Inventor 于洪巍叶丽丹边旗
Owner ZHEJIANG UNIV
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