Recombinant plasmid and recombinant yeast strain and establishing method and application thereof

A recombinant plasmid, paracoccus technology, applied in the field of genetic engineering, can solve the problems of high complexity and increased yield

Active Publication Date: 2016-08-17
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] For the Saccharomyces cerevisiae system, the astaxanthin synthesis gene crtS of Xanthophyllomycesdendrorhous and its corresponding P450 reductase crtR were expressed in Saccharomyces cerevisiae in 2009. After crtZ of Pantoea ananatis and crtW of Paracoccus sp. (NBRC101723), the yield increased slightly, reaching 29 μg/g; Two copies of hydroxylase and ketolase genes derived from Haematococcus pluvialis were introduced into the β-carotene yeast cells, and the astaxanthin yield was about 3.5mg/g during YPD fermentation, and then Introduce three copies of the hydroxylase and ketolase genes from Haematococcus pluvialis and add 0.52mM Fe during YPD fermentation 2+ , finally achieved 4.7mg/g astaxanthin synthesis (without adding Fe 2+ The output per hour is only about 4.2mg/g), which is the highest yield k

Method used

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  • Recombinant plasmid and recombinant yeast strain and establishing method and application thereof
  • Recombinant plasmid and recombinant yeast strain and establishing method and application thereof
  • Recombinant plasmid and recombinant yeast strain and establishing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Acquisition of foreign genes

[0066] Find Aa crtZ, Asp crtZ, Eu crtZ, Pa crtZ, Ps crtZ, Ss crtZ, B.SD212crtZ, B.DC263crtZ, HpChyb, Aa crtW, Asp crtW, CrBKT, Gv crtW, Np73102crtW, Nsp7120crtW, BSD212crtW, The corresponding amino acid sequences of B.SD212, B.DC263crtW, ReSH019crtO, and SDC18crtW were optimized, and the codons were optimized. Symmetrical BsaI and NotI restriction sites were introduced at both ends, namely NotI-BsaI-crtZ / crtW-BsaI-NotI. EcoR V was blunt-ended into pUC57 or pUC57-Kan vector to obtain pUC57-Kan / pUC57-crtZ and pUC57-Kan / pUC57-crtW, which were synthesized by GenScript.

Embodiment 2

[0067] Example 2: Acquisition of ADH1t-FBA1p / TEF1p-TDH3p-TDH2t Fragment

[0068] ADH1t, FBA1p, TDH3p, TDH2t and TEF1p were amplified by PCR using the genome of Saccharomyces cerevisiae BY4741 as a template, and then spliced ​​by OE-PCR to obtain ADH1t-FBA1p / TEF1p-TDH3p-TDH2t with PstI and BamHI restriction sites at both ends Fragment, in which a BsmBI restriction site was added between FBA1p / TEF1p and ADH1t, and a BsaI restriction site was added between TDH3p and TDH2t; then digested with restriction endonucleases PstI and BamHI, and then connected to the PLD2 vector to obtain PLD2-ADH1t-FBA1p / TEF1p-TDH3p-TDH2t spare.

Embodiment 3

[0069] Example 3: Insertion of foreign genes

[0070] The pUC57-Kan / pUC57-crtW in Example 1 was digested with BsaI and then connected to the PLD2-ADH1t-FBA1p / TEF1p-TDH3p-TDH2t vector that was also digested with BsaI to obtain PLD2-ADH1t-FBA1p / TEF1p-TDH3p- crtW-TDH2t, then pUC57-Kan / pUC57-crtZ in Example 1 was digested with BsaI and then connected to PLD2-ADH1t-FBA1p / TEF1p-TDH3p-crtW-TDH2t digested with BsmBI in turn to obtain PLD2-ADH1t -crtZ-FBA1p / TEF1p-TDH3p-crtW-TDH2t.

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Abstract

The invention relates to the technical field of gene engineering, and discloses a recombinant plasmid and a recombinant yeast strain and an establishing method and application thereof. The recombinant plasmid comprises a gene segment which is formed by sequentially splicing a terminator ADH1t, a beta-carotene hydroxylase gene crtZ, a promoter FBA1p or TEF1p, a promoter TDH3p, a beta-carotene ketolase gene crtW and a terminator TDH2t. By selecting the specific terminators, the specific promoters and the exogenous genes crtZ and crtW, the gene segment capable of producing astaxanthin is formed; thus, the recombinant plasmid is obtained and converted into the specific brewing yeast strain, the high yield of astaxanthin is ensured, and a more economical, more efficient and simpler mode is provided for heterologous microorganism production of astaxanthin.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and more specifically relates to a recombinant plasmid, a recombinant yeast strain and a construction method and application thereof. Background technique [0002] Astaxanthin (3,3'-dihydroxy-4,4'-diketo-β,β'-astaxanthin) is a member of the isoquinoid family. As a natural red pigment, it is widely found in birds, fish and crustaceans. Due to its strong antioxidant activity and singlet oxygen quenching ability, astaxanthin can slow down aging, maintain skin health, enhance the immune system, and prevent cardiovascular diseases, and has potential market value in the field of food, medicine and cosmetics. At present, commercial astaxanthin is mainly derived from chemical synthesis, but because of the configuration and food safety issues of synthetic astaxanthin, it can only be used in the field of coloring in aquaculture. However, the extraction of natural astaxanthin from Haematococcu...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C12N15/66C12P23/00C12R1/865
CPCC12N9/0069C12N9/0071C12N15/66C12N15/81C12P23/00
Inventor 肖文海王瑞钊王颖元英进
Owner TIANJIN UNIV
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