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

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...

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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 known so far; however, this technical method needs to be realized through a relatively complicated and cumbersome gene integration method, and at the same time, multiple copies need to be introduced in order to increase the output of astaxanthin. The degree of cumbersomeness is higher; Ni Mengxiang from China Pharmaceutical University et al performed crtZ from Brevundimonas, Xanthobacter autotrophicus, Paracoccussp.N81106, Pantoea ananatis, Pantoea ananatis LMG 20103 and from Nostocpunctiforme, Parvularculaber mudensis, Paracoccus sp.N81106, Brevundimonassp. Tried different combinations and found that astaxanthin can only be produced when crtZ from Brevundimonasd and crtW from Nostoc punctiforme or Brevundimonas sp.SD212 are expressed together, while other combinations can only produce intermediate metabolites

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