Rhodobacter sphaeroides engineering strain for producing beta-carotene and construction method thereof

A technology of Rhodobacter sphaeroides and carotene, applied in the field of metabolic engineering, can solve the problems of long cycle, instability, complex fermentation process, etc.

Active Publication Date: 2018-12-28
SHAANXI HEALTHFUL BIOLOGICAL ENG
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Problems solved by technology

[0004] At present, the only strain used for industrial production of β-carotene is Blakesleatrispora, but Blakesleatrispora lacks effective molecular manipulation technology, and its bacteria are divided

Method used

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  • Rhodobacter sphaeroides engineering strain for producing beta-carotene and construction method thereof

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Embodiment 1

[0078] according to figure 1 The β-carotene biosynthetic pathway in Rhodobacter sphaeroides shown, constructs the engineering strain of Rhodobacter sphaeroides producing β-carotene, and the specific construction method is as follows:

[0079] 1. Optimizing the lycopene cyclase gene crtY derived from Pantoea agglomerans Pa codons and promoters

[0080] Lycopene cyclase gene crtY from Pantoea agglomerans Pa After codon optimization, the nucleotide sequence is obtained as opt crtY shown in SEQID NO:1 Pa Gene (synthesized by Nanjing GenScript Biotechnology Co., Ltd.). The episomal plasmid pIND containing 5 different promoters BBa_J95025, BBa_J95027, BBa_J95026, tac and prrnB 4 -opt crtY Pa (synthesized by Nanjing GenScript Biotechnology Co., Ltd.) were heat-shocked and transformed into S17-1 competent cells. The transformation operation method was as follows: take 1 μL of plasmid with a pipette gun and mix gently with S17-1 competent cells, and put them in an ice bath. Place...

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Abstract

The invention discloses a rhodobacter sphaeroides engineering strain for producing beta-carotene and a construction method thereof. The method includes first optimizing a codon of a lycopene cyclase gene crtYPa derived from pantoea agglomerans to obtain an opt crtYPa gene, then replacing an endogenous phytoene three-step dehydrogenase gene crtI3 of rhodobacter sphaeroides with the opt crtYPa genecontaining a promoter prrnB and a phytoene four-step dehydrogenase gene crtIPa gene derived from the pantoea agglomerans in a traceless mode, then knocking out an endogenous neurosporene hydroxylase gene crtC of the rhodobacter sphaeroides and a 6-phosphoglucose dehydrogenase gene zwf and finally knocking out an endogenous 1-deoxyxylulose-5-phosphate synthase gene dxs of the rhodobacter sphaeroides with integration expression at zwf position to obtain the rhodobacter sphaeroides engineering strain for producing the beta-carotene. The strain is cultured by fermentation, and the content of the beta-carotene can reach 30mg/g DCW.

Description

technical field [0001] The invention belongs to the technical field of metabolic engineering, and in particular relates to a method for constructing a β-carotene-producing strain by transforming Rhodobacter sphaeroides with metabolic engineering. Background technique [0002] β-carotene is widely found in carrots, wolfberry and other green vegetables and yellow and orange fruits. Due to its unsaturated structure, it has strong antioxidant activity and the ability to scavenge free radicals, which can effectively improve the immune function of the body , has been widely used in functional food, medicine and health care and cosmetics and other fields, and the international market demand is increasing day by day. [0003] Natural β-carotene mainly comes from plant extraction and microbial fermentation, but the production cost of plant-derived β-carotene remains high due to the limitation of raw materials, while chemically synthesized β-carotene is mostly all-trans structure, bio...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N1/21C12P23/00C12R1/01
CPCC12N9/001C12N9/1022C12N9/90C12N15/74C12P23/00C12Y103/99031C12Y202/01007C12Y505/01018
Inventor 孟永宏强珊苏安平陈芝李颖
Owner SHAANXI HEALTHFUL BIOLOGICAL ENG
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