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Recombinant bacteria used for producing beta-carotene and construction method utilizing Crispr-Cas9 technology

A technology of carotene and recombinant bacteria, applied in the field of metabolic engineering and combinatorial biology, can solve the problems of low yield, long time-consuming, and low efficiency of molecular technology

Pending Publication Date: 2019-04-23
SHAANXI NORMAL UNIV
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  • Abstract
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Problems solved by technology

[0010] The purpose of the present invention is to provide a stable high-yield β-carotene in view of the problems of low efficiency, high cost and long time consumption of traditional molecular techniques, as well as low stability and low yield in existing methods for producing β-carotene. - A recombinant bacterium of carotene, and a method for constructing the recombinant bacterium using Crispr-Cas9 technology

Method used

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  • Recombinant bacteria used for producing beta-carotene and construction method utilizing Crispr-Cas9 technology

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Embodiment 1

[0078] according to figure 1 The β-carotene biosynthesis pathway in Yarrowia lipolytica is shown, and the recombinant bacteria producing β-carotene is constructed, and the specific construction method is as follows:

[0079] 1. Knockout of ku70 gene in Yarrowia lipolytica

[0080] Using the gene of Yarrowia lipolytica Po1f as the template, the primers were designed according to the principle of overlapping extension PCR primer design, using primers ku70-up-F and ku70-up-R, the upstream homology arm of ku70 was amplified by fidelity enzyme pfu PCR (800bp), PCR amplification system: 5×FastPfu Buffer 10μL, ku70-up-F (10μM) 1μL, ku70-up-R (10μM) 1μL, 2.5mM dNTPs 4μL, Po1f gene 1μL, FastPfu DNA Polymerase 1μL, ddH 2 O 32μL; using primers ku70-down-F and ku70-down-R, PCR amplification of the downstream homology arm (800bp) of ku70 with fidelity enzyme pfu, PCR amplification system: 5×FastPfu Buffer 10μL, ku70-down -F (10μM) 1μL, ku70-down-R (10μM) 1μL, 2.5mM dNTPs 4μL, Po1f gene 1...

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Abstract

The invention discloses recombinant bacteria used for producing beta-carotene and a construction method utilizing a Crispr-Cas9 technology. A Ku70 gene is subjected to knockout from yarrowia lipolytica, and then phytoene synthase / lycopene cyclase (carRA), phytoene desaturase (carB), geranyl pyrophosphate synthetase (GGS1) and a 3-hydroxy-3-methyl glutaryl coenzyme A reductase (tHMG) gene take snfas a target point and are integrated in a yarrowia lipolytica gene set after knockout of the ku70 gene, wherein the phytoene synthase / lycopene cyclase (carRA) and phytoene desaturase (carB) are from blakeslea trispora, and the geranyl pyrophosphate synthetase (GGS1) and the 3-hydroxy-3-methyl glutaryl coenzyme A reductase (tHMG) gene are from the yarrowia lipolytica; the Crispr-Cas9 technology isutilized for regulating the copy number of the carRA, the carRA, the GGS1 and the tHMG, and the recombinant bacteria capable of producing the high-yield beta-carotene is constructed. After the recombinant bacteria is fermented, cultured, extracted and separated, the content of the beta-carotene can reach the dry cell weight of 35 mg / g, and the bacterial strain stability is high.

Description

technical field [0001] The invention belongs to the technical field of metabolic engineering and combinatorial biology, and in particular relates to a method for constructing a recombinant bacterium that utilizes Crispr-Cas9 technology to improve the yield of beta-carotene. Background technique [0002] β-Carotene is the most common and stable carotenoid in nature, and has antioxidant function due to the existence of a large number of double bonds in its structure. In the human body, β-carotene is mainly used as a precursor of vitamin A (pro-vitamin A), and is a Class A nutritional food fortifier recognized by the WHO and FAO Food Additives Joint Expert Committee. Studies have shown that beta-carotene has the functions of preventing cancer, anti-tumor and enhancing human immune function. In addition, β-carotene can act as an antioxidant for low-density lipoprotein (LDL), and the oxidation of LDL is closely related to the development of atherosclerosis, so supplementation of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/90C12P23/00C12R1/645
CPCC12N15/905C12P23/00C07K14/37C07K14/39C12N2800/80C12N2810/10
Inventor 刘梁孟永宏屈玉玲杨凡钱亚丹
Owner SHAANXI NORMAL UNIV
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