Genetically engineered bacterium for producing alpha-carotene and construction method and application of genetically engineered bacterium

A technology of genetically engineered bacteria and carotene, which is applied in the field of genetic engineering and achieves good application prospects

Active Publication Date: 2016-11-16
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, β-carotene and lycopene have been successfully synthesized in yeast or Escherichia coli using plant-derived genes,...

Method used

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  • Genetically engineered bacterium for producing alpha-carotene and construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing alpha-carotene and construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing alpha-carotene and construction method and application of genetically engineered bacterium

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Embodiment 1

[0019] The applicant found that compared with other plants, banana (Musa nana Lour.) can accumulate a high proportion of α-carotene, and cloned banana tomato red from banana to α-carotene produced by cyclization of δ-carotene β-cyclase gene (MaLCYB), so the expression vector of this gene was constructed and transformed into engineering bacteria containing pAc-Delta plasmid to obtain the genetically engineered bacteria producing α-carotene that can accumulate a large amount of α-carotene.

[0020] The concrete steps of embodiment are described below:

[0021] a) extraction of banana RNA and obtaining of cDNA:

[0022] RNA extraction was performed using the CTAB method. The specific steps are: take 1 g of banana fruit ground with liquid nitrogen, add 4 mL of CTAB solution (100 mM Tris-HCl (pH 8.0), 2M NaCl, 25 mM EDTA, 2% CTAB, 2% PVP, 0.5 g / L spermidine (Spermidine)) and 70 μL β-mercaptoethanol were mixed, added 4 mL of chloroform / isoamyl alcohol (24:1), vortexed for 20 seco...

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Abstract

The invention discloses a genetically engineered bacterium for producing alpha-carotene and a construction method and application of the genetically engineered bacterium. The construction method includes the steps that a banana lycopene beta-cyclase gene is connected with an expression carrier and then converted into an engineered bacterium containing pAC-Delta plasmids, and the genetically engineered bacterium for producing alpha-carotene is obtained, wherein the nucleotide sequence of the banana lycopene beta-cyclase gene is shown as SEQ ID NO.1. The yield of alpha-carotene can reach up to 17.21 mg/L after the genetically engineered bacterium is subjected to fermentation culture for 3 days. High-purity and high-yield alpha-carotene can be directly obtained from thalli of the genetically engineered bacterium, mass production of high-pruity alpha-carotene can be achieved, and the genetically engineered bacterium has good application prospects.

Description

Technical field: [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetically engineered bacterium producing α-carotene, a construction method and application thereof. Background technique: [0002] α-carotene can be used as an important provitamin A, and recent series of studies have found that it can also effectively inhibit cancer metastasis, and can be used as an anti-metastasis agent for cancer, or as an adjuvant for anticancer drugs. The accumulation of β-carotene is common in most plants and microorganisms, but the proportion of α-carotene is relatively low. Moreover, the existing microbial synthesis is mainly aimed at β-carotene, and there is no effective synthesis of α-carotene in microorganisms. The α-carotene obtained commercially is very expensive and the production cost is high. Sigma officially sells α-carotene with a purity of more than 95% at a price of 4389 yuan / mg. In addition, the chemical synthes...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/61C12N15/70C12N1/21C12P23/00
CPCC12N9/90C12P23/00C12Y505/01019
Inventor 傅秀敏杨子银程思华杜冰蒋跃明段学武
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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