Genetic engineering bacteria producing beta-carotene, and application thereof

A technology of genetically engineered bacteria and carotene, which is applied in the fields of metabolic engineering and synthetic biology, can solve the problems of low production cost, decreased expression, and low enzyme activity, and achieve the effects of simplifying the production process, increasing yield, and reducing production costs

Active Publication Date: 2020-05-01
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, β-carotene is a fat-soluble compound stored in liposomes, and Saccharomyces cerevisiae is a non-oleaginous yeast, which limits the application of Saccharomyces cerevisiae as an expression host
In addition, heterologous expression of β-carotene-producing genes in Saccharomyces cerevisiae,...

Method used

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  • Genetic engineering bacteria producing beta-carotene, and application thereof
  • Genetic engineering bacteria producing beta-carotene, and application thereof
  • Genetic engineering bacteria producing beta-carotene, and application thereof

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Experimental program
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Embodiment approach

[0033] As mentioned above, many excellent characteristics make Saccharomyces cerevisiae often used as an ideal host for heterologous protein expression and construction of metabolic pathways. However, β-carotene is a fat-soluble compound stored in liposomes, and Saccharomyces cerevisiae is a non-oleaginous yeast, which limits the application of Saccharomyces cerevisiae as an expression host. In addition, heterologous expression of β-carotene-producing genes in Saccharomyces cerevisiae may result in decreased expression or low enzyme activity due to differences among species, ultimately leading to low production of β-carotene. In view of this, the present inventors have conducted a lot of research on the construction of β-carotene-producing genetically engineered bacteria using Saccharomyces cerevisiae as a host.

[0034] The inventors found that, using Saccharomyces cerevisiae as the expression host, the strong promoter P TDH3 and P TEF1 By bidirectionally expressing the het...

Embodiment 1

[0062] Embodiment 1: the construction of the basic strain of yeast producing β-carotene

[0063] according to figure 2 shown by P TDH3 and P TEF1 Plasmid map of heterologous genes controlled by bidirectional promoters, respectively integrating heterologous genes into the yeast genome. tHMG1, which is the catalytic domain of 3-hydroxy-3-methylglutaryl reductase HMGR, a plasmid containing a single-copy heterologous gene crtE expressed from a bidirectional promoter and the catalytic domain (tHMG1) gene of the rate-limiting step HMG1 was treated with XbaI Restriction enzymes for linearization. The specific operation method of the linearized plasmid is as follows:

[0064] Take 30 μL of plasmids containing crtE and tHMG1 genes, 5 μL of CutSmart solution, 1 μL of XbaI restriction endonuclease, and 14 μL of double distilled water, making a total of 50 μL of the system.

[0065] The linearized plasmid was concentrated by ethanol precipitation. The specific method is as follows:...

Embodiment 2

[0081] Example 2: Using CRISPR-Cas9 technology to knock out phospholipid phosphatase genes PAH1, DPP1 and LPP1 in one step Using the pCas9-lacZ plasmid as a template, the Golden Gate method was used to construct a multi-fragment plasmid. BsaI is a special enzyme that cuts a few bases after the recognition site GGTCTC, so a specific cohesive end is designed to achieve seamless splicing of multiple fragments.

[0082] The primer sequences used to construct the recombinant plasmid pCas9-pdl are as follows:

[0083] pah1.1F aaaggtctcaGATCCTGGACAAGCTGATTCCACGGTTTTTAGAGCTAGAAATAGCAAGTTA

[0084] dpp1.1R aaaggtctcaCTGATGGGTGACTTGCGCAAGCCCGGAATCGAACCGGG

[0085] dpp1.2F aaaggtctcaTCAGAGGATCCGTTTTTAGAGCTAGAAATAGCAAGTTAA

[0086] lpp1.3R aaaggtctctAAACAGCGGACATTCAAGTTTCATTGCGCAAGCCCGGAATCGAACCGGG

[0087] Using primers pah1.1F and dpp1.1R, and primers dpp1.2F and lpp1.3R, two fragments containing three gRNAs were amplified with NEB Q5 enzyme, respectively.

[0088]The PCR amplificat...

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Abstract

The invention belongs to the technical field of metabolic engineering and synthetic biology, and relates to genetic engineering bacteria producing beta-carotene. The genetic engineering first uses wild-type yeast as host bacteria, and strong promoter-expressed heterologous genes crtYB, crtI, crtE, and a catalytic region (tHMG1) gene of a rate-limiting step HMG1 are separately inserted into a genome of the host bacteria to construct initial recombinant saccharomyces cerevisiae producing beta-carotene; and then sterol acyltransferase genes (ARE1 and ARE2 genes) are overexpressed in the initial recombinant saccharomyces cerevisiae, and genes (PAH1, DPP1 and LPP1 genes) encoding phosphatidic acid phosphatase activity are knocked out to perform chassis microorganism transformation on the recombinant saccharomyces cerevisiae. The obtained recombinant saccharomyces cerevisiae can produce beta-carotene with relatively high yield and relatively low cost, the strain is stable during subculture,and no reverse mutations can appear.

Description

technical field [0001] The invention belongs to the technical field of metabolic engineering and synthetic biology, and relates to genetically engineered bacteria producing beta-carotene and applications thereof. Background technique [0002] Carotenoids are a class of tetraterpenoid fat-soluble compounds containing 40 carbon atoms. They have rich and bright colors and are widely used in food, cosmetics, medicine and health care. It has attracted considerable attention due to its potential benefits to human health and several industrial applications. β-carotene is a kind of carotenoid and one of the precursors of vitamin A. It has good antioxidant properties and can reduce the damage caused by active oxygen to cells or tissues. It is widely used. β-carotene crystals are generally orange or red, belonging to fat-soluble pigments, stored in liposomes, easily soluble in petroleum ether and phenol, slightly soluble in ethanol, insoluble in water, and its concentration in petrol...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12P23/00C12R1/865
CPCC12N1/18C12P23/00C12N9/1029
Inventor 刘子鹤延斯·尼尔森赵一瑾张跃平
Owner BEIJING UNIV OF CHEM TECH
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