Method for producing beta-carotene, and genetically engineered bacterium thereof

A technology of genetically engineered bacteria and carotene, applied in microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve problems such as low stability and complex operation

Active Publication Date: 2015-07-29
SHANGHAI INST OF PHARMA IND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The technical problem to be solved in the present inven

Method used

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  • Method for producing beta-carotene, and genetically engineered bacterium thereof
  • Method for producing beta-carotene, and genetically engineered bacterium thereof
  • Method for producing beta-carotene, and genetically engineered bacterium thereof

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0065] Example 1 Construction of recombinant strain pET32a(+)-dxs-idi-ispDF-pACYC184-M-crt

[0066] 1.1 Construction of upstream plasmid pET32a(+)-dxs-idi-ispDF

[0067] 1.1.1 Cloning of key enzyme genes in the MEP pathway

[0068] 1.1.1.1 Escherichia coli deoxyxylulose-5-phosphate synthase gene (dxs) cloning

[0069] Extract Escherichia coli (Escherichia coli) K12MG1655 (purchased from the National Culture Collection Center, ie ATCC NO.10798) genomic DNA, design primers according to the vector pET32a(+), and then PCR amplify the deoxyxylulose-5-phosphate synthase gene (dxs), GenBank accession number: 945060, and then use the gel recovery kit to recover the target gene fragment.

[0070] The amplification primer sequences are:

[0071] dxs-upstream: catg CCATGG CTAGTTTTGATATTGCCAAATACCCG (SEQ ID NO. 1);

[0072] Downstream of dxs: CG GAATTC TTATGCCAGCCAGGCCTTGATTTTG (SEQ ID NO. 2).

[0073] 1.1.1.2 Escherichia coli isovaleryl pyrophosphate isomerase gene (idi) cloning...

Embodiment 2

[0107] Example 2 Construction of recombinant strain pTrcHis2B-dxs-idi-ispDF-pACYC184-M-crt

[0108] 2.1 Construction of upstream plasmid pTrcHis2B-dxs-idi-ispDF

[0109] 2.1.1 Cloning of key enzyme genes in the MEP pathway

[0110] 2.1.1.1 Escherichia coli deoxyxylulose-5-phosphate synthase gene (dxs) cloning

[0111] Genomic DNA of Escherichia coli K12MG1655 was extracted, primers were designed according to the vector pTrcHis2B (purchased from Shanghai Karma Biotech), and then deoxyxylulose-5-phosphate synthase gene (dxs) was amplified by PCR, GenBank accession number: 945060, and recovered by gel The kit recovers the target gene fragment.

[0112] The amplification primer sequences are:

[0113] dxs-upstream: catg CCATGG CTAGTTTTGATATTGCCAAATACCCG (SEQ ID NO. 7);

[0114] Downstream of dxs: CCG CTCGAG TTATGCCAGCCAGGCCTTGATTTTG (SEQ ID NO. 8).

[0115] 2.1.1.2 Escherichia coli isovaleryl pyrophosphate isomerase gene (idi) cloning

[0116] Genomic DNA of Escherichia ...

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Abstract

The invention discloses a method for producing beta-carotene, and a genetically engineered bacterium thereof. The method comprises the following steps: culturing an Escherichia coli genetically engineered bacterium, and obtaining beta-carotene from a fermentation solution, wherein the Escherichia coli genetically engineered bacterium is Escherichia coli containing an exogenous expression vector containing four beta-carotene synthesis genes, and the four beta-carotene synthesis genes are crtE, crtI, crtB and crtY respectively, the Escherichia coli genetically engineered bacterium also contains an expression vector containing four MEP pathway genes, and the four MEP pathway genes are dxs, idi, ispD and ispF respectively. Four enzymes in MEP pathways are over-expressed to construct the MEP pathways with different intensity promoters in order to improve the output of beta-carotene.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for producing beta-carotene and genetically engineered bacteria. Background technique [0002] β-carotene belongs to terpenoids and is a member of the carotenoid family. It is widely used in food, medicine, cosmetics and health products. [0003] At present, the production methods of β-carotene include the production of chemical synthesis and the production of natural products, and the chemical synthesis products are all trans structures, and the natural products are cis and trans mixed types. Chemical synthesis as intermediate C in the synthesis of vitamin A 14 Aldehyde and vitamin A are used as raw materials to start synthesis, and the production method of natural products is mainly extracted from natural plants, silkworm excrement, salina and microbial fermentation broth. Due to people's requirements for the pharmacological activity of β-carotene, natural products have ...

Claims

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Application Information

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IPC IPC(8): C12P23/00C12N1/21C12N15/70C12R1/19
Inventor 胡又佳谢丽萍陈习平张琪朱宝泉刘艳
Owner SHANGHAI INST OF PHARMA IND
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